Regenerative Neurology and Regenerative Cardiology: Shared Hurdles and Achievements.

From the first success in cultivation of cells in vitro, it became clear that developing cell and/or tissue specific cultures would open a myriad of new opportunities for medical research. Expertise in various in vitro models has been developing over decades, so nowadays we benefit from highly specific in vitro systems imitating every organ of the human body. Moreover, obtaining sufficient number of standardized cells allows for cell transplantation approach with the goal of improving the regeneration of injured/disease affected tissue. However, different cell types bring different needs and place various types of hurdles on the path of regenerative neurology and regenerative cardiology. In this review, written by European experts gathered in Cost European action dedicated to neurology and cardiology-Bioneca, we present the experience acquired by working on two rather different organs: the brain and the heart. When taken into account that diseases of these two organs, mostly ischemic in their nature (stroke and heart infarction), bring by far the largest burden of the medical systems around Europe, it is not surprising that in vitro models of nervous and heart muscle tissue were in the focus of biomedical research in the last decades. In this review we describe and discuss hurdles which still impair further progress of regenerative neurology and cardiology and we detect those ones which are common to both fields and some, which are field-specific. With the goal to elucidate strategies which might be shared between regenerative neurology and cardiology we discuss methodological solutions which can help each of the fields to accelerate their development.

Combined segmentation and classification-based approach to automated analysis of biomedical signals obtained from calcium imaging.

Automated screening systems in conjunction with machine learning-based methods are becoming an essential part of the healthcare systems for assisting in disease diagnosis. Moreover, manually annotating data and hand-crafting features for training purposes are impractical and time-consuming. We propose a segmentation and classification-based approach for assembling an automated screening system for the analysis of calcium imaging. The method was developed and verified using the effects of disease IgGs (from Amyotrophic Lateral Sclerosis patients) on calcium (Ca2+) homeostasis. From 33 imaging videos we analyzed, 21 belonged to the disease and 12 to the control experimental groups. The method consists of three main steps: projection, segmentation, and classification. The entire Ca2+ time-lapse image recordings (videos) were projected into a single image using different projection methods. Segmentation was performed by using a multi-level thresholding (MLT) step and the Regions of Interest (ROIs) that encompassed cell somas were detected. A mean value of the pixels within these boundaries was collected at each time point to obtain the Ca2+ traces (time-series). Finally, a new matrix called feature image was generated from those traces and used for assessing the classification accuracy of various classifiers (control vs. disease). The mean value of the segmentation F-score for all the data was above 0.80 throughout the tested threshold levels for all projection methods, namely maximum intensity, standard deviation, and standard deviation with linear scaling projection. Although the classification accuracy reached up to 90.14%, interestingly, we observed that achieving better scores in segmentation results did not necessarily correspond to an increase in classification performance. Our method takes the advantage of the multi-level thresholding and of a classification procedure based on the feature images, thus it does not have to rely on hand-crafted training parameters of each event. It thus provides a semi-autonomous tool for assessing segmentation parameters which allows for the best classification accuracy.

MK-801 effect on regional cerebral oxidative stress rate induced by different duration of global ischemia in gerbils.

We investigated MK-801 effect on ischemia-induced oxidative stress-the most important factor that exacerbates brain damage by reperfusion. The common carotid arteries of gerbils were occluded for 5, 10, or 15 min. Immediately after the occlusion, MK-801 (3 mg/kg i.p.) or saline were given in normothermic conditions. The MK-801 effects were followed in vivo by monitoring the neurological status of animals and at the intracellular level by standard biochemical assays. We investigated nitric oxide levels, superoxide production, superoxide dismutase activity, index of lipid peroxidation (ILP), and reduced glutathione content in hippocampus, striatum, forebrain cortex, and cerebellum. The measurements took place at different times (1, 2, 4, 7, 14, and 28 days) after reperfusion. Increased duration of cerebral ischemia resulted in a progressive induction of oxidative stress. Our results revealed pattern of dynamic changes in each oxidative stress parameter level which corresponded with ischemia duration in all tested brain structures. Most sensitive oxidative stress parameters were ILP and superoxide production. Our study confirmed spatial distribution of ischemia-induced oxidative stress. Tested brain structures showed different sensitivity to each oxidative stress parameter. As judged by biochemical and neurological data, applied MK-801 showed neuroprotective efficiency by reduction of ischemia-induced oxidative stress in brain.

Heterogeneity in brain distribution of activated microglia and astrocytes in a rat ischemic model of Alzheimer's disease after 2 years of survival.

The present study was designed to follow neuroinflammation after ischemic brain injury in the long-term survival rat model. Immunohistochemistry was performed 2 years after 10 min global brain ischemia due to cardiac arrest. For the visualization of the cellular inflammatory reaction microglial marker Iba1 and astrocyte marker GFAP were used. In post-ischemic animals our study revealed significant activation of astrocytes in all tested brain regions (hippocampal CA1 and CA3 areas and dentate gyrus, motor and somatosensory cortex, striatum and thalamus), while microglial activation was only found in CA1 and CA3 areas, and the motor cortex. In the specifically sensitive brain areas microglia and astrocytes showed simultaneously significant activation, while in the resistant brain areas only astrocytes were activated. Thus, there was clear evidence of less intensive neuroinflammation in brain areas resistant to ischemia. Such neuroinflammatory processes are backed by microglia and astrocytes activity even up to 2 years after ischemia-reperfusion brain injury. Our study thus revealed a chronic effect of global cerebral ischemia on the neuroinflammatory reaction in the rat brain even 2 years after the insult.

Temporal patterns of soluble adhesion molecules in cerebrospinal fluid and plasma in patients with the acute brain infraction.

The aim of this study was to define concentration changes of soluble adhesion molecules (sICAM-1, sVCAM-1 and sE-Selectin) in cerebrospinal fluid and plasma, as well as, number of peripheral blood leukocytes and the albumin coefficient in the patients with the acute brain infarction. We also, analyzed the correlation between the measured levels, the infarct volume and the degree of neurological and the functional deficit. The study included 50 patients with the acute cerebral infarction and the control group consisted of 16 patients, age and sex matched. Obtained results showed significant increase in number of leukocytes, the albumin coefficient and the level of soluble adhesion molecules within the first seven days in patients. The highest values of measured parameters were noted within the third and the fourth day after the insult, which is the suggested period of maximal intensity of inflammatory reactions. Significant correlation was found between measured parameters and the infarct volume, the degree of neurological and the functional deficit. The results suggest that investigated parameters in CSF and blood represent a dynamic index of inflammatory events as one of the fundametal mechanisms responsible for neuron damage during acute phase of brain infarction.

Effect of continuous exposure to alternating magnetic field (50 Hz, 0.5 mT) on serotonin and dopamine receptors activity in rat brain.

External magnetic fields (MFs) have the ability to modify motor activity of animals, complex type of behaviour connected with dopaminergic and serotonergic neurotransmissions in the brain. Thus, the purpose of this study was to examine MF-induced changes in the activity of serotonin 5-HT(2A) receptors in the prefrontal cortex, as well as dopamine D(1) and D(2) receptors in the striatum of adult Wistar rats, considering their involvement in motor behavior regulation. Experimental animals were continuously exposed to extremely low frequency MF (ELF-MF, 50 Hz, 0.5 mT) for 1, 3, and 7 days. Subsequently, binding properties (K(d) and B(max)) of receptors were determined by in vitro radioligand receptor binding assays. It was shown that the affinity of serotonin 5-HT(2A) receptors decreased and their density increased in the prefrontal cortex of rats after ELF-MF exposure. Regarding affinity, this effect was duration-dependent and most prominent after 7-day of ELF-MF exposure. In contrast to serotonin 5-HT(2A) receptors in the prefrontal cortex, ELF-MF had no significant effect on the affinity and density of dopamine D(1) and D(2) receptors in the striatum. We can conclude that continuous exposure to ELF-MF up to 7 days affects cortical serotonergic neurotransmission, whereby intensity of these changes depends on ELF-MF exposure duration.

Temporal patterns of motor behavioural improvements by MK-801 in Mongolian gerbils submitted to different duration of global cerebral ischemia.

The purpose of this study was to investigate the temporal pattern of NMDA receptors antagonist-MK-801 on motor behaviour parameters in gerbils submitted to different duration of global cerebral ischemia. The common carotid arteries of gerbils were occluded for 5, 10 or 15min. Gerbils were given MK-801 (3mg/kg i.p.) or saline immediately after the occlusion in normothermic conditions prior to testing. Motor activity was registered 1, 2, 4, 7, 14, 21 and 28 days after reperfusion during 60min by open field test. At the same time, the effect of NMDA receptor blockade was followed in vivo by monitoring the neurological status of whole animals or at the cellular level by standard light and confocal microscopy on brain slices. Post-ischemic gerbils quickly developed hypermotor response with the most intensity in animals submitted to 15min ischemia. MK-801 administrated immediately after ischemia significantly decreased this hyperactivity. In all ischemic-treated animals, behavioural suppression by MK-801 was observed already 1 day after occlusion and was lasting as far as observed ischemia-dependent hypermotor responses. Beneficial effect of MK-801 was also confirmed by morphological and neurological status data. These findings suggest that sustained ischemia-induced hyperactivity is related to abnormalities in NMDA glutamatergic function, as well as its manifestation could be completely abolished by NMDA receptor blockade immediately after ischemic insult.

The extracellular matrix glycoprotein tenascin-C and matrix metalloproteinases modify cerebellar structural plasticity by exposure to an enriched environment.

The importance of the extracellular matrix (ECM) glycoprotein tenascin-C (TnC) and the ECM degrading enzymes, matrix metalloproteinases (MMPs) -2 and -9, in cerebellar histogenesis is well established. This study aimed to examine whether there is a functional relationship between these molecules in regulating structural plasticity of the lateral deep cerebellar nucleus. To this end, starting from postnatal day 21, TnC- or MMP-9-deficient mice were exposed to an enriched environment (EE). We show that 8 weeks of exposure to EE leads to reduced lectin-based staining of perineuronal nets (PNNs), reduction in the size of GABAergic and increase in the number and size of glutamatergic synaptic terminals in wild-type mice. Conversely, TnC-deficient mice showed reduced staining of PNNs compared to wild-type mice maintained under standard conditions, and exposure to EE did not further reduce, but even slightly increased PNN staining. EE did not affect the densities of the two types of synaptic terminals in TnC-deficient mice, while the size of inhibitory, but not excitatory synaptic terminals was increased. In the time frame of 4-8 weeks, MMP-9, but not MMP-2, was observed to influence PNN remodeling and cerebellar synaptic plasticity as revealed by measurement of MMP-9 activity and colocalization with PNNs and synaptic markers. These findings were supported by observations on MMP-9-deficient mice. The present study suggests that TnC contributes to the regulation of structural plasticity in the cerebellum and that interactions between TnC and MMP-9 are likely to be important for these processes to occur.

Time course of motor behavior changes in Mongolian gerbils submitted to different durations of cerebral ischemia.

In addition to morphological changes, global cerebral ischemia leads to functional changes that can be assessed by behavioral examination. The purpose of this study was to investigate the impact of the duration of global cerebral ischemia on the time course of a comprehensive set of motor behaviors in Mongolian gerbils. The common carotid arteries of gerbils were occluded either for 5 min, 10 min, or 15 min. Gerbil motor behavior was recorded in the open field at 24 h, 48 h, 4 days, 7 days, 14 days, 21 days, and 28 days after reperfusion. Each session lasted for 60 min and was composed of six intervals of 10 min. Our results revealed that ischemic gerbils quickly develop locomotor and stereotypic hyperactivity, with the expected decrease of resting time. The most evident effect was observed in gerbils submitted to a 15 min ischemia, whose locomotor activity returned to nearly normal values after 7 days. In contrast, the duration of global cerebral ischemia had no effects on rearing, clockwise, or counter-clockwise rotation. These findings indicate that exposure to global cerebral ischemia induces changes in locomotion, stereotypy, and resting time. The magnitude and duration of these effects depend on the duration of ischemia.

Effects of cooling on the response of the snail bursting neuron to acetylcholine.

The Br-type neuron of the snail Helix pomatia, involved in neuronal regulation of various homeostatic and adaptive mechanisms, represents an interesting model for studying effects of temperature change on neuronal activity of poikilotherms. Acetylcholine induces a transient, inward dose-dependent current in the identified Br neuron. In the work presented, we analyzed the effects of cooling on the acetylcholine-induced inward current. The amplitude of acetylcholine-induced inward current was markedly decreased after cooling, and the speed of the decay of acetylcholine response was decreased.

Mitochondrial superoxide production and MnSOD activity after exposure to agonist and antagonists of ionotropic glutamate receptors in hippocampus.

Competitive glutamate receptor antagonists APV and CNQX were injected with kainate unilaterally into CA3 region of the rat hippocampus. They both expressed sufficient neuroprotection in the sense of decreasing superoxide production and increasing MnSOD levels, but with differential effect in mechanisms and time dynamics. Our findings suggest that NMDA and AMPA/kainate receptors are differentially involved in superoxide production, and they also impose different neuroprotective effects on the induction of MnSOD activity in hippocampus.

Effects of some heavy metals on the action potentials of an identified Helix pomatia photosensitive neuron.

In the photosensitive MB neuron in the left parietal ganglion of Helix pomatia, the onset of light prolongs significantly (by about 40%) the duration of the action potential. The broadening of the action potential after the onset of light was found to be due to its calcium component and could not be induced after blocking Ca(2+) channels by Cd(2+) and Pb(2+) and in absence of Ca(2+) in medium. The blocking effect of both compounds was reversible. It was found that CdCl(2) exhibited a more intense blocking effect than PbCl(2).

The role of autophagy and lipolysis in survival of astrocytes under nutrient deprivation.

Astrocytes can survive nutrient deprivation (ND) for days. However, the pro-survival strategy of astrocytes under such a metabolic challenge is still not clear. In the present study, we examined the effects of inhibition of two potential steps in energy acquisition during ND: autophagy (using chloroquine) and lipolysis (using orlistat). The inhibition of autophagy did not show significant effects on cell viability until 8-9h of ND. From that point onwards, the number of dead cells gradually increased, reaching ∼60% between 10 and 12h of ND. In addition, early inhibition of autophagy made astrocytes more vulnerable to the latter ND. The inhibition of lipolysis decreased the viability of cells exposed to ND, but this appeared much later compared to the inhibition of autophagy. The application of orlistat prevented ND-related hyperpolarization of the mitochondrial membrane, and mitochondria became swollen. This study clearly shows that autophagy and lipolysis are essential for the survival of astrocytes under ND conditions, which might be related to their role as neuron-supporting cells.

Novel molecular biomarkers at the blood-brain barrier in ALS.

Recently neuroinflammation has gained a particular focus as a key mechanism of ALS. Several studies in vivo as well as in vitro have nominated immunoglobulin G (IgG) isolated from ALS patients as an active contributor to disease onset and progression. We have shown that ALS IgG affects astroglial Ca(2+) excitability and induces downstream activation of phosphatidylinositol 3-kinase. These studies were hampered by a lack of knowledge of the pathway of entry of immune factors in the CNS. Our MRI data revealed the blood-brain barrier BBB leakage and T cell infiltration into brain parenchyma in ALS G93A rats. Since astrocyte ensheathes blood vessel wall contributing to BBB stability and plays an important role in ALS pathogenesis, we have studied astrocytic membrane proteins water channel aquaporin-4 and the inwardly rectifying potassium channel. In this review, we will summarize data related to BBB disruption with particular emphasis on impaired function of astrocytes in ALS. We will discuss implication of membrane proteins expressed on astrocytic endfeet, aquaporin-4, and inwardly rectifying potassium channel in the pathology of ALS. In addition to ALS-specific IgGs, these membrane proteins are proposed as novel biomarkers of the disease.

Neural ECM molecules in synaptic plasticity, learning, and memory.

Neural extracellular matrix (ECM) molecules derived from neurons and glial cells accumulate in the extracellular space and regulate synaptic plasticity through modulation of perisomal GABAergic inhibition, intrinsic neuronal excitability, integrin signaling, and activities of L-type Ca(2+) channels, NMDA receptors, and Rho-associated kinase. Genetic or enzymatic targeting of ECM molecules proved to bidirectionally modulate acquisition of memories, depending on experimental conditions, and to promote cognitive flexibility and extinction of fear and drug memories. Furthermore, evidence is accumulating that dysregulation of ECM is linked to major psychiatric and neurodegenerative diseases and that targeting ECM molecules may rescue cognitive deficits in animal models of these diseases. Thus, the ECM emerged as a key component of synaptic plasticity, learning, and memory and as an attractive target for developing new generation of synapse plasticizing drugs.

Extremely low frequency magnetic field (50 Hz, 0.5 mT) reduces oxidative stress in the brain of gerbils submitted to global cerebral ischemia.

Magnetic field as ecological factor has influence on all living beings. The aim of this study was to determine if extremely low frequency magnetic field (ELF-MF, 50 Hz, 0.5 mT) affects oxidative stress in the brain of gerbils submitted to 10-min global cerebral ischemia. After occlusion of both carotid arteries, 3-month-old gerbils were continuously exposed to ELF-MF for 7 days. Nitric oxide and superoxide anion production, superoxide dismutase activity and index of lipid peroxidation were examined in the forebrain cortex, striatum and hippocampus on the 7(th) (immediate effect of ELF-MF) and 14(th) day after reperfusion (delayed effect of ELF-MF). Ischemia per se increased oxidative stress in the brain on the 7(th) and 14(th) day after reperfusion. ELF-MF also increased oxidative stress, but to a greater extent than ischemia, only immediately after cessation of exposure. Ischemic gerbils exposed to ELF-MF had increased oxidative stress parameters on the 7(th) day after reperfusion, but to a lesser extent than ischemic or ELF-MF-exposed animals. On the 14(th) day after reperfusion, oxidative stress parameters in the brain of these gerbils were mostly at the control levels. Applied ELF-MF decreases oxidative stress induced by global cerebral ischemia and thereby reduces possible negative consequences which free radical species could have in the brain. The results presented here indicate a beneficial effect of ELF-MF (50 Hz, 0.5 mT) in the model of global cerebral ischemia.

Neuroprotective efficiency of tetanus toxin C fragment in model of global cerebral ischemia in Mongolian gerbils.

The tetanus toxin C (TTC) fragment capacity of being transported in a retrograde way through motoneurons and its nontoxic nature opens the door to a new promising therapeutic strategy for neurodegenerative diseases. In this study, the TTC effect was tested for the first time in animal model of global cerebral ischemia induced by 10-min occlusion of both common carotid arteries. The aim was to evaluate the effect of TTC gene therapy treatment on the development and expression of global cerebral ischemia/reperfusion-induced oxidative stress and motor hyperactivity in Mongolian gerbils. Several oxidative stress and motor behavioral parameters were investigated between 2 h and 14 days after reperfusion. Neuroprotective efficiency of TTC was observed in the forebrain cortex, striatum, hippocampus, and cerebellum at the level of each examined oxidative stress parameter (nitric oxide level, superoxide production, superoxide dismutase activity, and index of lipid peroxidation). Additionally, TTC significantly decreased ischemia-induced motor hyperactivity based on tested parameters (locomotion, stereotypy, and rotations). As judged by biochemical as well as behavioral data, treatment with TTC for the first time showed neuroprotective efficiency by reduction of ischemia-induced oxidative stress and motor hyperactivity and can be a promising strategy for ischemia-induced neuronal damage treatment.

Astrocytic mitochondrial membrane hyperpolarization following extended oxygen and glucose deprivation.

Astrocytes can tolerate longer periods of oxygen and glucose deprivation (OGD) as compared to neurons. The reasons for this reduced vulnerability are not well understood. Particularly, changes in mitochondrial membrane potential (Δψ(m)) in astrocytes, an indicator of the cellular redox state, have not been investigated during reperfusion after extended OGD exposure. Here, we subjected primary mouse astrocytes to glucose deprivation (GD), OGD and combinations of both conditions varying in duration and sequence. Changes in Δψ(m), visualized by change in the fluorescence of JC-1, were investigated within one hour after reconstitution of oxygen and glucose supply, intended to model in vivo reperfusion. In all experiments, astrocytes showed resilience to extended periods of OGD, which had little effect on Δψ(m) during reperfusion, whereas GD caused a robust Δψ(m) negativation. In case no Δψ(m) negativation was observed after OGD, subsequent chemical oxygen deprivation (OD) induced by sodium azide caused depolarization, which, however, was significantly delayed as compared to normoxic group. When GD preceded OD for 12 h, Δψ(m) hyperpolarization was induced by both GD and subsequent OD, but significant interaction between these conditions was not detected. However, when GD was extended to 48 h preceding OGD, hyperpolarization enhanced during reperfusion. This implicates synergistic effects of both conditions in that sequence. These findings provide novel information regarding the role of the two main substrates of electron transport chain (glucose and oxygen) and their hyperpolarizing effect on Δψ(m) during substrate deprivation, thus shedding new light on mechanisms of astrocyte resilience to prolonged ischemic injury.

Neural ECM proteases in learning and synaptic plasticity.

Recent studies implicate extracellular proteases in synaptic plasticity, learning, and memory. The data are especially strong for such serine proteases as thrombin, tissue plasminogen activator, neurotrypsin, and neuropsin as well as matrix metalloproteinases, MMP-9 in particular. The role of those enzymes in the aforementioned phenomena is supported by the experimental results on the expression patterns (at the gene expression and protein and enzymatic activity levels) and functional studies, including knockout mice, specific inhibitors, etc. Counterintuitively, the studies have shown that the extracellular proteolysis is not responsible mainly for an overall degradation of the extracellular matrix (ECM) and loosening perisynaptic structures, but rather allows for releasing signaling molecules from the ECM, transsynaptic proteins, and latent form of growth factors. Notably, there are also indications implying those enzymes in the major neuropsychiatric disorders, probably by contributing to synaptic aberrations underlying such diseases as schizophrenia, bipolar, autism spectrum disorders, and drug addiction.

Response of hippocampal neurons and glial cells to alternating magnetic field in gerbils submitted to global cerebral ischemia.

The purpose of this study was to determine whether exposure to an extremely low-frequency magnetic field (ELF-MF, 50 Hz) affects the outcome of postischemic damage in the hippocampus of Mongolian gerbils. After 10-min bilateral carotid occlusion, the gerbils were continuously exposed to ELF-MF (average magnetic induction at the center of the cage was 0.5 mT) for 7 days. The impact of ELF-MF was estimated immediately (the 7th day after reperfusion) and 7 days after cessation of exposure (the 14th day after reperfusion) compared with ischemic gerbils without ELF-MF exposure. Applying stereological methods, histological evaluation of changes in the hippocampus was done for determining its volume, volume densities of degenerating neurons and astrocytes, as well as the number of microglial cells per unit area. ELF-MF per se did not induce any morphological changes, while 10-min global cerebral ischemia led to neuronal death, especially in CA1 region of the hippocampus, as expected. Ischemic gerbils exposed to ELF-MF had significantly a lower degree of cell loss in the examined structure and greater responses of astrocytes and microglial cells than postischemic gerbils without exposure on the seventh day after reperfusion (immediate effect of ELF-MF). Similar response was observed on the 14th day after reperfusion (delayed effect of ELF-MF); however, differences in measured parameters were low and insignificant. Applied ELF-MF has possible neuroprotective function in the hippocampus, as the most sensitive brain structure in the model of global cerebral ischemia, through reduction of neuronal death and activation of astrocytes and microglial cells.