Gabriele Röhn

Polyamine metabolism in reversible cerebral ischemia: effect of α-difluoromethylornithine

Abstract Severe forebrain ischemia was produced in rats by occluding both carotid and vertebral arteries. Following 30 min ischemia brains were recirculated for 8 or 24 h. Twelve animals subjected to 8 or 24 h recirculation ( n = 6, each group) were given α-difluoromethylornithine (DFMO; injected intraperitoneally) immediately before samples were taken from the cerebellum, cortex, caudatoputamen and hippocampus. Samples from the left hemisphere were used for measuring ornithine decarboxylase (ODC) activity, and those from the right hemisphere for determining putrescine profiles. During recirculation ODC activity increased markedly in all brain structures, the most pronounced change being in the caudatoputamen after 8 h recirculation. Putrescine increased drastically after 8 h and even more after 24 h recirculation. DFMO-treatment significantly reduced ODC activity after 8 h recirculation and following 24 h recirculation. Putrescine, however, was significantly reduced following 24 h but not after 8 h recirculation. The discrepancy between reduction in ODC activity and putrescine levels in DFMO-treated animals was not prominent in the hippocampus after 8 h recirculation: here DFMO reduced ODC activity to control values without affecting putrescine levels. The results suggest that the observed overshoot in putrescine formation following ischemia is only partly caused by activation of ODC.

Epigenetic silencing of the candidate tumor suppressor gene PROX1 in sporadic breast cancer

Extensive hypermethylation and consecutive transcriptional silencing of tumorsuppressor genes have been documented in multiple tumor entities including breast cancer. In a microarray based genome‐wide methylation analysis of five sporadic breast carcinomas we identified a hypermethylated CpG island within the first intron of the prospero related homeobox gene 1 (PROX1). We, therefore, investigated CpG island methylation of PROX1 in a series of 33 pairs of primary breast cancer and corresponding normal tissue samples by bisulfite sequencing and COBRA analyses. Seventeen of these (52%) breast cancer samples revealed a significant accumulation of methylated CpG sites along with a significant reduction of PROX1 transcription compared to normal breast tissues of the same patients. Frequent methylation was also observed in brain metastases from primary breast cancer (21/37 = 57% of cases). Secondary, we analysed 38 brain metastases of primary breast carcinomas and detected a significantly reduced expression of PROX1 compared to normal breast tissue (p < 0.001) and primary breast carcinomas (p < 0.05), respectively. Additionally, treatment of breast cancer cell lines with demethylating agents could reactivate PROX1 transcription. In summary, we have identified PROX1 as a novel target gene that is hypermethylated and transcriptionally silenced in primary and metastatic breast cancer.

Relationship between putrescine content and density of ischemic cell damage in the brain of mongolian gerbils: effect of nimodipine and barbiturate

SummaryTwenty mongolian gerbils were anesthetized (1.5% halothane) and severe forebrain ischemia was produced in 15 animals by occluding both common carotid arteries. After 5 min ischemia brains were recirculated spontaneously. Immediately after ischemia nimodipine (1.5 mg/kg) or pentobarbital (50 mg/kg) was injected intraperitoneally into five animals. Four days later animals were reanesthetized (1.5% halothane); the brains were frozen with liquid nitrogen and cut in a cryostat. Ten-micrometer-thick coronal cryostat sections were stained with cresyl violet to assess the extent of ischemic cell damage in the lateral striatum, the CA1-layer of the hippocampus, and the thalamus. In addition, tissue samples (about 4 mg each) were taken from the lateral striatum, CA1 layer of the hippocampus and the thalamus. Putrescine levels were measured in these samples using reversed-phase high performance liquid chromatography and fluorescence detection. Reversible cerebral ischemia produced a significant increase in putrescine in the lateral striatum (from 11.15±0.79 to 44.83±11.76 nmol/g,P≤0.05), the CA1 subfield of the hippocampus (from 11.27±0.64 to 41.80±3.62 nmol/g,P≤0.05) and less so in the thalamus (from 11.28±0.70 to 16.50±1.71 nmol/g). Both postischemic nimodipine and barbiturate treatment of animals markedly reduced this increase in the lateral striatum to 14.09±1.41 and 15.75±1.38 nmol/g, respectively (P≤0.05 cf. untreated animals), to 29.82±6.04 and 23.21±3.12 nmol/g in the CA1-subfield of the hippocampus (P≤0.05 barbiturate-treated cf. untreated animals), and to 11.92±1.37 and 11.76±0.64 in the thalamus (P<0.05 barbiturate-treated cf. untreated animals). Severe neuronal necroses were apparent in the lateral striatum in four out of five animals but in none of the nimodipine- or barbiturate-treated animals. In the CA1 subfield of the hippocampus the number of necrotic cells/mm stratum pyramidale amounted to 202.1±9.8, 141.9±4.2 and 78.0±33.4 in untreated, nimodipine- or barbiturate-treated animals, respectively (P≤0.05 barbiturate-treated cf. control animals). It is suggested that putrescine, produced during recirculation following ischemia, contributes to the manifestation of ischemic cell injury. Putrescine may thus be taken as a significant biochemical correlate of ischemic cell damage.

Polyamine metabolism in transient focal ischemia of rat brain

Polyamine metabolism was studied in rat brains subjected to 30 min transient cerebral ischemia by measuring the activity of the key enzyme ornithine decarboxylase (ODC) and levels of the polyamines putrescine, spermidine and spermine. A transient increase in ODC activity was apparent after 4 h of recirculation in the ipsilateral cortex and striatum (P less than 0.05). Putrescine levels were significantly increased in the ipsilateral striatum after 4 h of recirculation, and after 24 h of recirculation in both the ipsilateral cortex and striatum. During ischemia spermidine levels were significantly reduced in the ipsilateral hemisphere and spermine levels in the ipsilateral cortex. It is suggested that during ischemia polyamines are released from neurons into the extracellular compartment and cleared into the blood.

Ornithine decarboxylase in reversible cerebral ischemia: an immunohistochemical study

SummaryAnesthetized Mongolian gerbils were subjected to 5-min ischemia and 8 h of recirculation. Vibratiom sections were taken for studying changes in ornithine decarboxylase (ODC) immunoreactivity using an antiserum to ODC, and tissue samples were taken for measuring ODC activity. After 5-min ischemia and 8-h recirculation ODC activity increased 11.5-, 5.9-, and 7.9-fold in the cerebral cortex, striatum and hippocampus, respectively (P≤0.05 to 0.01). In the cortex, striatum and hippocampus of control animals immunoreactivity was low but clearly above the detection limit. The reaction was confined to neurons. After 5-min ischemia and 8-h recirculation a sharp increase in immunoreactivity was observed confined to neurons, indicating that the postischemic activation of polyamine metabolism is a neuronal response to ischemia. The immunoreactivity was markedly increased in the perinuclear cytoplasm and the dendrites. In the striatum the density of neurons exhibiting a sharp increase in immunoreactivity was more pronounced in the lateral than in the ventral part. In the hippocampus a strong reaction was present in all subfields but the CA1 subfield was particularly affected. The present study demonstrates for the first time that biosynthesis of a protein is markedly activated during the first 24 h of recirculation after 5-min cerebral ischemia of gerbils even in the vulnerable CA1 subfield, in which the overall protein synthesis is sharply reduced at the same time. Studying polyamine metabolism after ischemia may, thus, provide new information about the basic molecular mechanisms responsible for the altered gene expression after metabolic stress.

Cerebral polyamine metabolism in reversible hypoglycemia of rat: relationship to energy metabolites and calcium.

Abstract: Thirty minutes of insulin‐induced reversible hypoglycemic coma (defined in terms of cessation of EEG activity) was produced in anesthetized rats. At the end of the hypoglycemic coma or after recovery for 3, 24, or 72 h induced by glucose infusion, the animals were reanesthetized and their brains frozen in situ. Two control groups were used: untreated controls without prior manipulations, and insulin controls, which received injections of insulin followed by glucose infusion to maintain blood glucose within the physiological range. The brains of these latter animals were frozen 3, 24, or 72 h after glucose infusion. Tissue samples from the cortex, striatum, hippocampus, and thalamus were taken to measure ornithine decarboxylase (ODC) activity, and putrescine and spermidine levels, as well as phosphocreatine (PCr), ATP, glucose, and lactate content. In addition, 20‐μm thick coronal sections taken from the striatum and dorsal hippocampus were used for histological evaluation of cell damage and also stained for calcium. Insulin in the absence of hypoglycemia produced a significant increase in ODC activity and putrescine level but had no effect on the profiles of energy metabolites or spermidine. During hypoglycemic coma, brain PCr, ATP, glucose, and lactate levels were sharply reduced, as expected. Energy metabolites normalized after 3 h of recovery. In the striatum, significant secondary decreases in PCr and ATP contents and rises in glucose and lactate levels were observed after 24 h of recovery. ODC activity, and putrescine and spermidine levels were unchanged during hypoglycemic coma. After 3 h of recovery, ODC activity increased markedly throughout the brain, except in the striatum. After 24 h of recovery, ODC activity decreased and approached control values 2 days later. Putrescine levels increased significantly throughout the brain after reversible hypoglycemic coma, the highest values observed after 24 h of recovery (p≤ 0.001, compared with controls). After 72 h of recovery, putrescine levels decreased, but still significantly exceeded control values. Reversible hypoglycemic coma did not produce significant changes in regional spermidine levels except in the striatum, where an approximately 30% increase was observed after 3 and 72 h of recovery (p≤ 0.01 and p≤ 0.05, respectively). Twenty‐four hours after hypoglycemic coma, intense calcium staining was apparent in layer III of the cerebral cortex, the lateral striatum, and the crest of the dentate gyrus. After 72 h of recovery, the intense calcium staining included also cortical layer II, the septal nuclei, the subiculum, and the hippocampal CA1‐subfield. Changes in polyamine metabolism thus preceded the intense calcium staining in the brain. The results indicate that reversible hypoglycemic coma induces a sharp increase in putrescine level comparable to that observed previously after cerebral ischemia. We, therefore, conclude that the increase in putrescine content is an early biochemical marker of delayed neuronal cell necrosis irrespective of the pathogenesis of this injury. The possible role of polyamines in the manifestation of neuronal necrosis following hypoglycemic coma is discussed.

Protein synthesis in the hippocampal slice: Transient inhibition by glutamate and lasting inhibition by ischemia

Protein synthesis was measured in hippocampal slices which were exposed to glutamate (1 mM or 10 mM) or which were deprived of glucose and oxygen (‘in vitro ischemia’) for 15 min. Glutamate at 1 mM, a concentration estimated to occur duringin vivo ischemia did not affect protein synthesis. Ten mM glutamate inhibited protein synthesis immediately after exposure (50% of control values) and reduced ATP levels to about 30% of the control. After two hours, slices fully recovered their protein synthesis and energy metabolism. The effect of 10 mM glutamate was not receptor-mediated, as NMDA, AMPA, or metabotropic receptor antagonists failed to block the glutamate effect. Immediately after ischemia, protein synthesis was reduced to 30% of control values, and 2 hours later it was still depressed to one-half of control values. Energy charge, however, recovered completely. Ischemic inhibition of protein synthesis was not reversed by glutamate receptor antagonists. The data indicate that inhibition of protein synthesis in hippocampal slices during ischemia is not glutamate-dependent.

Ornithine Decarboxylase Activity and Putrescine Levels in Reversible Cerebral Ischemia of Mongolian Gerbils: Effect of Barbiturate

Reversible cerebral ischemia was produced in anesthetized Mongolian gerbils by occluding both common carotid arteries. After 5 min of ischemia, brains were recirculated for 8 or 24 h. Treated animals received a single intraperitoneal injection of pentobarbitol (50 mg/kg) immediately after the anuerysm clips were removed. At the end of the experiments, animals were reanesthetized and their brains frozen in situ. Tissue samples were taken from the cerebral cortex, lateral striatum, CA1 subfield of the hippocampus, thalamus, and cerebellum for measuring ornithine decarboxylase (ODC) activity and putrescine levels. In addition, 20-μm-thick coronal tissue sections were taken from the level of the striatum and stained with hematoxylin/eosin for evaluating the extent of ischemic neuronal necrosis in the lateral striatum. In control animals ODC activity and putrescine levels amounted, respectively, to 0.32 ± 0.03 nmol/g/h and 10.2 ±0.5 nmol/g in the cerebral cortex; 0.34 ± 0.02 nmol/g/h and 12.8 ± 0.5 nmol/g in the lateral striatum; 0.58 ± 0.05 nmol/g/h and 10.5 ± 0.7 nmol/g in the hippocampal CA1 subfield; 0.35 ± 0.01 nmol/g/h and 9.8 ± 0.4 nmol/g in the thalamus; and 0.25 ± 0.01 nmol/g/h and 8.3 ± 0.6 nmol/g in the cerebellum. After 5 min cerebral ischemia and 8 h recirculation, a significant 7- to 16-fold increase in ODC activity was observed in all forebrain structures studied. Following 24 h recirculation, ODC activity normalized in the cortex, striatum, and thalamus but was still significantly above control values in the hippocampal CA1 subfield. In the cerebellum ODC activity did not change significantly. Putrescine levels were significantly increased in all forebrain structures after 8 h (two- to threefold) and even more after 24 h recirculation (up to fivefold). In barbiturate-treated animals, ODC activity was not significantly changed in relation to untreated ones. There was, however, a trend to higher activity in the cerebral cortex, lateral striatum, and hippocampal CA1 subfield. Barbiturate did not produce a significant effect on postischemic putrescine levels except in the CA1 subfield. Here the putrescine content of treated animals was significantly below that found in untreated ones. In the lateral striatum, severe cell damage (>90% of neurons were necrotic) was observed in 5 of 12 untreated animals but in none of the barbiturate-treated ones (<10% of neurons necrotic). In animals with severe cell necrosis in the lateral striatum, putrescine levels amounted to 70.9 ± 3.4 nmol/g but to only 32.0 ± 2.9 nmol/g in animals in which <10% of neurons were affected (p ⩽ 0.001).

Ganglioside profiles in human gliomas: quantification by microbore high performance liquid chromatography and correlation to histomorphology and grading.

Summary¶ The composition and the content of gangliosides changes during physiological growth and differentiation as well as in neoplastic cell transformation. In order to determine if ganglioside profiles correlate with brain tumour malignancy, the ganglioside distribution was determined in 31 gliomas of astrocytic origin and in non-tumour tissue by a recently developed microbore high performance liquid chromatography (HPLC) method. Glioma malignancy was graded according to the grading system proposed by the World Health Organization (WHO) in 1993. In general, an increase of GD3 and a decrease of normal brain gangliosides correlated with a higher grade of malignancy. Pilocytic astrocytomas Grade I had a distinctive ganglioside profile, histologically as well as biochemically. Although they are low-grade gliomas, the pilocytic astrocytomas exhibited a GD3 content comparable to anaplastic gliomas and could only be biochemically distinguished from other tumour grades by relatively high type “b” ganglioside levels. Thus, ganglioside composition not only reflects anaplasia but can also be used to indicate biological characteristics of tumours of different histogenetic origin.

ISCHEMIA - INDUCED DISTURBANCES OF POLYAMINE SYNTHESIS

Publisher Summary This chapter discusses ischemia-induced disturbances in polyamine synthesis. It discusses the periods of time during which changes take place and the relationship of changes in polyamine synthesis to the duration of ischemia. Pharmacological interventions reducing ischemia-induced disturbances in polyamine synthesis are discussed. Emphasis is put on the possible role of ischemia-induced alterations in polyamine metabolism in the process of recovery from the metabolic stress produced either by cerebral ischemia or in the manifestation of neuronal necrosis. Transient cerebral ischemia causes a disturbance of polyamine synthesis that is characterized by a sharp increase in ornithine decarboxylase and decrease in S-adenosylmethionine decarboxylase activity. The result is an overshoot in putrescine formation and a reduction in spermine levels. In addition, evidence is presented that polyamines are released from the intracellular compartment during ischemia and following prolonged recirculation in severely damaged areas. Because of the main features of ischemia-induced disturbances in polyamine synthesis and the reactions known to be influenced by these altered polyamine profiles it is suggested that these changes play a role in the manifestation of ischemia-induced neuronal necrosis.