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Introduction: Uncoupling proteins (UCPs) are a family of transmembrane anion transporters present in the inner mitochondrial membrane. UCP-2, which exhibits the widest distribution in various tissues, plays an important role in many physiological processes. Human UCP-2 studies have been hampered by the lack of a method for measuring this protein in an easily accessible human tissue, e.g., blood. The aim of this study was to develop such a method and test its utility by comparing UCP-2 levels in smokers and non-smokers. Material and Methods: Venous blood samples from 10 smoking and seven non-smoking volunteers were used for the study; lymphocytes were isolated employing Lymphoprep. UCP-2 levels were measured by Western blotting combined with chemoluminescence detection. Results: Total lymphocyte homogenates were found useless for measuring UCP-2 levels, but it was possible to measure UCP-2 in homogenates of purified lymphocyte mitochondria. There was a significant, though moderate, linear correlation between UCP-2 level and daily cigarette use. UCP-2 level in peripheral blood lymphocytes from smokers was higher than that in non-smokers. Conclusion: The method for measuring UCP-2 in peripheral blood lymphocytes opens the possibility of UCP-2 screening studies in humans and thus may be useful for studying the role of the protein in human physiology and pathology.

Unilateral surgical brain injury of the rat is a model of surgery-related brain damage of humans. Our preliminary experiments showed two phenomena within the damaged cortical region of rat brain. Those were: degeneration and death of neurons and massive gliosis. In the present study we aimed to investigate the mechanisms of neuronal death following brain injury and to characterize responses of glial cells to the damage. We analyzed the morphological changes and alterations of immunochemical profile of cells localized in the brain areas adjacent to the lesion. Our data show the massive neuronal death following the lesion. Neurons undergo necrosis and apoptosis, but on the 4th day following the operation apoptosis prevails. Apoptotic cells showed heavy immunostaining for proapoptotic Bax and caspase 3. This result suggests the involvement of these proteins in neuronal apoptosis in our experimental model. Neuronal death is accompanied by the induction of astrogliosis in the perilesional cortical area. Astrocytes became hypertrophic. We did not detect any dying astrocytes at the investigated time point, but there is a possibility that apoptosis may occur in astroglia during another time period following the damage. This question requires further studies in our experimental model.

To estimate protective potential of citicoline in a model of birth asphyxia, the drug was given to 7-day old rats subjected to permanent unilateral carotid artery occlusion and exposed for 65 min to a hypoxic gas mixture. Daily citicoline doses of 100 or 300 m/kg, or vehicle, were injected intraperitoneally for 7 consecutive days beginning immediately after the end of the ischemic-hypoxic insult, and brain damage was assessed by gross zorphology score and weight deficit two weeks after the insult. Caspase-3, alpha-fodrin, Bcl-2, and Hsp70 levels were assessed at 0, 1, and 24 h after the end of the hypoxic insult in another group of rat pups subjected to the same insult and given a single dose of 300 m/kg of citicoline or the vehicle. Citicoline markedly reduced caspase-3 activation and Hsp70 expression 24 h after the insult, and dose-dependently attenuated brain damage. In the context of the well-known excellent safety profile of citicoline, these data suggest that clinical evaluation of the efficacy of the drug in human birth asphyxia may be warranted.

Carbohydrate (CHO)-restricted diets have been recommended for weight loss and to prevent obesity, but their long-term effects have not been fully elucidated. This study was designed to evaluate the effect of long-term (>1 year) consumption of a low-CHO high-fat diet ("The optimal diet," developed by Dr Kwaśniewski referenced herein) on lipid profile, glycemic control, and cardiovascular disease risk factors in healthy subjects. Of 31 "optimal" dieters enrolled in the study (17 women and 14 men, aged 51.7+/-16.6 years), 22 declared adherence to the diet for more than 3 years. Average energy intake and principal nutrients consumed were assessed from 6-day dietary records provided by the participants. In most dieters, concentrations of beta-hydroxybutyrate, free fatty acids, total cholesterol, and low-density lipoprotein cholesterol exceeded the upper limits of the reference ranges for nonstarved subjects. The metabolic profiles of most subjects were positive for several indicators, including relatively low concentrations of triacylglycerols, high levels of high-density lipoprotein cholesterol (HDL-C), and normal ratios of low-density lipoprotein cholesterol/HDL-C and total cholesterol/HDL-C. In most subjects, plasma concentrations of glucose, insulin, glucagon, cortisol, homocysteine, glycerol, and C-reactive protein were within reference ranges. Notably, in all but one subject, the homeostasis model assessment index of insulin resistance remained below the threshold for diagnosis of insulin resistance. These results indicate that long-term (>1 year) compliance with a low-CHO high-fat "optimal diet" does not induce deleterious metabolic effects and does not increase the risk for cardiovascular disease, as evidenced by maintenance of adequate glycemic control and relatively low values for conventional cardiovascular risk factors.

Cytidine-5-diphosphocholine (CDP-choline, citicoline) is an endogenous nucleoside involved in generation of phospholipids, membrane formation and its repair. It demonstrates beneficial effects in certain central nervous system injury models, including cerebral ischaemia, neurodegenerative disorders and spinal cord injury. Defective neuronal and/or glial glutamate transport is claimed to contribute to progressive loss of motor neurons (MNs) in amyotrophic lateral sclerosis (ALS). Our previous ultrastructural studies, performed on an organotypic tissue culture model of chronic glutamate excitotoxicity, documented a subset of various modes of MN death including necrotic, apoptotic and autophagocytic cell injury. The aim of this ultrastructural study was to determine the potential neuroprotective effect of CDP-choline on neuronal changes in a glutamate excitotoxic ALS model in vitro. Organotypic cultures of the rat lumbar spinal cord subjected to 100 microM DL-threo-beta-hydroxyaspartate (THA) were pretreated with 100 microM of CDP-choline. The exposure of spinal cord cultures to CDP-choline and THA distinctly reduced the development of typical apoptotic changes, whereas both necrotic and autophagocytic THA-induced MN injury occurred. These results indicate that CDP-choline treatment might exert a neuroprotective effect against neuronal apoptotic changes in a model of chronic excitotoxicity in vitro.

We present the results of biochemical and electron microscopic (EM) examinations of the spinal cord myelin from SOD1G93A transgenic Sprague Dawley rats in the early and late symptom-free period of the disease (60 and 93 days of life) and after four-leg paralysis has occurred (120 days of life). Biochemical and ultrastructural changes of myelin started already in the symptom-free period and become most pronounced in the paralyzed animals. Biochemical examinations indicated a decrease of lipids, phospholipids, cholesterol and cerebrosides. The pattern of particular phospholipids was in the normal range. A progressive decrease of the percentages of proteolipid, DM-20 and Wolfgram proteins was evident. Myelin basic proteins I and II were less affected. In EM,massive myelin disorganization was observed.

Stroke is a syndrome characterized by a sudden neurological deficit caused by cerebral ischemia. Computed tomography (CT) plays a crucial role in the evaluation of stroke patients even though it is not sufficient enough to extract the hypodense area corresponding to the infracted cerebral tissues in hyperacute stage. Careful selection of patients for thrombolytic therapy is fundamental to improve safety and efficacy; therefore the authors propose an additional, wavelet-based post-processing method for extracting hypodensity in CT scans in hyperacute stroke patients.A retrospective evaluation of 52 sets of examinations conducted in patients admitted with symptoms suggestive of stroke was undertaken by four radiologists unaware of the final clinical findings. All of the selected cases were considered as having no direct signs of hyperacute ischemia in the localization corresponding with clinical manifestation and follow-up studies. In the first stage, only CTs performed at admission were evaluated; a month later the same scans were reevaluated with additional use of a "stroke monitor". All sets were evaluated independently. Follow-up CT exam and/or the clinical picture confirmed or excluded the diagnosis.Higher AUC values were found for the "stroke monitor"-aided radiological diagnosis for all the radiologists and the differences were significant for all subgroups (p0.05) apart from the subgroup in which CT scans of patients with significant cerebral atrophy were excluded.Combining the results of CT and the "stroke monitor" provided a better diagnosis of stroke, especially in atrophic brains. Planned prospective studies will allow evaluation of the impact on the further treatment of hyperacute stroke patients.

Sir, I read with great interest the article by Skripuletz et al. (2015), who showed beneficial effects of exogenously applied CDP-choline (citicoline) in murine myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) and in cuprizone-induced mouse model of demyelination. I would like to congratulate the authors for their report, which already has been recognized as the best basic science paper published in the multiple sclerosis research area in 2014 (Hutchinson, 2015). In the discussion of their findings, Skripuletz et al. (2015) stated that, although several previous studies indicated that CDP-choline may have the capacity to protect cells such as neurons, …

An analysis of the dynamics of histological and immunocytochemical changes in the CNS of a transgenic rat model of fALS in various periods of life was performed. Material was obtained from animals on the 60th day of age (4), 93rd day of age (3) and 120th presymptomatic day and from 3 animals in paretic stage of the disease. Formalin-fixed and paraffin-embedded slices were stained with HE and Klüver-Barrera method. Immunoreactions to GFAP, S-100, ferritin, neurofilament, ubiquitin, synaptophysin and tau protein were also performed. Within the brain tissues patchy neuronal loss and dark or ischaemic neurons were dispersed in cortical layers, CA1, CA3 and CA4 hippocampal areas and structures of the hemispheres and brain stem. In the spinal cord, numerous alpha motoneurons were dark or ischaemic. Vacuoles or small pale spots were visible in their cytoplasm. Microspongiosis surrounded some motoneurons, particularly cells subjected to neuronophagy. Neuronophagy, sporadically observed at the age of 60th day, was more extensive on the 93rd day of age, and at the age of 120 days already involved all interneurons of the anterior and posterior horns. In the immune reaction to neurofilament numerous fibres, often thick, fragmented or rosary-like, were observed. They were located within subcortical white matter, external and internal capsules, anterior horns of the spinal cord. Changes became more intensive with age. Astrocytic reactivity was weak in animals on the 60th and 93rd day of life. Non-numerous cells were immunoreactive to GFAP and S-100, although an increase of astrocytic nuclei was observed. On the 120th day of age and in symptomatic stage astrocytic hypertrophy and proliferation were intensive. But from the 60th day of age ubiquitin and tau protein immunopositive material was accumulated in the perinuclear area of astroglial cytoplasm. Immunoreaction of nerve cells to these proteins was negative.1) In the subclinical stage of the disease the pathological process within the CNS takes place already on the 60th day of age and its intensity increases with age. 2) Morphological changes are not limited to motor neuronal cells. Various structures of the CNS are damaged. 3) Weak astroglial reaction probably depends on pathological accumulation of ubiquitin and tau protein in cytoplasm. 4) Astroglial cells are probably also a "target" for pathogenic factors in the rat model of fALS.

The purpose of this study was to determine structural changes which trigger the onset and progression of amyotrophic lateral sclerosis in rats expressing a human SOD1 transgene with mutation G93A. Lumbar spinal cord of affected rats in early and late presymptomatic (PM, 60 and 93 days of age) and symptomatic (S, 120 days of age) stage of the disease were analyzed ultrastructurally. At 60 days the structure of lumbar spinal cord as well as alpha motoneurons type S and F appeared normal; however, careful examination revealed that approximately 15% of axons were filled with mitochondria that were abnormal in number, size and morphology. Grossly swollen mitochondria with disrupted cristae were a prominent feature in all large axons at 93 days of age. At this time swelling and dilated mitochondria were observed also in type S motoneurons, while type F had small, well preserved mitochondria. At symptomatic stage the alpha motoneurons showed moderate neuronal loss, mainly of the S type. The most interesting finding at this stage was the occurrence of motoneurons with morphological signs of apoptotic-like degeneration. Such apoptotic-like motoneurons were characterized by nuclear and cytoplasmic condensation, chromatin compaction and formation of uniformly dense, dark structures. Numerous axons with very dark, compact interior as well as apoptotic bodies were irregularly scattered throughout the neuropil. Our ultrastructural study indicates that dying motoneurons in transgenic mutant SOD1G93A rats exhibit reminiscent apoptotic morphology which is preceded by significant mitochondrial abnormalities mainly in proximal axons and S motoneurons. Different reaction of slow and fast motoneurons to degenerating factors requires further analysis.

A colony of transgenic rats expressing the human mutant Cu,Zn superoxide dismutase gene (hSOD1G93A) that is associated with some cases of familial form of amyotrophic lateral sclerosis (ALS) has been maintained in the Animal House of the Polish Academy of Sciences Medical Research Centre since 2003. This transgenic model, generated by Howland et al. (Proc Natl Acad Sci USA 2002; 99: 1604-1609), has been obtained under the material transfer agreement from Wyeth Corporation. The transgenic SOD1G93A (or 'Howland') rats develop neurological and neuropathological symptoms reminiscent of human ALS, i.e. progressive loss of motoneurons leading to paralysis and death. This paper describes maintenance of the transgenic rat colony, and general procedures used in experiments with these animals (i.e. genotyping, neurological observations, anaesthesia, etc.). At the beginning of the colony, up to the 3rd generation of the rats, symptoms of the model disease appeared at 95-125 days of age, and the animals survived till 120-145 days of age. Thereafter a gradual change in the disease phenotype occurred, and in the 8th generation approximately 1/3 of the rats displayed much slowed disease progression.

Morphological alterations and the course of changes in motoneuron counts were studied by light microscopy (cresyl violet staining) in the L2/L3 region of the spinal cord of hemizygotic transgenic rats carrying the amyotrophic lateral sclerosis-associated mutant human gene for Cu,Zn superoxide dismutase (hSOD1G93A) and of their non-transgenic littermates. In 60-day old transgenic rats, a few ischaemic-looking alpha-motoneurons and occasional vacuolization and accumulation of tigroid in some of the cells were apparent. On day 93 of life more distinct cellular pathology was found in transgenic rats, including moderate gliosis, neuronophagy of alpha-motoneurons, and occasional neuronophagy of gamma-motoneurons. In 120-day-old transgenic rats, abundant gliosis and profound neuronophagy of alpha-motoneurons were observed combined with occasional neuronophagy of other cells. Some loss of alpha-motoneurons was also apparent in 120-day-old non-transgenic littermates of the transgenic rats. No difference in alpha-motoneuron and gamma-motoneuron counts was found between the rats on day 60 of life (early presymptomatic stage of the model disease in the transgenic rats). At 93 days of age (late presymptomatic stage), alpha-motoneuron count, but not gamma-motoneuron count, tended to be lower (p=0.06) in the transgenic rats. On day 120 of life (symptomatic stage), alpha-motoneuron count in the transgenic rats was about half that in their nontransgenic littermates (p0.001); at this time point the relative decline in alpha-motoneuron number in the former was 57% (day 120 versus day 60; p0.001). A smaller decline in alpha-motoneuron count was also found in nontransgenic rats (day 120 vs day 60: 24%, p0.05); this was not associated with the emergence of neurological symptoms or distinct changes in the cell morphology of the spinal cord region studied.

Tezacitabine (FMdC) is a new cytostatic/cytotoxic agent widely investigated in clinical trials and on the cellular level. In a previous paper (3) we worked on human and murine leukemia (L-1210, HL-60, and MOLT-4) cells, and in this paper we investigated the influence of FMdC on the cell cycle and apoptosis in vitro of three other leukemias (CCRF-SB, KG-1, and Jurkat), and human solid tumor (carcinoma) cell lines (COLO-205, MCF-7, and PC-3). We found that FMdC induces the G1 (at concentrations higher than 10 nM). and S-phase (at low concentration) leaky block of the cell cycle. FMdC also effectively induces apoptotic death of cells by the caspase 3/7 pathway. We found also that FMdC induces intensive changes in the protein metabolism. These changes are correlated with the cell death.

Immune system activation and oxidative stress are involved in the pathogenesis of heart failure (HF). We aimed to test the hypothesis that upgrading from right ventricular pacing (RVp) to biventricular pacing (BiVp) can counteract these phenomena.28 HF patients, with BiVp were switched to RVp for one week, and then returned to BiVp. Immediately prior to, and 48 h after the return to BiVp, left ventricular (LV) systolic function was evaluated by echocardiography, and serum N-terminal pro-brain natriuretic peptide (NTproBNP), C-reactive protein (CRP), tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL6), nitric oxide metabolites (NO(x)) and malondialdehyde (MDA) were assayed.LV systolic function significantly improved 48 h after switching from RVp to BiVp: Ao-VTI (p0.001), SV (p0.001) and CO (p0.001), and mitral regurgitation significantly decreased (p=0.003). At the same time, indices of peripheral immune activation decreased: TNF-alpha (p=0.02) and IL6 (p0.001). MDA decreased (p0.001), whereas NO(x) increased (p=0.04). NTproBNP and CRP did not change. In addition, in "responders" (i.e. CO increase10% during BiVp vs. RVp) NTproBNP decreased and NO(x) increased. However, during BiVp, the decreases in TNF-alpha, IL6, and MDA occurred both in responders and in non-responders and were accompanied by a reduction in mitral regurgitation.The beneficial effect of BiVp compared to RVp extends beyond improving cardiac haemodynamics and comprises a decrease in immune activation accompanied by an increase in serum NO(x) and decrease in serum MDA.

Streptozotocin (STZ) is a bacterial toxin which selectively damages both insulin-producing cells and insulin receptors. Injections of STZ into the cerebral ventricles of experimental animals are followed by sustained biochemical, metabolic and behavioral effects resembling those which are found in human brains afflicted by Alzheimer's disease. The aim of the present study was to assess the effects of double intracerebroventricular application of STZ on the ultrastructure of rat frontoparietal cortical neurons. The most prominent change, seen 3 weeks after STZ injection, was a significant enlargement of the Golgi apparatus caused by expansion of the trans-Golgi segment of the cellular protein secretory pathway. Morphometric analysis revealed that the area of the trans part of the Golgi complex in neuronal cells was increased more than two-fold (median values: 312 x 10(3) nm in 14 neurons from control animals, and 846 x 10(3) nm3 in 19 neurons from STZ-treated animals, P = 0.0012), whereas that of the cis part did not significantly change. The effects of STZ did not resemble Golgi atrophy and fragmentation described in neurons from disease-prone brain structures of patients with Alzheimer's disease, but were similar to that observed after intravenous application of a non-metabolizable glucose analog 2-deoxyglucose. Considering that proamyloidogenic processing of beta-amyloid precursor protein may occur preferentially in the trans-Golgi segment, the observed early response of neuronal ultrastructure to desensitization of insulin receptors may predispose cells to form beta-amyloid deposits.

Hyperglycemia and type 2 diabetes are an independent risk factor of cardiovascular (CV) disorders of atherosclerotic etiology. Proatherosclerotic sequelae of hyperglycemia comprise oxidative stress caused by increased production of free radicals, activation of leucocyte-endothelium interactions, and protein glycosylation. According to current reccomendations diabetes is diagnosed when plasma glucose concentration exceeds 10.0 mmol/L in 2-hour standard oral glucose tolerance test, whereas the result of 6.7-9.9 mmol/L speaks for asymptomatic impaired glucose tolerance (IGT) which does not require treatment. However, several data sets and their meta-analyses are suggestive of that not only diabetes, but also IGT is associated with significantly increased risk of cardiovascular death. A recent report indicated also that glycosylated hemoglobin HbA(1c) level is a continuous and very strong risk factor for CV death. In view of these data therapeutic interventions for IGT shall be considered.

Delta-aminolevulinic acid (ALA) is a precursor of the synthesis of porphyrins including heme produced in all mammalian cells. Exogenous ALA induces selective accumulation of the other heme precursor, protoporphyrin IX (PpIX), in neoplastic cells, such as those of malignant gliomas. Upon exposure to violet-blue light PpIX becomes activated, which results in red-light fluorescence as well, as in photodynamic oxidations which may be lethal to the cells. In neurosurgery ALA is used for intraoperative labeling of the border regions of malignant gliomas infiltrated by alive clonogenic tumor cells (ALA-PDD), and is helpful in precise resection of these regions. Clinical data indicate that ALA-PDD-assisted resection of malignant gliomas may result in statistically significant prolongation of postoperative survival. Ongoing research concentrates also on the use of ALA for a selective elimination of glioma cells in situ, and on lipophilic ALA derivatives with more favorable pharmacokinetic properties.

Mice of the DBA/2J strain spontaneously develop complex ocular abnormalities, including glaucomatous loss of retinal ganglion cells (RGC). In the present study ultrastructural features of retinal neurodegeneration in DBA/2J mice of different age (3, 6, 8 and 11 months) are described. By 3 months, RGC apoptosis characterized by electron-dense karioplasm and cytoplasm of ganglion cells was observed. The occurrence of apoptotic ganglion cells peaked at the age of 6 months. Past this age, necrosis characterized by swelling and electron-rare cytoplasm appeared to be the prevailing form of cell death. Muller glia activation increased with age, but there were no signs of leukocyte infiltration. At 8 and 11 months, signs of neoangiogenesis were found both at the ultrastructural level and in clinical examinations. In these older animals myelin-like bodies, most probably representing the intracellular aggregates of phospholipids in irreversibly injured cells, were also seen. Photoreceptor cells were not affected at any age. Our observations suggest that retinal degeneration in the DBA/2J mice does not involve recruitment of blood-borne inflammatory/phagocytosing cells, and that apoptosis is gradually replaced by necrosis as the predominant pathway of RGC death. Retinal degeneration in 3- to 11-month-old DBA/2J mice partially resembles human pigment dispersion syndrome and pigmentary glaucoma with characteristic anterior segment changes and elevation of intraocular pressure. However, neovasculogenesis and myelin-like bodies are observed during aging. Therefore, the DBA/2J model requires judicious interpretation as a glaucoma model.

Standard therapeutic modalities including surgery, radio- and/or peripheral chemotherapy are ineffective in malignant gliomas. This is due to infiltration of distant brain areas by glioma cells and to the blood-brain barrier limiting penetration of anticancer drugs. One of the most promising experimental methods of glioma therapy is intracerebral implantation of biodegradable polymers containing cytotoxic compounds. This method allows to avoid peripheral toxicity of drugs. Considering that practically it is only malignant cells that proliferate in the brain, the use of drugs displaying selective toxicity toward DNA-replicating cells might help to avoid central toxicity as well. Several nucleoside analogs display selective cytotoxic and/or radiosensitizing effects on proliferating cells. Despite encouraging results obtained in the in vitro and animal models of gliomas, peripheral administration of these drugs turned out to be ineffective in the clinical settings. Intracerebral implantation of nucleoside analogs-containing biodegradable polymers may be much more efficacious, especially when combined with radiotherapy. Appropriate nucleoside analogs may also be employed in cell-selective radiotherapy and gene therapy of malignant gliomas.

Proton magnetic spectroscopy of brain tumor extracts, and more recently magic angle spinning proton magnetic resonance spectroscopy (MAS 1H-MRS) of intact cooled tissue specimens have been used for tumor classification. The aim of this study was to obtain MAS 1H-MRS spectra of lyophilised and pulverised specimens of various brain tumors and to assess their utility for tumor type recognition.Tissue samples of intracranial tumors were obtained at surgery and frozen immediately in liquid nitrogen. A part of each sample was subjected to a routine histopathological examination, while the rest was lyophilised, pulverised, and subjected to high-speed magic angle spinning MRS using Bruker DSX-400 WB AVANCE spectrometer and rotation speed up to 33 kHz.MAS 1H-MRS spectra of 39 samples (including 16 glioblastomas or gliosarcomas, 10 meningiomas and 5 craniopharyngiomas) were obtained. Invariably the spectra were dominated by broad lipid resonances, reflecting "NMR visible" protons in methyl,methylene and methine groups of fatty acid chains. Resolution of the spectra was markedly higher for glioblastomas/gliosarcomas and craniopharyngiomas than for meningiomas.Lyophylization of brain tumor specimens eliminates signals of watersoluble metabolites and results in a loss of discriminative power of the 1H-MRS analysis. Resolution of the spectra is increased for tumors that accumulate lipids extracellularly, but is not related to tumor histological grade.

Intracerebral implantation of biodegradable polymers loaded with cytotoxic or radiosensitising nucleoside analogues is a promising treatment strategy for malignant gliomas, which are currently intractable. The aim of the study was to develop biodegradable polymers containing nucleosides which could be implanted intracerebrally. Methods of synthesis were developed for the copolymers composed of D,L-lactide, glycolide and caprolactone in different proportions, as well as a novel method of introducing nucleosides to these copolymers at the polymerisation step. Upon degradation in an aqueous medium some of these copolymers emit nucleosides in micromolar concentration over several months. Their in situ degradation and biocompatibility with brain tissues was assessed by means of scanning and transmission electron microscopy. At the ultrastructural level tissue responses to the copolymer implantation closely resembled the responses to mechanical trauma.

Citicoline (exogenous cytidine-5′-diphosphocholine) was reported to enhance dopaminergic neurotransmission in the brain. A few clinical studies showed beneficial effects of this drug on the function of the visual pathway in patients with glaucoma or amblyopia. The present study was aimed at determining whether citicoline could influence retinal catecholamine levels in adult male Albino rabbits. The animals received the drug (50 mg/kg i.p., twice daily) or vehicle for 7 days, and retinal catecholamine concentrations were determined by HPLC. Compared to vehicle-treated controls, citicoline-treated animals displayed a significantly higher retinal dopamine concentration and a tendency toward an increase in adrenaline concentration, while the noradrenaline concentration remained unchanged. It is, therefore, conceivable that citicoline reinforces dopaminergic transmission in the retina.

Biopsies of 6 malignant gliomas (grade 3 or 4) and 11 low-grade meningiomas were extracted with perchloric acid or methanol/ water, and the fully-relaxed 1H-MRS spectra of the extracts containing water-soluble metabolites and a concentration and chemical shift standard were recorded at 11.4 T. The resonance signals assigned to inositol (Ino), glycerophospho- and phosphocholine (GPC + PC), choline (Cho), creatine and phosphocreatine (Cr + PCr), glutamate (Glu), acetate (Ac), alanine (Ala) and lactate (Lac) were integrated, and analyzed by two methods. First, the concentrations of the aforementioned substances in the bioptates were estimated from their resonance signals in the extracts. Second, these signals were normalized to the Cr + PCr resonance signal. The Mann-Whitney U-test was used to verify statistical significance between the data sets obtained for gliomas and meningiomas. When the first method of analysis was used, the only difference was in the Ala concentration, which in meningiomas was on average 4 times higher than in gliomas (P < 0.01). However, when the second method of analysis was applied, gliomas expressed lower normalized resonance signals of Ala and Glu (P < 0.001, ranges not overlapping), Lac (P < 0.005), as well as Ino and GPC + PC (P < 0.05). In proton MR spectra of brain tumor tissue extracts containing water soluble metabolites, the resonance signals normalized to that of total creatine may provide a very good discrimination between malignant gliomas and low-grade meningiomas.

Post-surgical radiation therapy is a routine procedure in the treatment of primary malignant brain tumors. Along with modest therapeutic effects conventional fractionated radiotherapy, in spite of any modifications, produces damage to non-malignant brain tissues lying within the treatment volume, the extent of which depends on radiation dose. Serial 1H-MRS allows non-invasive investigation of tissue metabolic profiles. In the present study the ratios of resonance signals assigned to the major 1H-MRS-visible metabolites (N-acetylaspartate, choline, creatine, inositol, lactate and lipid methylene group) were evaluated before, during and after post-surgical fractionated radiotherapy in brain regions close to and more distant from the tumor bed, receiving different radiation exposures (60 and

Treatment with oral coenzyme Q10 (CoQ10, 10 mg/kg per day for 6 days) was compared with no treatment in a previously described rabbit model of symptomatic cerebral vasospasm [Endo et al. (1988) Stroke 19: 1420-1425]. The treatment was initiated within 1-2 h after injection of autologous blood into the subarachnoid space. In CoQ10-untreated rabbits, moderate to severe neurological deficits developed, and multiple focal ischemic lesions were found in the brain regions with compromised blood supply, i.e., in the regions normally supplied by common carotid arteries which are subject to ligation in this model. CoQ10 treatment prevented the development of both the neurological deficits and histologically detectable brain tissue damage. In both CoQ10-treated and -untreated rabbits, infiltration of mononuclear cells was evident in the brain stem, although this region did not show signs of ischemic damage. The findings indicate that the histological and neurological correlates of brain tissue damage in this rabbit model of symptomatic cerebral vasospasm develop via mechanism(s) involving free radical-mediated oxidation of plasma lipoproteins. Similar mechanisms may play a role in the development of brain damage attributed to cerebral atherosclerosis.

We have previously shown that the novel immunosuppressive agent cladribine (CDA) inhibits human T and B cell lymphoproliferative responses and immunoglobulin synthesis in vitro, yet appears to be particularly efficacious as an inhibitor of B cell responses. We now report the effects of CDA on the human mixed lymphocyte reaction and on expression of T and B cell activation markers. CDA produced a significant inhibition of lymphocyte proliferation in human mixed lymphocyte reactions at a concentration of 10 nM. At concentrations of 10-100 nM the drug inhibited phytohaemagglutinin-induced expression of CD25 and HLA-D (by approximately 50%), but not phorbol myristate acetate-induced expression of CD69 on purified human T cells. At a concentration of 10 nM CDA totally abolished Staphylococcus aureus Cowan-induced expression of CD25 on purified B cells. These findings confirm that CDA is a potent immunosuppressive agent with some selectivity towards B cells. The drug may have potentially wide applications in clinical immunosuppression.

Twenty patients suffering from subarachnoid haemorrhage due to ruptured intracranial aneurysm and operated on within 72 h after SAH were treated with an experimental immunosuppressive drug 2-chlorodeoxyadenosine (2-CDA), dose 0.05 mg/kg/day i.v. for 7 days. The 2-CDA treatment was started immediately after angiographic confirmation of ruptured aneurysm, and the standard pharmacological treatment (nimodipine and steroids) was also given. 50% of patients were severely threatened by "delayed vasospasm" or late neurological deficit (Fisher's score 3 or 4). The neurological outcome (assessed 8-12 weeks after SAH) was good (GOS = 1) in 70%, and fair (moderate disability, GOS = 2) in 25%. A single case of severe disability (GOS = 3), as well as two cases of less than perfect outcome (GOS = 2), were related to unusual pre- or intraoperative complications. We conclude that the low doses of 2-CDA can be considered as a valuable adjunct to the standard pharmacotherapy of SAH patients operated on early.

We compared the morphology of the basilar artery walls in rabbits with experimental subarachnoid haemorrhage (SAH) without and with concomitant treatment with a novel immunosuppressive drug, 2-chloro-2′-deoxyadenosine (2 CdA). The treatment was successful in the prevention of the angiopathic changes, most likely due to its ability to inhibit lymphocyte activation in response to mitogenic signals. Since lymphocyte (and monocyte?) activation may play an important role in the development of the delayed neurological deficits in patients following SAH, the use of 2 CdA to prevent these deficits shall be considered.

The frequencies of the HLA-A, -B and -DR were determined in a group of 59 transplant donors who died from subarachnoid haemorrhage within three days following the rupture of intracranial aneurysm (the SAH group) and compared with those of a control group consisting of 389 donors who died from other causes. The only significant difference was in the increased frequency in the SAH group of non-typed ("empty")-DR loci in association with the DR7 phenotype. The most probable explanation of this finding is that in the SAH group the frequency of DR7 homozygotes is several times higher than in the general population, and that bearing the DR7 allele in homozygotic form is associated with a very high risk of developing potentially fatal intracranial aneurysmal haemorrhage.

Purpose Increased expression of glial fibrillary acidic protein (GFAP) is a characteristic of gliotic activation (Müller cells and astrocytes) in the retina. This study assessed vitreous body GFAP levels in various forms of retinal pathology. Methods This prospective study included 82 patients who underwent vitrectomy (46 retinal detachments (RDs), 13 macular hole (MHs), 15 epiretinal glioses (EGs), 8 organ donors). An established enzyme–linked immunosorbent assay (ELISA, SMI26) was used for quantification of GFAP. Results The highest concentration of vitreous body GFAP in organ donors was 20 pg/mL and it was used as the cutoff. A significant proportion of patients suffering from RD (65 %) to EG (53 %) had vitreous body GFAP levels above this cutoff when compared to organ donors (0 %, p

In classical textbooks of physiology the network generating respiratory rhythm was described as an “oscillator”, and it used to be modelled “cybernetically” by analogy to electronic oscillators. Such an interpretation resulted from the then-actual concept of synaptic transmission as a mere continuation of nerve conduction.

The effects of citicoline and/or low dose of MK-801 (sufficient to prevent the development of seizures) on survival, neurological and behavioral recovery following transient hyperglycemic–oligemic–hypoxic insult have been evaluated in mice. Neurological recovery was assessed semi-quantitatively on the third and the 10th day after the insult, and behavioral tests evaluating spontaneous locomotor activity, motor coordination and spontaneous alternation performance were performed on day 10. Neither drug given alone did influence survival rate, but the combination of MK-801 and higher citicoline dose decreased mortality on day 10. Behavioral performance was markedly compromised by the insult. Citicoline, but not MK-801, slightly but significantly improved behavioral outcome in all three tests. Conclusion: when brain ischemic insult is complicated with acute hyperglycemia, post-treatment with citicoline combined with MK-801 in low anti-convulsive dose improves survival and neurological recovery, and citicoline but not MK-801 enhances behavioral recovery.

BACKGROUND (E)-2'-deoxy-2'-(fluoromethylene)-cytidine (FMdC), an irreversible inhibitor of ribonucleotide reductase, displays a strong toxicity towards many cell lines derived from human solid tumors, while its activity on leukemia lines is less well-known. The aim of this study was to assess the effect of FMdC on the cell cycle and cell death of human leukemia lines HL-60 and MOLT-4, and murine leukemia L-1210 in vitro. It has been assumed that a prerequisite of FMdC cytotoxicity is intracellular phosphorylation by deoxycytidine kinase (dCK). METHODS Cell cultures in the exponential phase of growth were exposed to different concentrations of FMdC (10 nM to 10 microM) for 6 and 24 hours. In a parallel set of experiments 1 mM deoxycytidine was added to prevent phosphorylation of the drug by dCK. The DNA and protein content in the cells, as well as Annexin V/PI binding were assessed by flow cytometry. The cell cycle was analyzed by the MacCycle software. RESULTS The cytotoxic effects of FMdC, i.e., G(1)/S block and cell death were observed, associated with pronounced changes in the protein content. These effects were of variable intensity among the cell lines studied (HL-60 being the most susceptible), and in some cases, were not completely reversed by deoxycytidine excess. CONCLUSIONS FMdC is a potent cytotoxic/cytostatic agent against human leukemia cell lines in vitro. It also changes the cellular protein content. Unphosphorylated FMdC may slightly influence the cell cycle of some leukemic lines.

The purpose of our study was to determine the efficacy of 2-chlorodeoxyadenosine (2-CdA) administered in 2-hour intravenous infusions in previously treated patients with low grade non-Hodgkin's lymphoma (LGNHL). We treated 94 LGNHL patients with 2-CdA at a dosage of 0.12 mg/kg/24h in 2-hour intravenous infusion for 5 consecutive days. The treatment consisted of from 1 to 7 courses (median 3), repeated usually at monthly intervals. All patients were refractory to or relapsed after standard chemotherapy. Of these 94 patients 78 (83%) had clinical stage IV of the disease. Complete response (CR) was obtained in 12 (12.8%) and partial response (PR) in 36 (38.3%) giving an overall response rate of 51.1%. In 12 (12.8%) grade 4 thrombocytopenia with haemorrhagic diathesis was noted, grade 4 neutropenia was observed in 12 (12.8%) and infections complicated the course of treatment in 38 (40.4%) patients. 2-CdA treatment was the cause of death of 3 patients. The results of our study show that 2-CdA given in 2-hour infusions is an effective agent in advanced, heavily pretreated patients with LGNHL.