Time-Resolved Spectroscopy of mitochondria,cells and tissues under normal and pathological conditions

In this study, the detailed dependence of light scattering on tissue architecture and intracellular composition has been investigated. Firstly, we simulated the reduced scattering coefficient (_s) of the rat liver using the Mie theory, the Rayleigh-Debye-Gans approximation and electron microscopy data. Then, the reduced scattering coefficient of isolated rat liver mitochondria, isolated hepatocytes and various rat tissues (i.e. perfused liver, brain, muscle, tumors) was measured at 780 nm by using time-resolved spectroscopy and a sample-substitution protocol. The comparison of the isolated mitochondria data with the isolated hepatocyte and whole liver measurements suggests that the mitochondrial compartment is the primary factor for light propagation in hepatic tissue, thus strengthening the relevance of the preliminary theoretical study. Nevertheless, the possibility that other intracellular components, such as peroxisomes and lysosomes, interfere with light propagation in rat liver is discussed. Finally, we demonstrate that light scattering in normal rat tissues and tumors is roughly proportional to the mitochondrial content, according to estimates of the mitochondrial protein content of the tissues.     

Ischemic Postconditioning Decreases Cerebral Edema and Brain Blood Barrier Disruption Caused by Relief of Carotid Stenosis in a Rat Model of Cerebral Hypoperfusion

Background and Purpose

Complications due to brain edema and breakdown of blood brain barrier are an important factor affecting the treatment effects of patients with severe carotid stenosis. In this study, we investigated the protective effects of ischemic postconditioning on brain edema and disruption of blood brain barrier via establishing rat model of hypoperfusion due to severe carotid stenosis.

Methods

Wistar rat model of hypoperfusion due to severe carotid stenosis was established by binding a stainless microtube to both carotid arteries. Ischemic postconditioning procedure consisted of three cycles of 30 seconds ischemia and 30 seconds reperfusion. Brain edema was evaluated by measuring cerebral water content, and blood brain barrier permeability was assayed by examining cerebral concentration of Evans'' Blue (EB) and fluorescein sodium (NaF). ELISA was used to analyze the expression of MMP-9, claudin-5 and occludin. The activity and location of MMP-9 was analyzed by gelatin zymography and in situ zymography, respectively. The distribution of tight junction proteins claudin-5 and occludin was observed by immunohistochemistry.

Results

The increased brain water content and cerebral concentration of EB and NaF were suppressed by administration of ischemic postconditioning prior to relief of carotid stenosis. Zymographic studies showed that MMP-9 was mainly located in the cortex and its activity was significantly improved by relief of carotid stenosis and, but the elevated MMP-9 activity was inhibited markedly by ischemic postconditioning. Immunohistochemistry revealed that ischemic postconditioning improved the discontinuous distribution of claudin-5 and occludin. ELISA detected that the expression of up-regulated MMP-9 and down-regulated claudin-5 and occludin caused by carotid relief were all attenuated by ischemic postconditioning.

Conclusions

Ischemic postconditioning is an effective method to prevent brain edema and improve BBB permeability and could be used during relief of severe carotid stenosis.     

A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

Background

Alpha-synuclein (asyn) has been shown to play an important role in the neuropathology of Parkinson’s disease (PD). In the diseased brain, classic intraneuronal inclusions called Lewy bodies contain abnormal formations of asyn protein which is mostly phosphorylated at serine 129 (pS129 asyn). This suggests that post-translational modifications may play a role in the pathogenic process. To date, several uniplex assays have been developed in order to quantify asyn not only in the brain but also in cerebrospinal fluid and blood samples in order to correlate asyn levels to disease severity and progression. Notably, only four assays have been established to measure pS129 asyn specifically and none provide simultaneous readout of the total and pS129 species. Therefore, we developed a sensitive high-throughput duplex assay quantifying total and pS129 human asyn (h-asyn) in the same well hence improving accuracy as well as saving time, consumables and samples.

Results

Using our newly established duplex assay we measured total and pS129 h-asyn in vitro showing that polo-like kinase 2 (PLK2) can phosphorylate asyn up to 41 % in HEK293 cells and in vivo the same kinase phosphorylated h-asyn up to 17 % in rat ventral midbrain neurons. Interestingly, no increase in phosphorylation was observed when PLK2 and h-asyn were co-expressed in rat striatal neurons. Furthermore, using this assay we investigated h-asyn levels in brain tissue samples from patients with PD as well as PD dementia and found significant differences in pS129 h-asyn levels not only between disease tissue and healthy control samples but also between the two distinct disease states especially in hippocampal tissue samples.

Conclusions

These results demonstrate that our duplex assay for simultaneous quantification is a useful tool to study h-asyn phosphorylation events in biospecimens and will be helpful in studies investigating the precise causative link between post-translational modification of h-asyn and PD pathology.

     

Age-related features of protein synthesis rhythm. Effects of extracellular medium

Cell interactions have been studied in cultures pf hepatocytes from young and old rats. The rhythm of protein synthesis is an index of cell interaction and synchronization in culture, while the amplitude of oscillations characterized cell cooperation in an aggregate rhythm. The mean rhythm amplitude in the culture of hepatocytes from old rats is twice lower than that from young rats. Gangliosides (mixture, bovine brain gangliosides) and ₁-adrenomimetic phenylephrine enhanced synchronization of cultures of the cells from old rats and increased the amplitude of oscillations to the level of young animals. Addition of rat blood serum (10%) to the medium revealed the rhythm of protein synthesis in the culture, asynchronous in the control, i.e., led to their synchronization. In media with young and old rat blood sera, oscillations were intense, with high amplitudes, and low, respectively. Addition of bovine brain gangliosides to a medium with old rat blood serum increased the amplitudes of oscillations to a level of the rhythm stimulated by the young rat serum. Thus, the cells of old animals can fully perceive synchronizing factors and, in the case of their increased concentration, the rhythm of protein synthesis in old animals did not differ from that in young rats. Current data on biochemical mechanisms underlying intercellular cooperation in the formation of population rhythm of protein synthesis have been discussed.Translated from Ontogenez, Vol. 36, No. 1, 2005, pp. 9–17.Original Russian Text Copyright © 2005 by Brodsky, Nechaeva, Zvezdina, Novikova, Gvazava, Fateeva, Malchenko.     

Spermine partially normalizes in vivo antioxidant defense potential in certain brain regions in transiently hypoperfused rat brain

Activities of the antioxidant enzymes such as superoxide dismutase (Cu,Zn-SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R) as well as the level of reduced glutathione and the concentration of thiobarbituric acid—reactive substance (TBARS) in brain regions in transiently hypoperfused rat brain with or without intravenous infusion of spermine were evaluated. Cerebral hypoperfusion was induced by temporary occlusion of common carotid arteries for 30 min and subsequently, by reperfusion for 60 min. Infusion of spermine reversed the decrease in SOD activity in the cerebral cortex, striatum, hippocampus, hypothalamus and midbrain, and amounted to 50.1 U, 61.5 U, 50.3 U, 30.0 U, 38.0 U, respectively, while GSH-Px restored to normal values only in the cerebral cortex and striatum and amounted to 100 u and 110 U, respectively. During hypoperfusion/reperfusion and after use of spermine no changes in GSSG-R were seen in the hypothalamus and midbrain. The activity of GSSG-R was in accordance with the control for the striatum and amounted to 39.0 IU after using spermine. GSH content returned to normal values in the striatum and midbrain after i.v. use of spermine and amounted to 210 and 240 nmol/g of wet tissue, respectively. In addition, the production of TBARS dropped markedly (P<0.05) in the hippocampus and midbrain and amounted to 100 and 105 μmol/g of wet tissue, respectively. Partially beneficial effect of spermine could result from the inhibition of free radical generation and capability of chelate formation with iron ions.     

Chronic Cerebral Hypoperfusion Induces Transient Reversible Monoaminergic Changes in the Rat Brain

Chronic restriction of cerebral blood flow in hypoperfused Wistar rats has been proposed as a new model of cerebrovascular-type dementia. Using this model, we have investigated central monoaminergic neuronal systems that are closely related to higher brain function. Monoamine and monoamine-metabolite levels were determined, as relative monoaminergic markers, at 1 day and 1,3,6 and 12 weeks after the bilateral occlusion of common carotid arteries. Dopaminergic changes in the frontal cortex and striatum were observed in hypoperfused rats at 1–3 weeks following occlusion. Serotonergic changes were recognized at four brain regions examined (frontal cortex, hippocampus, striatum and thalamus+midbrain). In particular, the immediate enhancement of serotonin turnover in the striatum appeared to influence the reaction to the acute ischemic attack such as vasoconstriction produced by hypoperfusion. Our findings suggest that chronic cerebral hypoperfusion induces transient reversible changes in central monoaminergic neuronal function within three weeks of ligation of carotid arteries. This time interval seems to represent a turning point in the process of chronic cerebral hypoperfusion-induced progressive brain injury.     

The Nootropic Drug Vinpocetine Inhibits Veratridine-Induced [Ca2+]i Increase in Rat Hippocampal CA1 Pyramidal Cells

The alkaloid derivative vinpocetine (14-ethoxycarbonyl-(3,16-ethyl)-14,15-eburnamine; Cavinton) has a well known beneficial effect on brain function in hypoxic and ischemic conditions. While it increases CNS blood flow and improves cellular metabolism, relatively little is known about vinpocetine's underlying molecular mechanisms on the single cell level. Since apoptotic and necrotic cell damage is always preceded by an increase in [Ca²⁺]_i, this study investigated the effect of vinpocetine on [Ca²⁺]_i increases in acute brain slices. Sodium influx is an early event in the biochemical cascade that takes place during ischemia. The alkaloid veratridine can activate this Na⁺ influx, causing depolarization and increasing [Ca²⁺]_i in the cells. Therefore, it can be used to simulate an ischemic attack in brain cells. Using a cooled CCD camera-based ratio imaging system and cell loading with fura 2/AM, the effect of vinpocetine on [Ca²⁺]_i changes in single pyramidal neurons in the vulnerable CA1 region of rat hippocampal slices was investigated. Preperfusion and continuous administration of vinpocetine (10 M) significantly inhibited the elevation in [Ca²⁺]_i induced by veratridine (10 M). When the drug was administered after veratridine, it could accelerate the recovery of cellular calcium levels. Piracetam, another nootropic used in clinical practice, could attenuate the elevation of [Ca²⁺]_i only at a high, 1 mM, concentration. We have concluded that vinpocetine, at a pharmacologically relevant concentration, can decrease pathologically high [Ca²⁺]_i levels in individual rat hippocampal CA1 pyramidal neurons; this effect might contribute to the neuroprotective property of the drug.     

Comparison of an endogenous protein kinase C substrate in rat aorta with rat brain MARCKS

We have compared the properties of a rat aorta-derived protein kinase C substrate (p75) with those of 80 kDa kinase C substrates from rat brain (MARCKS) and rabbit aorta (p80). Rat aortic p75 appeared to be closely related to rat brain MARCKS on the basis of: solubility in perchloric acid and trichloroacetic acid, heat stability, isoelectric point (pI 4.2), overall V8 protease phosphopeptide map, and immunocrossreactivity with an antibody directed against the N-terminal domain of MARCKS. However, p75 could be distinguished from rat brain MARCKS and from the rabbit aorta-derived p80 on the basis of its consistently more rapid electrophoretic mobility in SDS-containing gels, and in terms of a unique proteolytic phosphopeptide found in MARCKS but not in aortic p75. We conclude that p75 probably belongs to the family of protein kinase C substrates represented by MARCKS, and that differences in post-translational processing (glycosylation) or mRNA processing may account for the unique properties of the p75 protein in rat aortic tissue.Abbreviations p75 75,000 Da protein - MARCKS Myristoylated Alanine-Rich C Kinase Substrate     

A possible mechanism for cholesteryl ester formation during demyelination

Lecithin: cholesterol acyltransferase (LCAT) activity has been examined in the rat by using a brain homogenate preparation as the phospholipid substrate and blood plasma as the enzyme source. LCAT activity was detected on using 60 l of serum onwards. Successive experiments have also shown that LCAT activity is present in the edematous rat brain tissue homogenate when incubated with inactivated rat plasma as substrate. The results are discussed in relation to cholesteryl ester accumulation in brain during demyelinating diseases.     

Fluorescence and Fourier-transform infrared spectroscopic studies on the role of disulfide bond in the calcium binding in the 33 kDa protein of Photosystem II

The 33 kDa protein of Photosystem II has one intrachain disulfide bond. Fluorescence spectroscopy shows that the major groups in the protein that bind to Ca²⁺ should be the carboxylic side groups of glutamic acid and/or aspartic acid. Fluorescence and Fourier-transform infrared (FTIR) spectroscopic studies indicate that the conformation of the 33 kDa protein is altered upon reduction, while the reduced protein still retains the secondary structure. FTIR spectroscopy also shows that the metal ions induce a relative decrease of unordered structure and -sheet, and a substantial increase of -helix in both the intact and the reduced 33 kDa protein. This indicates that the addition of cations results in a much more compact structure and that both the intact and the reduced 33 kDa proteins have the ability to bind calcium. The above results may suggest that the disulfide bridge is not essential for calcium binding.Abbreviations CD circular dichroism - FTIR Fourier transform infrared - La lanthanum - PS photosystem - Tb terbium     

Activation of BDNF mRNA and protein after seizures in hyperbaric oxygen: implications for sensitization to seizures in re-exposures

Previous studies have demonstrated that exposure to convulsive doses of hyperbaric oxygen (HBO) increases sensitivity to seizures in re-exposures. Because brain derived neurotrophic factor (BDNF) is induced after a variety of seizures and increases cell excitability, it may contribute to the mechanism of sensitization. In this study, a fast induction in BDNF mRNA 2 hr after seizures and a temporary increase in BDNF protein 1 day after seizures induced by 100% O₂ at 5 atm (gauge pressure) were demonstrated in the rat cortex. To determine whether an elevation in BDNF protein level can modify sensitivity to the toxic effect of HBO, recombinant BDNF (12 g) was injected into cerebral ventricles 30 min prior to exposure. Administration of exogenous BDNF significantly shortened latent time to seizures in HBO exposures. We propose that upregulation of BDNF expression in the brain after seizures may contribute to sensitization to HBO toxicity.     

Administration of Myo-inositol Plus Ethanolamine Elevates Phosphatidylethanolamine Plasmalogen in the Rat Cerebellum

Plasmalogens are ether-linked phospholipids highly abundant in nervous tissue. Previously we demonstrated that acute administration of myo-inositol (myo-Ins) + [2-¹³C] ethanolamine ([2-¹³C]Etn) significantly elevated phosphatidylethanolamine plasmalogen (PlsEtn) in rat whole brain. Current experiments investigated the effects of acute myo-Ins+[2-¹³C]Etn administration on [PlsEtn] and the biosynthesis of new Etn lipids using NMR spectroscopy in rat cerebral cortex, hippocampus, brainstem, midbrain and cerebellum. Treated rats received a single dose of myo-Ins+[2-¹³C]Etn and controls received saline rather than myo-Ins. Data reveal that the cerebellum is the brain region most affected by treatment, which resulted in a 22% increase in [PlsEtn] and 89% increase in newly synthesized Etn lipids relative to controls (P 0.05). Furthermore, the cerebellar PlsEtn/phosphatidylethanolamine ratio and molar percentage of PlsEtn were significantly elevated by 12% and 8%, respectively (P 0.05). These data suggest that myo-Ins influences Etn lipid metabolism in brain, particularly in the cerebellum where there is a stimulation in the biosynthesis of new Etn lipids with a preference towards PlsEtn.     

Enzyme activities of monoamine oxidase,cathechol-o-methyltransferase and γ-aminoubutyric acid transaminase in primary astroglial cultures and adult rat brain from different brain regions

The activities of monoamine oxidase (MAO), cathechol-O-methyltransferase (COMT) and -aminobutyric acid transaminase (GABA-T) were measured in primary cultures from newborn rat cultivated from 6 different brain regions. These primary cultures contained mostly astroglial cells, evaluated by the presence of the glial fibrillary acidic protein (GFAp, -albumin) and the S-100 protein. The enzyme activities in the corresponding brain areas from adult rat were also quantified. MAO activities were on the same level in 14-day old cultures and in adult rat brain homogenates, with significantly lower values in brain stem as compared to the other brain regions examined. COMT activities were on a higher level in the cultures than in adult rat brain homogenates. Astroglial cells from hippocampus were found to have the highest and those from brain stem the lowest COMT-activities. GABA-T activities were lower in the cultures than in adult rat homogenates. No significant differences were seen in the various astroglial cultures. Accumulation of [³H]dopamine and [³H]-aminobutyric acid (GABA) visualized by autoradiography showed only a slight uptake of dopamine in comparison with the uptake of GABA. It is concluded that astroglial cells in culture have enzymatic properties similar to those of astroglial cells in different brain regions of adult rat brain. Studies are in progress to evaluate if the regional heterogeneity observed among cultivated astroglial cells is affected by in vivo differentiation until cultivation and/or time in culture.     

Purification and characterization of the high molecular weight microtubule associated proteins from neonatal rat brain

The changes in the levels of microtubule-associated proteins (MAPs) during advanced embryonic stages, neonatal and adult organisms reflect the importance of these cytoskeletal proteins in relation to the morphogenesis of the central nervous system. MAP-1B is found in prenatal brains and it appears to have the highests levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast procedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal rat brains was designed, based on the differential capacity of poly L-aspartic acid to release MAPs during temperature-dependent cycles of microtubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular protein polymerization in the presence of low concentrations of polyaspartic acid. Instead, MAP-2 and a 180 kDa protein with characteristics of MAP-3 remained associated to the polymer after the assembly. Further purification of MAP-1B was attained after phosphocellulose chromatography. Isolation of MAP-2 isoforms together with MAP-3 was achieved on the basis of their selective interactions with calmodulin-agarose affinity columns. In addition, MAP-2 and MAP-3 were also purified on the basis of their capacities to interact with the tubulin peptide -II (422–434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the -II tubulin fragment, but it showed interaction with the Affigel-conjugated -I (431–444) tubulin peptide. The different MAPs componentes were characterized by western blots using specific monoclonal antibodies. A salient feature of neonatal rat brain MAP-3 was its interactions with site-directed antibodies that recognize binding epitopes on the repetitive sequences of tau and MAP-2. However, these site-specific antibodies did not interact with MAP-1B from the neonatal rat brain tissue.Abbreviations PAA poly (L-aspartic acid) - HMW-MAPs high molecular weight microtubule associated proteins     

Chronic Cerebral Hypoperfusion Induces Striatal Alterations Due to the Transient Increase of NO Production and the Depression of Glutathione Content

We examined the effects of chronic cerebral hypoperfusion on the endogenous oxidative stress-related indices, nitrite and nitrate (NOx) concentration, glutathione (GSH) content, superoxide dismutase and catalase activities, and thiobarbituric acid-reactive substances level in the rat striatum, to clarify the participation of oxidative stress in the chronic cerebral hypoperfusion-induced alterations. Our present results indicate that chronic cerebral hypoperfusion produces oxidative stress and disturbs intracellular redox regulation in two distinct phases: at 1 day, acute and at 6 weeks, chronic alterations after the operation. Therefore, striatal neural cell damage may be mainly attributed to the transient increase of NOx production at 1 day after, and the delayed reduction of muscarinic acetylcholine receptor binding in the striatum may be mostly attributed to the continuous depression of GSH content from the 1st to the 6th postoperative week. In particular, the continuous GSH depression may be considered to accompany the pathophysiology of chronic cerebral hypoperfusion.     

Responses of muscle sympathetic nerve activity to static handgrip exercise after 14 days of exposure to simulated microgravity

Decrease in muscle perfusion affects on cardiovascular response to exercise. Muscle hypoperfusion enhances the increase in blood pressure responses to exercise. Muscle perfusion depends not only on central blood pressure but also how fit the active muscle is above or below the heart level; muscle perfusion decreases as arm is elevated. Static exercise increases muscle sympathetic nerve activity (MSNA) innervating vessels in non-active muscles. The exercise-induced increase in MSNA is mainly mediated by stimulating chemosensitive muscle afferents in active muscles. However, the effect of arm elevation on MSNA during forearm exercise is not examined. On the other hand, space flight and simulated microgravity exposure causes reduction in muscle blood flow, suggesting chronic muscle hypoperfused condition during simulated microgravity. Therefore, there is a possibility that arm elevation after microgravity exposure alters MSNA responsiveness during exercise. However, arm elevation effect after exposure to simulated microgravity is not examined.     

Barriers in the Immature Brain

1. The term blood–brain barrier describes a range of mechanisms that control the exchange of molecules between the internal environment of the brain and the rest of the body.2. The underlying morphological feature of these barriers is the presence of tight junctions which are present between cerebral endothelial cells and between choroid plexus epithelial cells. These junctions are present in blood vessels in fetal brain and are effective in restricting entry of proteins from blood into brain and cerebrospinal fluid. However, some features of the junctions appear to mature during brain development.3. Although proteins do not penetrate into the extracellular space of the immature brain, they do penetrate into cerebrospinal fluid by a mechanism that is considered in the accompanying review (Dziegielewska et al., 2000).4. In the immature brain there are additional morphological barriers at the interface between cerebrospinal fluid and brain tissue: strap junctions at the inner neuroependymal surface and these and other intercellular membrane specializations at the outer (pia–arachnoid) surface. These barriers disappear later in development and are absent in the adult.5. There is a decline in permeability to low molecular weight lipid-insoluble compounds during brain development which appears to be due mainly to a decrease in the intrinsic permeability of the blood–brain and blood–cerebrospinal fluid interfaces.     

Brain 12-HETE formation in different species,brain regions,and in brain microvessels

We used gas chromatography/mass spectrometry to measure brain 12-HETE (12-Hydroxy-5,8,10,14-eicosatetraenoic acid) formation from endogenous arachidonic acid in different species and different brain regions and in isolated brain microvessels. When blood-free brain slices were incubated for 20 minutes we found that the rabbit and cat brain incubates contained little 12-HETE when compared to rat and mouse brain incubates. Further in vitro studies of various rat brain regions showed a generally even distribution of 12-HETE. When isolated rat or rabbit microvessels were incubated and analyzed, we found 1 and 0.25 g, respectively, of 12-HETE/mg of microvessel protein. Also, rabbit brain had limited or no capacity to actively metabolize tritiated 12-HETE. In summary, these studies show substantial species variation with respect to brain formation of 12-HETE and indicate that the vasculature is a potentially significant contributor to the 12-HETE found in whole brain tissue.     

Omega-Oxidation of Monocarboxylic Acids in Rat Brain

The accumulation of dicarboxylic acids is a prominent feature of inborn and toxin induced disorders of fatty acid metabolism which are characterized by impaired mental status. The formation of dicarboxylic acids is also a critical step in liver in the induction of intracellular fatty acid binding proteins and the proliferation of peroxisomes. In order to understand what potential roles dicarboxylic acids have in brain, we examined the extent of omega-oxidation in rat brain. Homogenates of rat brain catalyze the omega-oxidation of monocarboxylic acids with a specific activity of between 0.87 and 5.23 nmol/mg of post-mitochondrial protein/h, depending on the substrate. The activity is remarkably high, between one-fourth and 4 times the activity found in rat liver, depending on the chain length of the substrate. Specific activity increases with increasing chain length of the substrate. The omega-oxidation of palmitic acid is linear over a range of 0.125–3.0 mg of protein and 5–50 M substrate for up to 45 minutes of incubation. The product of omega-oxidation in brain is almost exclusively dicarboxylic acid. Cultured rat neurons, astrocytes, and oligodendrocytes all contain omega-oxidation activity. Western blots of rat brain homogenate demonstrate a protein that is recognized by antibody to rat liver CYP4A omega-hydroxylase. These results demonstrate that the omega-oxidative pathway is prominent in brain and could play a role in brain fatty acid metabolism.