Longitudinal Assessment of Blood-Based Inflammatory, Neuromuscular, and Neurovascular Biomarker Profiles in Intensive Care Unit-Acquired Weakness: A Prospective Single-Center Cohort Study.

BACKGROUND: The diagnosis of intensive care unit (ICU)-acquired weakness (ICUAW) and critical illness neuromyopathy (CINM) is frequently hampered in the clinical routine. We evaluated a novel panel of blood-based inflammatory, neuromuscular, and neurovascular biomarkers as an alternative diagnostic approach for ICUAW and CINM. METHODS: Patients admitted to the ICU with a Sequential Organ Failure Assessment score of ≥ 8 on 3 consecutive days within the first 5 days as well as healthy controls were enrolled. The Medical Research Council Sum Score (MRCSS) was calculated, and motor and sensory electroneurography (ENG) for assessment of peripheral nerve function were performed at days 3 and 10. ICUAW was defined by an MRCSS < 48 and CINM by pathological ENG alterations, both at day 10. Blood samples were taken at days 3, 10, and 17 for quantitative analysis of 18 different biomarkers (white blood cell count, C-reactive protein, procalcitonin, C-terminal agrin filament, fatty-acid-binding protein 3, growth and differentiation factor 15, syndecan 1, troponin I, interferon-γ, tumor necrosis factor-α, interleukin-1α [IL-1α], IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-13, and monocyte chemoattractant protein 1). Results of the biomarker analysis were categorized according to the ICUAW and CINM status. Clinical outcome was assessed after 3 months. RESULTS: Between October 2016 and December 2018, 38 critically ill patients, grouped into ICUAW (18 with and 20 without) and CINM (18 with and 17 without), as well as ten healthy volunteers were included. Biomarkers were significantly elevated in critically ill patients compared to healthy controls and correlated with disease severity and 3-month outcome parameters. However, none of the biomarkers enabled discrimination of patients with and without neuromuscular impairment, irrespective of applied classification. CONCLUSIONS: Blood-based biomarkers are generally elevated in ICU patients but do not identify patients with ICUAW or CINM. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02706314.

Mitochondrial Dysfunction in Intensive Care Unit-Acquired Weakness and Critical Illness Myopathy: A Narrative Review.

Mitochondria are key structures providing most of the energy needed to maintain homeostasis. They are the main source of adenosine triphosphate (ATP), participate in glucose, lipid and amino acid metabolism, store calcium and are integral components in various intracellular signaling cascades. However, due to their crucial role in cellular integrity, mitochondrial damage and dysregulation in the context of critical illness can severely impair organ function, leading to energetic crisis and organ failure. Skeletal muscle tissue is rich in mitochondria and, therefore, particularly vulnerable to mitochondrial dysfunction. Intensive care unit-acquired weakness (ICUAW) and critical illness myopathy (CIM) are phenomena of generalized weakness and atrophying skeletal muscle wasting, including preferential myosin breakdown in critical illness, which has also been linked to mitochondrial failure. Hence, imbalanced mitochondrial dynamics, dysregulation of the respiratory chain complexes, alterations in gene expression, disturbed signal transduction as well as impaired nutrient utilization have been proposed as underlying mechanisms. This narrative review aims to highlight the current known molecular mechanisms immanent in mitochondrial dysfunction of patients suffering from ICUAW and CIM, as well as to discuss possible implications for muscle phenotype, function and therapeutic approaches.

The Effect of Early Application of Synthetic Peptides 19-2.5 and 19-4LF to Improve Survival and Neurological Outcome in a Mouse Model of Cardiac Arrest and Resuscitation.

The synthetic antimicrobial peptides (sAMPs) Pep19-2.5 and Pep19-4LF have been shown in vitro and in vivo to reduce the release of pro-inflammatory cytokines, leading to the suppression of inflammation and immunomodulation. We hypothesized that intervention with Pep19-2.5 and Pep19-4LF immediately after cardiac arrest and resuscitation (CA-CPR) might attenuate immediate systemic inflammation, survival, and long-term outcomes in a standardized mouse model of CA-CPR. Long-term outcomes up to 28 days were assessed between a control group (saline) and two peptide intervention groups. Primarily, survival as well as neurological and cognitive parameters were assessed. In addition, systemic inflammatory molecules and specific biomarkers were analyzed in plasma as well as in brain tissue. Treatment with sAMPs did not provide any short- or long-term benefits for either survival or neurological outcomes, and no significant benefit on inflammation in the CA-CPR animal model. While no difference was found in the plasma analysis of early cytokines between the intervention groups four hours after resuscitation, a significant increase in UCH-L1, a biomarker of neuronal damage and blood-brain barrier rupture, was measured in the Pep19-4LF-treated group. The theoretical benefit of both sAMPs tested here for the treatment of post-cardiac arrest syndrome could not be proven.

Early Post-ischemic Brain Glucose Metabolism Is Dependent on Function of TLR2: a Study Using [18F]F-FDG PET-CT in a Mouse Model of Cardiac Arrest and Cardiopulmonary Resuscitation.

PURPOSE: The mammalian brain glucose metabolism is tightly and sensitively regulated. An ischemic brain injury caused by cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) affects cerebral function and presumably also glucose metabolism. The majority of patients who survive CA suffer from cognitive deficits and physical disabilities. Toll-like receptor 2 (TLR2) plays a crucial role in inflammatory response in ischemia and reperfusion (I/R). Since deficiency of TLR2 was associated with increased survival after CA-CPR, in this study, glucose metabolism was measured using non-invasive [18F]F-FDG PET-CT imaging before and early after CA-CPR in a mouse model comparing wild-type (WT) and TLR2-deficient (TLR2-/-) mice. The investigation will evaluate whether FDG-PET could be useful as an additional methodology in assessing prognosis. PROCEDURES: Two PET-CT scans using 2-deoxy-2-[18F]fluoro-D-glucose ([18F]F-FDG) tracer were carried out to measure dynamic glucose metabolism before and early after CPR. To achieve this, anesthetized and ventilated adult female WT and TLR2-/- mice were scanned in PET-CT. After recovery from the baseline scan, the same animals underwent 10-min KCL-induced CA followed by CPR. Approximately 90 min after CA, measurements of [18F]F-FDG uptake for 60 min were started. The [18F]F-FDG standardized uptake values (SUVs) were calculated using PMOD-Software on fused FDG-PET-CT images with the included 3D Mirrione-Mouse-Brain-Atlas. RESULTS: The absolute SUVmean of glucose in the whole brain of WT mice was increased about 25.6% after CA-CPR. In contrast, the absolute glucose SUV in the whole brain of TLR2-/- mice was not significantly different between baseline and measurements post CA-CPR. In comparison, baseline measurements of both mouse strains show a highly significant difference with regard to the absolute glucose SUV in the whole brain. Values of TLR2-/- mice revealed a 34.6% higher glucose uptake. CONCLUSIONS: The altered mouse strains presented a different pattern in glucose uptake under normal and ischemic conditions, whereby the post-ischemic differences in glucose metabolism were associated with the function of key immune factor TLR2. There is evidence for using early FDG-PET-CT as an additional diagnostic tool after resuscitation. Further studies are needed to use PET-CT in predicting neurological outcomes.

Fibroblast Growth Factor 21 as a Potential Biomarker for Improved Locomotion and Olfaction Detection Ability after Weight Reduction in Obese Mice.

Obesity is one of the most challenging diseases of the 21st century and is accompanied by behavioural disorders. Exercise, dietary adjustments, or time-restricted feeding are the only successful long-term treatments to date. Fibroblast growth factor 21 (FGF21) plays a key role in dietary regulation, but FGF21 resistance is prevalent in obesity. The aim of this study was to investigate in obese mice whether weight reduction leads to improved behaviour and whether these behavioural changes are associated with decreased plasma FGF21 levels. After establishing a model for diet-induced obesity, mice were subjected to three different interventions for weight reduction, namely dietary change, treadmill exercise, or time-restricted feeding. In this study, we demonstrated that only the combination of dietary change and treadmill exercise affected all parameters leading to a reduction in weight, fat, and FGF21, as well as less anxious behaviour, higher overall activity, and improved olfactory detection abilities. To investigate the interrelationship between FGF21 and behavioural parameters, feature selection algorithms were applied designating FGF21 and body weight as one of five highly weighted features. In conclusion, we concluded from the complementary methods that FGF21 can be considered as a potential biomarker for improved behaviour in obese mice after weight reduction.

Toll-like receptor 4 deficiency or inhibition does not modulate survival and neurofunctional outcome in a murine model of cardiac arrest and resuscitation.

BACKGROUND: Patients experiencing cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) often die or suffer from severe neurological impairment. Post resuscitation syndrome is characterized by a systemic inflammatory response. Toll-like receptor 4 (TLR4) is a major mediator of inflammation and TLR4 has been implicated in the pathogenesis of post-resuscitation encephalopathy. The aim of this study was to evaluate whether TLR4 deficiency or inhibition can modulate survival and neurofunctional outcome after CA/CPR. METHODS: Following intubation and central venous cannulation, CA was induced in wild type (C57Bl/6J, n = 38), TLR4 deficient (TLR4-/-, n = 37) and TLR4 antibody treated mice (5mg/kg MTS510, n = 15) by high potassium. After 10min, CPR was performed using a modified sewing machine until return of spontaneous circulation (ROSC). Cytokines and cerebral TNFalpha levels were measured 8h after CA/CPR. Survival, early neurological recovery, locomotion, spatial learning and memory were assessed over a period of 28 days. RESULTS: Following CA/CPR, all mice exhibited ROSC and 31.5% of wild type mice survived until day 28. Compared to wild type mice, neither TLR4-/- nor MTS510 treated wild type mice had statistically significant altered survival following CA/CPR (51.3 and 26.7%, P = 0.104 and P = 0.423 vs. WT, respectively). Antibody-treated but not TLR4-/- mice had higher IL-1ß and IL-6 levels and TLR4-/- mice had higher IL-10 and cerebral TNFalpha levels. No differences existed between mice of all groups in early neurological recovery, locomotion, spatial learning ability or remembrance. CONCLUSION: Therapeutic strategies targeting TLR4 may not be suitable for the reduction of mortality or neurofunctional impairment after CA/CPR.

Oral administration of the casein kinase 2 inhibitor TBB leads to persistent KCa2.2 channel up-regulation in the epileptic CA1 area and cortex, but lacks anti-seizure efficacy in the pilocarpine epilepsy model.

Temporal lobe epilepsy (TLE) is the most common epileptic syndrome in adults and often presents with seizures that prove intractable with currently available anticonvulsants. Thus, there is still a need for new anti-seizure drugs in this condition. Recently, we found that the casein kinase 2 inhibitor 4,5,6,7-tetrabromotriazole (TBB) prevented the emergence of spontaneous epileptic discharges in an acute in vitro epilepsy model. This prompted us to study the anti-seizure effects of TBB in the pilocarpine model of chronic epilepsy in vivo. To this end, we performed long-term video-EEG monitoring lasting 78-167 days of nine chronically epileptic rats and obtained a baseline seizure rate of 3.3 ± 1.3 per day (baseline of 27-80 days). We found a significant age effect with more pronounced seizure rates in older animals as compared to younger ones. However, the seizure rate increased to 6.3 ± 2.2 per day during the oral TBB administration (treatment period of 21-50 days), and following discontinuation of TBB, this rate remained stable with 5.2 ± 1.4 seizures per day (follow-up of 30-55 days). After completing the video-EEG during the follow-up the hippocampal tissue was prepared and studied for the expression of the Ca2+-activated K+ channel KCa2.2. We found a significant up-regulation of KCa2.2 in the epileptic CA1 region and in the neocortex, but in no other hippocampal subfield. Hence, our findings indicate that oral administration of TBB leads to persistent up-regulation of KCa2.2 in the epileptic CA1 subfield and in the neocortex, but lacks anti-seizure efficacy in the pilocarpine epilepsy model.

Interplay between interictal spikes and behavioral seizures in young, but not aged pilocarpine-treated epileptic rats.

Interictal spike activity is commonly observed in the EEG of patients with epilepsy, but the causal interrelationship between interictal spikes and behavioral seizures is poorly understood. We performed long-term video-EEG monitoring of 16 epileptic rats after pilocarpine-induced status epilepticus and five control animals. To quantify the interplay between periods of spikes and seizures, we calculated the time spent with spikes as well as the time spent with seizures for each animal. Within a given subject, we found a significant correlation between these two measures in 7/11 young epileptic rats (<400 days); this correlation was positive in six cases and negative in one. By contrast, none of five aged pilocarpine-treated animals exhibited significant correlation coefficients between spike periods and seizures (>600 days, P<0.05). Instead, aged epileptic rats showed a prominent predominance for either spike periods or seizures, which might explain the absence of significant correlation in this population. We found that there is a significant interplay between interictal periods of spikes and behavioral seizures in young epileptic animals, but this association is absent during aging.

Seizure frequency in pilocarpine-treated rats is independent of circadian rhythm.

Pilocarpine-induced status epilepticus (SE) results in chronic spontaneous recurrent seizures resembling human temporal lobe epilepsy. In this and other experimental models, behaviorally monitored seizure frequency was suggested to vary in a circadian fashion, and to increase with time. We re-addressed those hypotheses using continuous video-electroencephalography (EEG) telemetry in rats with SE at 30 days of age. In 11 chronically epileptic animals monitored up to 300 days after SE in a fixed 12 h light/dark cycle, we found that seizure frequency did not correlate with circadian rhythm.

High K+-induced contraction requires depolarization-induced Ca2+ release from internal stores in rat gut smooth muscle.

AIM: Depolarization-induced contraction of smooth muscle is thought to be mediated by Ca2+ influx through voltage-gated L-type Ca2+channels. We describe a novel contraction mechanism that is independent of Ca2+ entry. METHODS: Pharmacological experiments were carried out on isolated rat gut longitudinal smooth muscle preparations, measuring isometric contraction strength upon high K+-induced depolarization. RESULTS: Treatment with verapamil, which presumably leads to a conformational change in the channel, completely abolished K+-induced contraction, while residual contraction still occurred when Ca2+ entry was blocked with Cd2+. These results were further confirmed by measuring intracellular Ca2+ transients using Fura-2. Co-application of Cd2+ and the ryanodine receptor blocker DHBP further reduced contraction, albeit incompletely. Additional blockage of either phospholipase C (U 73122) or inositol 1,4,5-trisphophate (IP3)receptors (2-APB) abolished most contractions, while sole application of these blockers and Cd2+ (without parallel ryanodine receptor manipulation) also resulted in incomplete contraction block. CONCLUSION: We conclude that there are parallel mechanisms of depolarization-induced smooth muscle contraction via (a) Ca2+ entry and (b) Ca2+ entry-independent, depolarization-induced Ca2+-release through ryanodine receptors and IP3, with the latter being dependent on phospholipase C activation.

Functional significance of HCN2/3-mediated I(h) in striatal cells at early developmental stages.

Hyperpolarization-activated cAMP-gated cation currents (I(h)) were recently linked to pre- and postnatal developmental processes in several brain regions, including the ventral telencephalon. To evaluate the role of I(h) in striatal development, we used short-term cultured cells from the lateral ganglionic eminence at embryonic day 14 (E14) and postnatal days 1-3 (P1-3) as well as the embryonic striatal progenitor cell line ST14A. Western blot analysis of the I(h) underlying subunit proteins HCN1-4 revealed strong HCN2 expression in proliferating ST14A cells and weak expression in postmitotic ST14A cells and in cells from the developing brain. We also found HCN3 expression only in ST14A cells at both proliferative and nonproliferative stages but not in short-term cultured striatal cells. In all cases, HCN1 and HCN4 transcripts were below the detection level. Despite the selective protein expression, RT-PCR analysis showed stable expression of HCN2-4 but not HCN1 mRNA in all short-term-cultured striatal cells and in the ST14A cell line. Consistent with the strong protein expression, an I(h) was recorded with features of an HCN2-mediated current in ST14A cells at the proliferative stage and in short-term-cultured E14 cells. Of particular importance is that we detected no currents upon hyperpolarization in the ST14A cells at the nonproliferative stage when only HCN3 protein was present. These results suggest the potential importance of ST14A cells in defining the molecular mechanisms regulating I(h) expression and function.

Dopamine exerts no acute effects on Kv1.3 in activated encephalitogenic T cells.

Apart from a central function in the extrapyramidal motor system, dopamine has been suggested to play a role in neuroimmune interactions. Particularly in diseases of the central nervous system, such as multiple sclerosis, alterations in dopamine homeostasis might have immunological consequences. We investigated potential effects of dopamine stabilized by ascorbic acid on specifically activated encephalitogenic T cells at the peak of activation. Those cells exhibited an upregulation of voltage-sensitive K+ channels which play a role in many neurotransmitter responses of lymphocytes and fulfilled a prerequisite to respond to dopamine, i.e. stable expression of mRNA for dopamine receptors DRD1, DRD2 and DRD3. However, whole-cell and perforated whole-cell recordings revealed no change in voltage-sensitive K+ currents. Moreover, T cell proliferation was not changed in the presence of dopamine. Previously reported dopamine effects on T cells may be explained by a comparatively lower activation of the cells under investigation, suggesting an activation dependence of dopamine effects that may not be mediated by K+ channels. Alternatively, the occurrence of dopamine degradation products under unprotected conditions may account for the changes reported. Nevertheless, care should be taken when using the dopamine-protecting anti-oxidant ascorbic acid, since we found that it markedly inhibited both K+ currents and lymphocyte proliferation at higher concentrations.

An impaired neocortical Ih is associated with enhanced excitability and absence epilepsy.

Neuronal subthreshold excitability and firing behaviour are markedly influenced by the activation and deactivation of the somato-dendritic hyperpolarization-activated cation current (Ih). Here, we evaluated possible contributions of Ih to hyperexcitability in an animal model of absence seizures (WAG/Rij rats). We investigated pyramidal neurons of the somatosensory neocortex, the site of generation of spike-wave discharges. Ih-mediated functions in neurons from WAG/Rij rats, Wistar rats (sharing the same genetic background with WAG/Rij, but less epilepsy-prone) and ACI rats (an inbred strain, virtually free of seizures) were compared. We complemented whole-cell recordings from layer 2-3 pyramidal neurons with immunohistochemistry, Western blot and RT-PCR analysis of the h-channel subunits HCN1-4. The fast component of Ih activation in WAG/Rij neurons was significantly reduced (50% reduction in the h-current density) and four times slower than in neurons from nonepileptic Wistar or ACI rats. The results showing decreases in currents corresponded to a 34% reduction in HCN1 protein in the WAG/Rij compared to the Wistar neocortex, but HCN1 mRNA showed stable expression. The other three Ih subunit mRNAs and proteins (HCN2-4) were not affected. The alterations in Ih magnitude and kinetics of gating in WAG/Rij neurons may contribute to augmented excitatory postsynaptic potentials, the increase in their temporal summation and the facilitation of burst firing of these neurons because each of these effects could be mimicked by the selective Ih antagonist ZD 7288. We suggest that the deficit in Ih-mediated functions may contribute to the development and onset of spontaneously occurring hyperexcitability in a rat model of absence seizures.