Protein kinase C as an early and sensitive marker of ischemia-induced progressive neuronal damage in gerbil hippocampus

In the model of transient brain ischemia of 6-min duration in gerbils we have estimated:

1. The concentration of brain gangliosides: A significant decrease to about 70% of control was observed selectively in the hippocampus at 3 and 7 d after ischemia.
2. The activity of Na⁺,K⁺-ATPase: The enzyme activity was not affected in either hippocampus nor in cerebral cortex.
3. The malonylaldehyde (MDA) concentration: The levels of MDA had increased at 30 min after ischemia up to 123 and 129% of control in hippocampus and cerebral cortex, respectively.
4. Immunoreactivity of protein kinase C detected by Western blotting: In hippocampus the early translocation toward membranes was followed by a decrease in total enzyme content at 6, 24, 72, and 96 h of postischemic recovery. Also, a sharp increase of 50 kDa isoform (PKM) was noticed immediately and at the early recovery times.

The behavior of these biochemical markers of ischemic brain injury in the hippocampus after the short (6 min) insult was contrasted with their reaction in the cerebral cortex as well as after prolongation of the ischemia to 15 min. These results taken together indicate that an early increase in PKC translocation followed by a decrease is the most symptomatic for selective, delayed, postischemic hippocampal injury, resulting from short duration (6 min) ischemia of the gerbil brain.     

Stress-induced dynamic changes in mouse brain polyamines. Role in behavioral reactivity

Recent findings indicate that rapid and transient changes in polyamine metabolism, termed the polyamine-stress-response, may occur repeatedly in the brain after chronic intermittent stress. Here, we sought to examine the effects of chronic intermittent restraint stress, or of daily intraperitoneal dexamethasone injections on polyamine concentrations in the hippocampus of adult male C57BL/6 mice. Additionally, we studied the effects of alpha-difluoromethylornithine, an irreversible ornithine decarboxylase inhibitor, on stress-induced changes in polyamines and on behavioral reactivity to novelty stress measured in an open-field arena. As previously observed, following a single stress episode putrescine concentration increased transiently, but the polyamines spermidine and spermine remained unchanged. Following chronic restraint stress, putrescine concentration was increased after each daily stress episode with the largest increase observed after the 4th episode, while spermidine was increased just after the 2nd and 4th episodes and spermine only after the 4th daily episode. In contrast, all polyamine concentrations were increased after 10 injections of either dexamethasone or vehicle (0.9% NaCl). A 14-day course of alpha-difluoromethylornithine treatment resulted in a complete putrescine depletion and over 50% reduction in polyamines, and led to changes in open field activity indicative of altered emotional behavior. CONCLUSIONS: (a) while putrescine concentration increases in the hippocampus after each restraint stress episode, spermidine and spermine undergo a delayed but transient increase; (b) in contrast, chronic dexamethasone treatment may lead to a permanent increase in the concentrations of all polyamines and; (c) chronic alpha-difluoromethylornithine treatment reduces brain polyamine concentrations and modulates emotional reactivity to novelty stress. The study indicates that the brain polyamine-stress-response is a dynamic process that varies with the type, intensity and length of stressful stimuli, and implicates this response as an adaptive mechanism in the reaction to stressors.     

Polyamines in cerebral ischemia

The present series of experiments was designed to study regional profiles of polyamines (putrescine, spermidine, and spermine) in reversible cerebral ischemia produced in rats and Mongolian gerbils. Polyamine profiles did not change during ischemia, but did following recirculation. The most prominent changes were a dramatic postischemic increase in putrescine and a marked decrease in spermine in severely damaged regions. Within a given brain structure, the postischemic putrescine levels correlated closely with the density of ischemic cell injury and the time period of cerebral ischemia. Furthermore, putrescine was already considerably increased in the CA1-subfield of the hippocampus of gerbils after 8 h recirculation, i.e., at a time when the cells are still intact. The results indicate that putrescine may be viewed as an excellent biochemical correlate of ischemic cell injury. The postischemic changes in putrescine levels are discussed in relation to the known activities of this compound.     

Polyamine metabolism in different pathological states of the brain

Biosynthesis of the polyamines spermidine and spermine and their precursor putrescine is controlled by the activity of the two key enzymes ornithine decarboxylase (ODC) andS-adenosylmethionine decarboxylase (SAMDC). In the adult brain, polyamine synthesis is activated by a variety of physiological and pathological stimuli, resulting most prominently in an increase in ODC activity and putrescine levels. The sharp rise in putrescine levels observed following severe cellular stress is most probably the result of an increase in ODC activity and decrease in SAMDC activity or an activation of the interconversion of spermidine into putrescine via the enzymes spermidineN-acetyltransferase and polyamine oxidase. Spermidine and spermine levels are usually less affected by stress and are reduced in severely injured areas. Changes of polyamine synthesis and metabolism are most pronounced in those pathological conditions that induce cell injury, such as severe metabolic stress, exposure to neurotoxins or seizure. Putrescine levels correlate closely with the density of cell necrosis. Because of the close relationship between the extent of poststress changes in polyamine metabolism and density of cellular injury, it has been suggested that polyamines play a role in the manifestation of structural defects. Four different mechanisms of polyamine-dependent cell injury are plausible: (1) an overactivation of calcium fluxes and neurotransmitter release in areas with an overshoot in putrescine formation; (2) disturbances of the calcium homeostasis resulting from an impairment of the calcium buffering capacity of mitochondria in regions in which spermine levels are reduced; (3) an overactivation of the NMDA receptor complex caused by a release of polyamines into the extracellular space during ischemia or after ischemia and prolonged recirculation in the tissue surrounding severely damaged areas; (4) an overproduction of hydrogen peroxide resulting from an activation of the interconversion of spermidine into putrescine via the enzymes spermidineN-acetyltransferase and polyamine oxidase. Insofar as a sharp activation of polyamine synthesis is a common response to a variety of physiological and pathological stimuli, studying stressinduced changes in polyamine synthesis and metabolism may help to elucidate the molecular mechanisms involved in the development of cell injury induced by severe stress.     

Regional brain polyamine levels in permanent focal cerebral ischemia

Transient global cerebral ischemia has been shown to induce marked changes in the polyamine pathway with a significant increase in putrescine, the product of the ornithine decarboxylase reaction. This study examined the relationship between tissue and extracellular polyamines and regional cerebral blood flow and brain edema. Six hours of focal ischemia in cats (n=10) was produced by permanent middle cerebral artery occlusion. Extracellular polyamines were measured in extracellular fluid obtained by microdialysis. Regional cerebral blood flow using laser Doppler flowmetry and specific gravity, an indicator of brain edema, were measured in contralateral (non-ischemic), penumbra and densely ischemic brain regions. A significant increase in the tissue putrescine level was found in the penumbra but there was no difference in the putrescine levels between contralateral and densely ischemic regions. There was no significant change in the spermidine and spermine levels in the three regions. Extracellular levels of putrescine and spermidine were found to be significantly lower than the tissue levels and no change in polyamines was observed in any region. Significant edema formation was observed in densely ischemic and penumbra regions. This is the first demonstration that tissue putrescine is increased in the penumbra region, an area of incomplete ischemia that is developing brain edema.     

Regional profile of polyamines in reversible cerebral ischemia of Mongolian gerbils

Reversible cerebral ischemia was produced in Mongolian gerbils (Meriones unguicultatus) by occluding both common carotid arteries. After 5 min ischemia brains were recirculated, for 8, 24, 48, 72, or 96 h. An additional 6 animals were subjected to 10 min ischemia and 24 h recirculation. Sham-operated animals served as controls. At the end of the experiments, brains were frozenin situ and cut in a cryostat. Coronal sections, 10 μm thick, were taken for histological staining. In addition, tissue samples (2–4 mg each) were taken from the cortex, lateral caudoputamen, CA1-layer of the hippocampus, and thalamus. Polyamines (spermidine, spermine, and the precursor putrescine) were measured in these samples using reverse-phase HPLC and fluorescence detection after extraction and precolumn derivatization. Five-minute cerebral ischemia had no effect on the levels of putrescine, spermidine, or spermine. However, following recirculation, putrescine increased markedly with time, being most pronounced in the CA1-subfield of the hippocampus, less so in the cortex, and even less so in the thalamus. After prolonged recirculation, severe neuronal necroses could be observed only in regions exhibiting high putrescine levels. Spermidine or spermine did not change during recirculation, except in severely damaged regions: Here, spermine levels were markedly reduced following prolonged recirculation. The postischemic increase in putrescine is discussed in respect to the known multiple activities of putrescine.     

Picrotoxin and bicuculline have different effects on lumbar spinal networks and motoneurons in the neonatal rat

Polyamines (putrescine, spermidine and spermine) are ubiquitous cellular components, but their specific role in central nervous system (CNS) injury has yet to be characterized. CNS injury results in increased activities of ornithine decarboxylase and spermidine/spermine-N(1)-acetyltransferase, and accumulation of putrescine. The present study determined the polyamine profile in three models of CNS injury, in two different species (gerbil and rat) and two strains of rats (Sprague-Dawley and spontaneously hypertensive): (1) transient focal cerebral ischemia in spontaneously hypertensive rats (SHR); (2) traumatic brain injury in Sprague-Dawley rats; and (3) transient forebrain ischemia in gerbils. While there was a significant increase in putrescine in all three models, spermine and spermidine levels were unaltered in forebrain ischemia and traumatic brain injury. However, transient focal cerebral ischemia shows depletion of spermine and spermidine levels in injured hemisphere compared to contralateral region. Exogenous spermine significantly restored the spermine as well as spermidine levels in the ipsilateral hemisphere after transient focal cerebral ischemia, but did not alter putrescine levels or the ratio of spermidine to spermine. The loss of spermine in particular, may have several consequences that contribute to ischemic injury, including destabilization of chromatin, decreased mitochondrial Ca(2+) buffering capacity, and increased susceptibility to oxidative stress. Based on our and other studies, we propose a tentative antioxidant mechanism of spermine neuroprotection.     

Dopamine D2 receptor imaging with SPECT using IBZM in 16 patients with Parkinson disease

The striatal D2 receptor was investigated for variability of behavior in 16 patients with Parkinson disease by means of SPECT using 123I-IBZM, a recently synthesized radioligand with high affinity and specificity for this type of receptor. All the patients underwent routine laboratory tests, EEG and cranial CT scanning as well as SPECT. To ensure accurate clinical assessment we used the Hoehn and Yahr, and Webster scales, NUDS and Mini Mental State examination. Our preliminary data indicate:

- increased uptake by the striatal dopamine receptor in untreated patients with early PD compared with controls (striatum/cerebellum ratio of 1.77±0.12 vs 1.59±0.13);

- a slight but significant lowering of D2 receptor binding when L-Dopa is started (1.49±0.09);

- low D2 uptake values in the more severely affected patients;

- absence of asymmetries in hemiparkinsonism.

     

Neuronal and glial responses to polyamines in the ischemic brain

The polyamines, putrescine, spermidine and spermine are present in most living cells, with the essentiality for normal cell function, cellular growth and differentiation. In the mammalian brain, polyamines are also present at relatively high concentrations with different regional distribution profiles. Cerebral ischemia is a leading cause of disability and mortality in humans, and believed to yield a cascade of cytotoxic molecules responsible for the death of viable cells in the brain. Polyamines have been implicated in the pathogenesis of ischemic brain damage. For example, polyamine biosynthesis is increased after the onset of cerebral ischemia through an induction of ornithine decarboxylase, a key enzyme in the polyamine biosynthetic pathway. The administration of a drug that inhibits ornithine decarboxylase activity prevents the development of ischemic brain damage, suggesting a critical role of the accumulation of polyamines in the ischemic brain in the pathogenesis of stroke. Both spermine and spermidine are linked to the development of glutamate-mediated neurotoxicity, for they can bind to the N-methyl-D-aspartate (NMDA)-sensitive subtype of glutamate receptors to potentiate cellular responses to glutamate. Moreover, polyamines are metabolized by polyamine oxidases after acetylation to produce different cytotoxic aldehydes and reactive oxygen species such as hydrogen peroxide, which possibly damage proteins, DNA and lipids. Polyamines have been extensively studied in the ischemic brain, particularly with respect to neuronal responses such as NMDA receptor-mediated excitotoxicity. However, little is known about glial responses to polyamines in the ischemic brain to date. In this review, we would summarize previous studies related to neuronal and glial responses to polyamines in the ischemic brain.     

Polyamines affect growth of cultured rat cerebellar neurons in different sera

The study examines the effects of polyamines on growth of cultured neurons from 6-day-old rat cerebellar cortex, by means of: (a) irreversible inhibition of ornithine decarboxylase activity with alpha-difluoromethylornithine, and (b) treatment with the exogenous diamine putrescine and the polyamines spermidine and spermine, in the presence of sera from different sources. Inhibition of ornithine decarboxylase activity starting at plating time led after 24 hr to a partial inhibition of cell aggregation with a drastic (90%) inhibition of neurite formation. However, after 48 hr of enzyme inhibition aggregation and neurite formation increased to approach the 24 hr control values and eventually cultures fully recovered. Polyamines added at plating time in the presence of fetal calf serum led to a permanent dose-dependent inhibition of aggregation and neurite formation, spermine being effective at lower doses (spermine less than spermidine much less than putrescine). Adhesion of cells to polylysine coated surfaces was not affected by polyamines. Recovery from the cytostatic polyamine effect was observed after washing and addition of fresh medium. Prevention of the effect was achieved in the presence of aminoguanidine, an inhibitor of diamine and polyamine oxidases. The preventive effect of aminoguanidine was dose polyamine-dependent, with higher aminoguanidine concentrations needed to prevent the spermine effect (spermine much greater than spermidine greater than putrescine). The polyamine effects were observed in the presence of fetal calf, heat-inactivated fetal calf and human sera, but not with rat serum. Addition of polyamines to 24-hr-old cultured neurons, in the presence of fetal calf serum, led 12 hr later to cell death. This lethal effect could be inhibited by aminoguanidine. We conclude: (a) irreversible inhibition of ornithine decarboxylase activity delays but does not prevent neuronal growth in culture; (b) oxidation products of extracellular polyamines inhibit cell aggregation and neurite formation of cultured neuroblasts, and have lethal effects on growing neurons in culture, and (c) different pharmacological effects of polyamines can be expected in different species.     

The changes in the permeability of the blood-brain barrier when under neurosurgical intervention

The aim of this research was to determine the influence of anesthesia, drugs, and neurosurgical trauma on the permeability of the blood-brain barrier (BBB) for macromolecules. Protein markers of oligodendrocytes and astroglia were used. The research methods were unique. Two groups of patients were formed: with neurosurgical brain trauma and without it. Everyone in both groups was subjected to anesthesia. The results were unexpected and did not depend on surgery:

1. Neurosurgical brain trauma did not change the permeability of BBB.
2. Anesthesia resulted in increasing permeability of BBB with peak upon 24–48 h.
3. High blood levels of neurospecific proteins proved protein synthesis in glial cells.
4. Massive transferring of brain antigens into blood did not stimulate antibody synthesis during a period of 21 d.

According to the high blood levels of neural proteins, the antigen-antibody reaction took place in blood and autoimmune complexes were eliminated before penetration into the brain. A new mechanism of brain autoimmune safety is discussed.     

Polyamines can protect against ischemia-induced nerve cell death in gerbil forebrain

We have previously demonstrated that administration of the polyamines putrescine, spermidine, or spermine can prevent neuronal degeneration in rats during naturally occurring cell death or after injurious treatments such as nerve injury or monosodium glutamate neurotoxicity. The present study demonstrates that also in adult gerbils polyamine treatment can protect forebrain neurons from degeneration after ischemia. Neurons in the hippocampus and striatum were rescued from delayed cell death after brief (5 min) global ischemia in gerbils which were treated with daily injections (10 mg/kg) of polyamines. The evidence accrued, so far, indicates that systemic polyamines can protect a wide variety of central and peripheral neurons from natural or induced degeneration.     

Epilepsie in einem ländlichen Krankenhaus in Afrika

Purpose Epilepsy is the commonest brain disorder throughout the developing world. Classification of seizures in view of adequate treatment however has been difficult due to lack of diagnostic tools such as electroencephalograms and imaging. Our study was conducted with the aim of developing a classification system for seizures suitable for local circumstances in resource poor countries. Methods Based on clinical, diagnostic, prognostic and therapeutic needs of 337 people with epilepsy from a clinic in northern Tanzania, we adjusted the International Classification of Epileptic Seizures (ICES) and suggest the following clinically based classification:

1. Generalised types of seizures:
2. Generalised seizures within a specific age range (6-25 years)
3. Generalised seizures outside that specific age range
4. Partial types of seizures:
5. Generalised seizures with diffuse brain damage
6. Generalised seizures with focal signs
7. Complex partial seizures
8. Simple partial seizures

Results Genuine epilepsy was diagnosed in 337 out of 346 patients over a period of 26 months. Two hundred and six patients (59.6%) had primary generalised seizures, split into 147 with generalised seizures which started within a specific age range (42.5%) and 59 with generalised seizures outside that age range (17.1%). A history of focal seizures was found in 102 patients (29.4%) with generalised seizures with focal signs in 60 (17.3%) and generalised seizures with diffuse brain damage in 41 patients (11.8%); one patient had complex partial seizures. Other types of seizures were diagnosed in 29 patients (8.3%), 24 had two different types of seizures (6.9%) and in five (1.4%) no clear diagnosis could be reached due to lack of information. Provoked seizures were found in 9 cases (2.6%). ConclusionWe are suggesting an ICES adjusted and simplified classification system for seizures in developing countries. Based on our suggested diagnostic groups, we were able to classify all our patients but five.     

Interventional neuroradiology in children

Conclusions about pediatric interventional neuroradiology

? It deals with rare diseases that are mostly not treatable by a direct surgical approach. Results have been published that cannot be ignored today.

? The traditionally poor prognosis of vascular disease in young children cannot justify isolated heroic procedures, just because the tools are easy to use and to purchase.

? The low costs, clinical efficiency and low mortality-morbidity in cerebral intervention require a critical mass of patients per year.

? In a given place standards of quality should be guaranted before settling on pediatric interventional neuroradiology and not after ward.

? Although an interventional neuroradiologist is required for 5 million patients in the adult population, the vascular disease spread in pediatrics probably justifies European network coverage by a few centers to offer satisfactory training and the creation of new converging centers.

     

Determination of polyamines by precolumn derivatization with 9-fluorenylmethyl chloroformate and reverse-phase high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) method for the determination of picomole levels of polyamines (putrescine, spermidine, and spermine) is described. Amino groups in polyamines react with 9-fluorenylmethyl chloroformate (FMOC) to form stable and highly fluorescent derivatives which can be separated and quantitatively estimated by HPLC in about 12 min. The mean relative elution times (n = 14) for putrescine, spermidine and spermine are 4.21 +/- 0.02, 10.09 +/- 0.02 and 11.19 +/- 0.04 min, respectively. The method has been applied to determine polyamine concentration in rat dorsal root ganglia (DRG) without interference with endogenous amino acids. Polyamine content of individual rat DRG has been calculated and the values are as follows: putrescine, 36.8 +/- 2.01, spermidine, 1652 +/- 131.0 and spermine 388.5 +/- 38.4 pmol/DRG. Information on polyamine concentrations in DRG may be useful in understanding the mechanism of action of toxic chemicals on nervous system.     

Identification of novel catecholamine absorbing proteins in the central nervous system

Several pharmacologically active catecholamines have been shown to react covalently with CNS proteins, namely species of 47, 40, 22, and 20 kDa. Of these, the 47-kDa protein showed the greatest incorporation of tritium following treatment with [³H]dopamine, [³H]ADTN, or [³H]N-propyl-norapomorphine. Labeling was accomplished by incubating the tritiated ligands with crude membrane preparations in the absence of reducing agents. These proteins displayed several unique characteristics:

1. The proteins are distributed throughout the CNS, but no evidence was found for their presence in other tissues;
2. The proteins have a unique pharmacological profile, interacting with dopamine, ADTN, N-propylnorapomorphine, and apomorphine, but not with ligands specific for other proteins known to interact with these compounds;
3. The labeling of these proteins is not inhibited by several similar catecholamines and other catechols, suggesting specific structural requirements; and
4. These proteins exhibited stereoselectivity with respect to this labeling.

These results demonstrate the existence of novel CNS proteins capable of covalently absorbing several physiologically important catecholamines in vitro.     

Ornithine decarboxylase and polyamines in developing rat brain and heart: Effects of perinatal hypothyroidism

Hypothyroidism induced by perinatal administration of propylthiouracil (PTU) had profound effects on growth of the heart, with major organ weight deficits persisting well beyond the termination of drug treatment. These effects were preceded by disruption of the developmental patterns of cardiac ornithine decarboxylase (ODC) and the polyamines, which are thought to be intracellular modulators of cellular maturation. Activity of cardiac ODC was depressed in the PTU-treated group and putrescine and spermidine levels were markedly subnormal. PTU administration also affected brain growth, but much less so than in the heart. The disruption of the brain ODC/polyamine system was also less pronounced, with relatively small degrees of spermidine depletion and a slight elevation of ODC. For both tissues, the biochemical effects of perinatal hypothyroidism were opposite to those found previously for administration of exogenous thyroid hormones. These results support the views that:

1. (1) endogenous thyroid hormones contribute to the maintenance of normal developmental patterns of ODC and the polyamines, and

2. (2) the ODC/polyamine system participates in events modulating subsequent tissue growth.

Cellular Polyamines Promote Amyloid-Beta (Aβ) Peptide Fibrillation and Modulate the Aggregation Pathways

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Study of nystagmus suppression in the rotatory tests

Thirty one healthy subjects ranging in age between 21 and 50, participated voluntarily in the following experiment: they underwent programmed sequential alternating rotations, while their nystagmus was recorded by way of electronystagmography. The direction of rotation was changed every minute, and the total rotation time was 36 minutes. The subjects were rotated under external conditions which were altered every six minutes; these conditions included the following:

1. Rotation in the light with visual fixation and convergence.
2. Rotation in the light with visual fixation without convergence (one eye closed, the other looking at the target).
3. Rotation in the light without visual fixation (both eyes covered by a shade or by Frenzel's glasses).
4. Rotation in the dark without convergence (looking straight ahead).
5. Rotation in the dark with convergence (looking at the thumb, closely placed in front of the nose).

The purpose of the experiment was to study the phenomenon of nystagmus suppression observed with visual fixation and often attributed to cortical activity. The conclusion of the study is that nystagmus may be suppressed by light alone as well as by visual stimuli, convergence and proprioceptive stimuli. The inference is that the mechanism involves brain stem reflexes and not necessarily visual cortex activity.     

Effects of delayed administration of (-)-epigallocatechin gallate,a green tea polyphenol on the changes in polyamine levels and neuronal damage after transient forebrain ischemia in gerbils

(-)-Epigallocatechin gallate has a potent antioxidant property and can reduce free radical-induced lipid peroxidation as a green tea polyphenol. In previous study, systemic administration of (-)-epigallocatechin gallate immediately after ischemia has been shown to inhibit the hippocampal neuronal damage in the gerbil model of global ischemia. Polyamines are thought to be important in the generation of brain edema and neuronal cell damage associated with various types of excitatory neurotoxicity. We examined the effects of delayed administration of (-)-epigallocatechin gallate on the changes in polyamine levels and neuronal damage after transient global ischemia in gerbils. To produce transient global ischemia, both common carotid arteries were occluded for 3 min with micro-clips. The gerbils were treated with (-)-epigallocatechin gallate (50 mg/kg, i.p.) at 1 or 3 h after ischemia. The polyamines; putrescine, spermidine, and spermine levels were examined using high performance liquid chromatography in the cerebral cortex and hippocampus 24 h after ischemia. Putrescine levels in the cerebral cortex and hippocampus were increased significantly after ischemia and the delayed administrations of (-)-epigallocatechin gallate (1 or 3 h after ischemia) attenuated the increases. Only minor changes were noted in the spermidine and spermine levels after ischemia. In histology, neuronal injuries in the hippocampal CA1 regions were evaluated quantitatively 5 days after ischemia. (-)-Epigallocatechin gallate administered 1 h or 3 after ischemia significantly reduced hippocampal neuronal damage. The present results show that the delayed administrations of (-)-epigallocatechin gallate inhibit the transient global ischemia-induced increase of putrescine levels in the cerebral cortex and hippocampus. (-)-Epigallocatechin gallate is neuroprotective against neuronal damage even when administered up to 3 h after global ischemia. These findings suggest that (-)-epigallocatechin gallate may be promising in the acute treatment of stroke.