Pavle R. Andjus

Structural features of ischemic damage in the hippocampus.

Cerebral ischemic injury resulting from either focal or global circulatory arrests in the brain is one of the major causes of death and disability in the adult population. The hippocampus, playing important roles in learning and memory, is selectively vulnerable to ischemic insults. Distinct populations of hippocampal neurons are targeted by ischemia and multiple factors, including excitotoxicity, oxidative stress, and inflammation, are responsible for their damage and death. Modifications of synapses occur very early after ischemia, reflecting related changes in synaptic transmission. These modifications structurally relate to spatial patterns formed by synaptic vesicles, geometry of postsynaptic density, and so forth. Ischemia‐induced changes of synaptic contacts can be implicated in the mechanisms leading to delayed neuronal death. In this review, we summarize the available data on the structural aspects of ischemic injury of the hippocampus obtained in tissue culture and animal models and discuss pathways of neurodegeneration common for cerebral ischemia and various neurodegenerative disorders. Anat Rec, 292:1914–1921, 2009.

Changes in the astrocytic aquaporin‐4 and inwardly rectifying potassium channel expression in the brain of the amyotrophic lateral sclerosis SOD1G93A rat model

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting upper and lower motor neurons. Dysfunction and death of motor neurons are closely related to the modified astrocytic environment. Astrocytic endfeet, lining the blood–brain barrier (BBB), are enriched in two proteins, aquaporin‐4 (AQP4) and inwardly rectifying potassium channel (Kir) 4.1. Both channels are important for the maintainance of a functional BBB astrocytic lining. In this study, expression levels of AQP4 and Kir4.1 were for the first time examined in the brainstem and cortex, along with the functional properties of Kir channels in cultured cortical astrocytes of the SOD1G93A rat model of ALS. Western blot analysis showed increased expression of AQP4 and decreased expression of Kir4.1 in the brainstem and cortex of the ALS rat. In addition, higher immunoreactivity of AQP4 and reduced immunolabeling of Kir4.1 in facial and trigeminal nuclei as well as in the motor cortex were also observed. Particularly, the observed changes in the expression of both channels were retained in cultured astrocytes. Furthermore, whole‐cell patch‐clamp recordings from cultured ALS cortical astrocytes showed a significantly lower Kir current density. Importantly, the potassium uptake current in ALS astrocytes was significantly reduced at all extracellular potassium concentrations. Consequently, the Kir‐specific Cs+‐ and Ba2+‐sensitive currents were also decreased. The changes in the studied channels, notably at the upper CNS level, could underline the hampered ability of astrocytes to maintain water and potassium homeostasis, thus affecting the BBB, disturbing the neuronal microenvironment, and causing motoneuronal dysfunction and death.

In Vivo Morphological Changes in Animal Models of Amyotrophic Lateral Sclerosis and Alzheimer's‐Like Disease: MRI Approach

Magnetic resonance imaging (MRI) is the only noninvasive technique that provides structural information on both cell loss and metabolic changes. After reviewing all the results obtained in clinical studies, reliable biomarkers in neurological diseases are still lacking. Diffusional MRI, MR spectroscopy, and the assessment of regional atrophy are promising approaches, but they cannot be simultaneously used on a single patient. Thus, for further research progress, reliable animal models are needed. To this aim, we have used the clinical MRI to assess neurodegenerative processes in the hSOD‐1G93A ALS rat model and in the trimethyltin (TMT)‐treated model of Alzheimers‐like disease. T2‐weighted (T2W) hyperintensive neurodegenerative foci were found in the brainstem of the ALS rat with apparent lateral ventricle dilation (T1W—hypointensity vs. T2W—hyperintensity). Degenerative processes in these areas were also confirmed by confocal images of GFAP‐positive astrogliosis. MRI after i.v.i. of magnetic anti‐CD4 antibodies indicated an accumulation of inflammatory cells near dilated ventricles. TMT‐treated rats also revealed the dilation of lateral ventricles. Expected deterioration in the hippocampus was not observed by clinical MRI, but immunocytochemistry could reveal significant redistribution of macro‐ and microglia in this structure. In both models, Gd‐DTPA contrast revealed a compromised blood brain barrier that may serve as the passage for inflammatory immune cells in the vicinity of dilated lateral ventricles. Moreover, in both models the midbrain region of the dorsal hippocampus was the target of BBB compromise, thus revealing a potentially vulnerable point that can be the primary target of neurodegeneration in the central nervous system. Anat Rec, 292:1882–1892, 2009.

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.

Protective role of fructose in the metabolism of astroglial C6 cells exposed to hydrogen peroxide.

Astroglial cells represent the main line of defence against oxidative damage related to neurodegeneration. Therefore, protection of astroglia from an excess of reactive oxygen species could represent an important target of the treatment of such conditions. The aim of our study was to compare the abilities of glucose and fructose, the two monosaccharides used in diet and infusion, to protect C6 cells from hydrogen peroxide (H(2)O(2))-mediated oxidative stress. It was observed using confocal microscopy with fluorescent labels and the MTT test that fructose prevents changes of oxidative status of the cells exposed to H(2)O(2) and preserves their viability. Even more pronounced protective effects were observed for fructose 1,6-bis(phosphate). We propose that fructose and its intracellular forms prevent H(2)O(2) from participating in the Fenton reaction via iron sequestration. As fructose and fructose 1,6-bis(phosphate) are able to pass the blood-brain barrier, they could provide antioxidative protection of nervous tissue in vivo. So, in contrast to the well-known negative effects of frequent consumption of fructose under physiological conditions, acute infusion or ingestion of fructose or fructose 1,6-bis(phosphate) could be of benefit in the cytoprotective therapy of neurodegenerative disorders related to oxidative stress.

Tenascins and inflammation in disorders of the nervous system

In vitro and in vivo studies on the role of tenascins have shown that the two paradigmatic glycoproteins of the tenascin family, tenascin-C (TnC) and tenascin-R (TnR) play important roles in cell proliferation and migration, fate determination, axonal pathfinding, myelination, and synaptic plasticity. As components of the extracellular matrix, both molecules show distinct, but also overlapping dual functions in inhibiting and promoting cell interactions depending on the cell type, developmental stage and molecular microenvironment. They are expressed by neurons and glia as well as, for TnC, by cells of the immune system. The functional relationship between neural and immune cells becomes relevant in acute and chronic nervous system disorders, in particular when the blood brain and blood peripheral nerve barriers are compromised. In this review, we will describe the functional parameters of the two molecules in cell interactions during development and, in the adult, in synaptic activity and plasticity, as well as regeneration after injury, with TnC being conducive for regeneration and TnR being inhibitory for functional recovery. Although not much is known about the role of tenascins in neuroinflammation, we will describe emerging knowledge on the interplay between neural and immune cells in autoimmune diseases, such as multiple sclerosis and polyneuropathies. We will attempt to point out the directions of experimental approaches that we envisage would help gaining insights into the complex interplay of TnC and TnR with the cells that express them in pathological conditions of nervous and immune systems.

Amyotrophic lateral sclerosis immunoglobulins G enhance the mobility of Lysotracker-labelled vesicles in cultured rat astrocytes

Aim:  We examined the effect of purified immunoglobulins G (IgG) from patients with amyotrophic lateral sclerosis (ALS) on the mobility and exocytotic release from Lysotracker‐stained vesicles in cultured rat astrocytes.

Relevance of the ability of fructose 1,6-bis(phosphate) to sequester ferrous but not ferric ions

The cytoprotective activity of F16BP has been documented in severe conditions such as convulsions, reperfusion injury, septic shock, diabetic complications, hypothermia-induced injury, UV-provoked skin damage and in other processes including apoptosis and excitotoxicity. F16BP shows very efficient cytoprotective activity in astroglial cells exposed to H(2)O(2)-provoked oxidative stress and during neuronal injury caused by hypoxic conditions. As most of the aforementioned processes involve iron activity-related conditions, we investigated the ferric and ferrous iron binding properties of F16BP under physiological conditions using (31)P NMR and EPR spectroscopy. Our results indicate that cytoprotective F16BP activity is predominantly based on ferrous iron sequestration. (31)P NMR spectroscopy of F16BP employing paramagnetic properties of iron clearly showed that F16BP forms stabile complexes with Fe(2+) which was verified by EPR of another divalent cation-Mn(2+). On the other hand, F16BP does not sequester ferric iron nor does it increase its redox activity as shown by (31)P NMR and EPR spin-trapping. Therefore, F16BP may be beneficial in neurodegenerative and other conditions that are characterised by ferric iron stores and deposits.

Fluctuating vs. Continuous Exposure to H2O2: The Effects on Mitochondrial Membrane Potential, Intracellular Calcium, and NF-κB in Astroglia

The effects of H2O2 are widely studied in cell cultures and other in vitro systems. However, such investigations are performed with the assumption that H2O2 concentration is constant, which may not properly reflect in vivo settings, particularly in redox-turbulent microenvironments such as mitochondria. Here we introduced and tested a novel concept of fluctuating oxidative stress. We treated C6 astroglial cells and primary astrocytes with H2O2, using three regimes of exposure – continuous, as well as fluctuating at low or high rate, and evaluated mitochondrial membrane potential and other parameters of mitochondrial activity – respiration, reducing capacity, and superoxide production, as well as intracellular ATP, intracellular calcium, and NF-κB activation. When compared to continuous exposure, fluctuating H2O2 induced a pronounced hyperpolarization in mitochondria, whereas the activity of electron transport chain appears not to be significantly affected. H2O2 provoked a decrease of ATP level and an increase of intracellular calcium concentration, independently of the regime of treatment. However, fluctuating H2O2 induced a specific pattern of large-amplitude fluctuations of calcium concentration. An impact on NF-κB activation was observed for high rate fluctuations, whereas continuous and low rate fluctuating oxidative stress did not provoke significant effects. Presented results outline the (patho)physiological relevance of redox fluctuations.

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 (Δψm) 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 Δψm, 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 Δψm during reperfusion, whereas GD caused a robust Δψm negativation. In case no Δψm 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, Δψm 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 Δψm during substrate deprivation, thus shedding new light on mechanisms of astrocyte resilience to prolonged ischemic injury.