Neuroprotective role of ornithine decarboxylase activation in transient focal cerebral ischaemia: a study using ornithine decarboxylase‐overexpressing transgenic rats

Nuclear magnetic resonance imaging (MRI) was used to study dynamics of maturation and the size of ischaemic stroke lesions in rats with greatly increased activity of ornithine decarboxylase (ODC). Syngenic rats, either with or without chronic pre-ischaemic treatment with an ODC inhibitor, α-difluoromethylornithine (DFMO), as well as ODC-overexpressing transgenic rats were subjected either to transient middle cerebral artery (MCA) occlusion or permanent occlusion of the cortical branch of MCA. The two models were chosen to assess the role of ODC activity in damage caused by ischaemia and reperfusion, respectively. Diffusion of water was quantified by means of the trace of the diffusion tensor (D_av = Trace=D) to assess the extent of energy failure and cytotoxic oedema, whereas the spin–spin relaxation time (T₂) was used as a quantitative indicator of irreversible damage by MRI. Exposure to transient MCA occlusion resulted in significantly smaller stroke lesions in the ODC-overexpressing transgenic (246 ± 14 mm³) than in syngenic (320 ± 9 mm³) or DFMO-treated (442 ± 63 mm³) rats as determined 48 h after the occlusion. The differences in sizes were due to smaller lesions in the cortical tissue (transgenic vs. syngenic) or both in cortical and striatal regions (transgenic vs. DFMO-treated animals). The degree of irreversible oedema was greater in DFMO-treated rats than in syngenic or transgenic animals indicating accelerated development of a permanent damage in the absence of ODC induction. Cortical infarct following permanent MCA occlusion developed faster in the DFMO-treated than in syngenic or transgenic rats as the lesion sizes at 10 h were 26.2 ± 4.3 mm³, 14.2 ± 2.3 mm³ and 12.3 ± 1.9 mm³, respectively. However, the stroke volumes by 48 h were not statistically different in the three animal groups. The present data demonstrate that ODC activation is an endogenous neuroprotective measure in transient cerebral ischaemia.     

Ornithine decarboxylase knockdown exacerbates transient focal cerebral ischemia-induced neuronal damage in rat brain.

Transient cerebral ischemia leads to increased expression of ornithine decarboxylase (ODC). Contradicting studies attributed neuroprotective and neurotoxic roles to ODC after ischemia. Using antisense oligonucleotides (ODNs), the current study evaluated the functional role of ODC in the process of neuronal damage after transient focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats. Transient MCAO significantly increased the ODC immunoreactive protein levels and catalytic activity in the ipsilateral cortex, which were completely prevented by the infusion of antisense ODN specific for ODC. Transient MCAO in rats infused with ODC antisense ODN increased the infarct volume, motor deficits, and mortality compared with the sense or random ODN-infused controls. Results of the current study support a neuroprotective or recovery role, or both, for ODC after transient focal ischemia.     

Do catecholamines contribute to the effects of neonatal hypoxia on development of brain and heart? Influence of concurrent alpha-adrenergic blockade on ornithine decarboxylase activity

Hypoxia in the neonate releases catecholamines from the adrenal medulla, a response which is necessary to survive. This study examines whether a similar dependence exists for the ability of brain and heart tissue to recover from hypoxia-induced damage, as assessed by measurements of ornithine decarboxylase (ODC) activity. Hypoxia at either 1 day or 8 days of age produced a subsequent elevation of brain ODC which persisted for 1 week, a pattern known to be associated with recovery from tissue damage and delayed cellular maturation. Pretreatment of the rats with phenoxybenzamine, an alpha-receptor blocking agent, resulted in attenuation of the long-term ODC response, but did not interfere with effects on the enzyme during the hypoxia itself. In the heart, hypoxia at 8 days of age displayed similar effects, with long-term ODC elevations which were attenuated by phenoxybenzamine. Hypoxia at 1 day of age also produced long-term heart ODC stimulation, but in this case the effect was exacerbated by phenoxybenzamine, an effect consistent with the greater dependence of cardiac tissue on alpha-receptor-mediated responses to hypoxia at that age. These results suggest that alpha-receptor stimulation by catecholamines released during neonatal hypoxia play a role in the metabolic adjustment of brain and heart tissue to damage and may aid in subsequent recovery.     

Developmental profiles of ornithine decarboxylase activity in the hippocampus, neocortex and cerebellum: Modulation following lead exposure

Ornithine decarboxylase (ODC) is a growth-associated enzyme which is critical for cell growth and transformation. ODC activity follows a specific ontogenetic pattern of activity in distinct brain regions according to their developmental stage. Perturbations in the pattern of ODC activity have been associated with brain damage including arrested cerebral growth. Modulations in the pattern of ODC activity were examined in the hippocampus, neocortex and cerebellum of neonatal rats (PND 3, 6, 9, 15) exposed via the dam to 0.2% lead-acetate (Pb²⁺ prenatally (gestational day 13 to birth), postnatally (PND 1–15) or perinatally (gestational day 13 to PND 15). Prenatal exposure to Pb²⁺ perturbed the profile of ODC activity in all three brain regions examined, while postnatal exposure to Pb²⁺ resulted in prolonged stimulations of ODC activity in the cerebellum. Following prenatal exposure, these effects were manifested as a stimulation of ODC activity in the hippocampus, a repression of activity in the neocortex and a combination of these effects in the cerebellum. Perinatal exposure to Pb²⁺ transiently modulated the pattern of ODC activity similarly in all three brain regions, in a characteristic manner irrespective of their developmental stage. These Pb²⁺-induced modulations of ODC activity suggest that polyamine-dependent processes may play a significant role in the manifestation of Pb²⁺-induced neurotoxicity dependent upon developmental factors at specific exposure periods.     

Do catecholamines contribute to the effects of neonatal hypoxia on development of brain and heart? Influence of concurrent α-adrenergic blockade on ornithine decarboxylase activity

Hypoxia in the neonate releases catecholamines from the adrenal medulla, a response which is necessary to survive. This study examines whether a similar dependence exists for the ability of brain and heart tissue to recover from hypoxia-induced damage, as assessed by measurements of ornithine decarboxylase (ODC) activity. Hypoxia at either 1 day or 8 days of age produced a subsequent elevation of brain ODC which persisted for 1 week, a pattern known to be associated with recovery from tissue damage and delayed cellular maturation. Pretreatment of the rats with phenoxybenzamine, an α-receptor blocking agent, resulted in attenuation of the long-term ODC response, but did not interfere with effects on the enzyme during the hypoxia itself. In the heart, hypoxia at 8 days of age displayed similar effects, with long-term ODC elevations which were attenuated by phenoxybenzamine. Hypoxia at 1 day of age also produced long-term heart ODC stimulation, but in this case the effect was exacerbated by phenoxybenzamine, an effect consistent with the greater dependence of cardiac tissue on α-receptor-mediated responses to hypoxia at that age. These results suggest that α-receptor stimulation by catecholamines released during neonatal hypoxia play a role in the metabolic adjustment of brain and heart tissue to damage and may aid in subsequent recovery.     

Preserved induction of long-term potentiation in the stratum radiatum in the CA1 field of hippocampal slices from transgenic mice overexpressing ornithine decarboxylase and overproducing putrescine

The role of putrescine in synaptic neurotransmission and plasticity was studied using transgenic mice overexpressing ornithine decarboxylase (ODC), a polyamine-synthesizing enzyme. Transgenic mice were produced using the standard microinjection technique leading to elevated levels of putrescine in the periphery and in the brain. The experiments investigated whether or not ODC mice with elevated levels of putrescine show alterations in synaptic transmission and induction of long-term potentiation in the CA1 field of the hippocampus in vitro. Our results indicated that (1) putrescine levels in brain slices of the transgenic mice were more than ten times higher than those in fresh slices of control mice, although the absolute levels of putrescine and spermine decreased (by 15 and 40%, respectively) after 3–6 h incubation in vitro, while the levels of spermidine slightly increased (by 10%), (2) the excitatory synaptic response waveforms were wider (an increased half-width), and paired-pulse facilitation was somewhat reduced in ODC mice as compared to controls, and (3) potentiation of excitatory synaptic responses (measured 30–45 min after theta burst stimulation) did not differ between ODC and control mice. These results indicate that synaptic transmission is affected, but synaptic plasticity in the field CA1 assessed in vitro is not changed by elevated levels of intracellular putrescine. Synapse 28:288–293, 1998. © 1998 Wiley-Liss, Inc.     

Impaired neurogenesis by methylazoxymethanol in newborn rats results in transient reduction of ornithine decarboxylase and polyamines in the cerebellum,but not in the olfactory bulbs

Polyamines and the key enzyme for their biosynthesis, ornithine decarboxylase (ODC) play an important role in the control of neuronal proliferation and differentiation. Exposure to agents that interfere with normal cell maturation is expected to result in alteration of neuronal ODC developmental pattern. We have administered to newborn rats, about 6 and 30 hr after birth, 20 mg/kg of methylazoxymethanol acetate (MAM), an agent able to selectively kill dividing cells and we have evaluated ODC activity and polyamine levels in the cerebellum and ODC activity in the olfactory bulbs at various developmental stages starting from postnatal day 4 (PD 4) until PD 28. Cerebellar weight decreased by 22–50% at the different developmental stages in MAM-treated animals. A decline in ODC specific activity was observed at PD 4 and a decrease of putrescine levels at PD 4 and PD 6 in the cerebellum. At PD 10, however, both ODC activity and putrescine level were increased in MAM-treated animals. Spermidine levels were never affected by the treatment, while spermine was significantly decreased at PD 6 and PD 8. These results demonstrate that altered ontogenetic patterns of ODC activity and polyamine levels are the consequence of disturbance of the normal process of brain maturation. No significant differences in specific ODC activity were noticed in the olfactory bulbs of MAM-treated rats. This may be related to the more widespread time-span of neurogenesis in this region, a fact that is also revealed by the higher ODC activity constitutively expressed at times in which neurogenesis has ended in the rest of the brain.     

Polyamine biosynthetic decarboxylases in muscles of rats with different experimental myopathies

The activities of the two polyamine biosynthetic decarboxylases (PBD), L-ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (SAMD), have been measured in quadriceps femoris of rats killed at different times after the induction of calciphylaxis- or serotonin(5-HT)-induced myopathy. Decreases in both PBD levels were observed at early times after both myotoxic treatments. Subsequent progressive increases in both enzyme levels were observed to nearly control values by 4 days after 5-HT administration. In the 5-HT-treated rats, the effects on the myocardial PBD activities were different from those in skeletal muscle, with no effect on ODC but much on SAMD, when rats were killed shortly after 5-HT injection. These results demonstrate that the time-course of the changes in PBD activities in quadriceps femoris mirrors quite well the successive occurrence of degenerative and regenerative processes during the calciphylaxis-induced myopathy and the 5-HT-induced myopathy; it is 5-HT that is mainly responsible for the decreases in PBD levels observed in both experimental myopathies, since dihydrotachysterol alone was without any effect on PBD activity levels and 5-HT alone was effective; myocardial ODC reacts more slowly to 5-HT than quadriceps femoris ODC.     

Correlation between lead-induced changes in cerebral ornithine decarboxylase and protein kinase C activities during development and in cultured PC 12 CELLS

Exposure to lead (Pb) interferes with neurodevelopment and disturbs ornithine decarboxylase (ODC) activity. ODC the key regulatory enzyme of the polyamine pathway, is a potential substrate for protein kinase C (PKC). Therefore, we examined developmental changes in PKC activity and its relationship to ODC activity. Male rats were lactationally exposed to 0.2% Pb-acetate from birth to weaning. PKC and ODC activity were measured on postnatal days (PND) 3, 5, 10, 20 and 30. We found that the basal patterns of ODC and PKC activities resembled each other in both the neocortex and cerebellum and Pb-exposure attenuated both enzymes in a similar manner. To determine whether any link existed between these enzymes, ODC and PKC activities were induced to increase using nerve growth factor (NGF) in the presence and/or absence of ODC (difluoromethylornithine, DFMO) and PKC (staurosporine) inhibitors, in control and Pb-exposed Pheochromocytoma (PC-12) cells. Staurosporine decreased both ODC activity and PKC activity, while DFMO had no effect on PKC activity. These data suggest that ODC may be regulated by PKC and that Pb-induced developmental alterations in ODC activity may be secondary to changes in the integrity of PKC.     

Role of serotonin in the apomorphine-induced increase of adrenomedullary ornithine decar☐ylase activity

The role of serotonin in the regulation of adrenomedullary ornithine decar☐ylase (ODC) activity has been explored in rats after systemic administration of p-chlorophenylalanine (PCPA) and intraventricular 5,6-dihydroxytryptamine (DHT) or in animals with electrolytic lesions of the medial and dorsal raphe nuclei. None of the treatments produced any alteration in endogenous ODC activity. However, all except lesion of the dorsal raphe nucleus significantly potentiated the induction of adrenomedullary ODC produced by apomorphine (APM) administration. It is suggested that serotonergic fibers originating partly in the medial raphe nucleus exert a tonic inhibitory action over the APM-induced increase in adrenomedullary ODC activity.     

Role of serotonin in the apomorphine-induced increase of adrenomedullary ornithine decarboxylase activity

The role of serotonin in the regulation of adrenomedullary ornithine decar?ylase (ODC) activity has been explored in rats after systemic administration of p-chlorophenylalanine (PCPA) and intraventricular 5,6-dihydroxytryptamine (DHT) or in animals with electrolytic lesions of the medial and dorsal raphe nuclei. None of the treatments produced any alteration in endogenous ODC activity. However, all except lesion of the dorsal raphe nucleus significantly potentiated the induction of adrenomedullary ODC produced by apomorphine (APM) administration. It is suggested that serotonergic fibers originating partly in the medial raphe nucleus exert a tonic inhibitory action over the APM-induced increase in adrenomedullary ODC activity.     

Ornithine decarboxylase in developing brain of hydrocephalic rats Effects of shunt operation

We examined the changes in ornithine decarboxylase (ODC) immunoreactivity in the hydrocephalic cerebral cortex of HTX rats after decompression by shunt operation. The ODC immunoreactivity reached a very low level after the completion of cortical layer formation, and only faint staining was found on postnatal day (Pd) 11. The ODC immunoreactivity re-appeared after the shunt operation when the operation was done in the early days of life: the ODC immunoreactivity was first found on day 2 after shunting and persisted until day 8 after shunting. However, this was not apparent when the operation was not performed until Pd 14. The re-expression of ODC in hydrocephalic brain after shunting appears to cause resumption of the developmental process by relieving neurons from increased hydrostatic pressure. The dependence of ODC re-expression on the timing of the operation indicates that there may be a period of neocortical decompression that is critical for effective compensatory development, so that when delayed, decompression fails to re-activate the ODC-dependent development.     

Mast cell adhesion induces cytoskeletal modifications and programmed cell death in oligodendrocytes

In this paper we show that rat peritoneal mast cells (RPMC) adhere to rat oligodendrocytes (ODC) in culture and switch on a bi-directional signal affecting both adhering cell and its target. Following heterotypic interaction, RPMC release granule content and ODC show morphological changes and enter the apoptotic programme. Altogether, these findings indicate that the interaction of MC with ODC could play a role in the mechanism of CNS damage induced by the inflammatory reaction.     

Ornithine decarboxylase is differentially induced by kainic acid during brain development in the rat

The induction of brain ornithine decarboxylase (ODC) as a consequence of systemic kainic acid administration was studied in the hippocampus and the olfactory cortex-amygdala area of 10-day-old rat pups and 30-day-old young rats. In pups, ODC levels were moderately increased (plus 50–80%) 4 h after kainic acid administration, coming back quickly to control levels afterwards. In young rats, instead, ODC levels were dramatically increased by 17–25-fold, 16 h after kainic acid administration and decreased towards basal levels 48–72 h after injection. The present results suggest that the process of excitotoxic ODC induction can be split in two phases: a first phase characterized by moderate induction and essentially linked to the overstimulation of brain circuits and a second phase, during which a dramatic enzyme stimulation is accompanied by the appearance of neurodegenerative pathology.     

Overexpression of 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase in Transgenic Mice Alters Oligodendrocyte Development and Produces Aberrant Myelination

The function of the intracellular protein 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) of oligodendrocytes (ODC) is unknown. We have now generated several homozygous transgenic mouse lines in which the human CNP gene is overexpressed up to sixfold, revealing new insights into early stages of myelinogenesis. Although no behavioral phenotype is immediately apparent, abnormalities of ODC and their myelin sheaths are striking. These are manifested as redundant myelin membrane and intramyelenic vacuoles, as well as lack of myelin compaction concordant with failure of the cytoplasmic leaflets of compact myelin to fuse. Further, ODC that overexpress CNP appear to mature earlier in development, resulting in earlier maximum gene expression for myelin basic proteins and proteolipid protein. These results indicate that CNP is an early expressed regulator of cellular events that culminate in CNS myelination.     

Effect of cold, restraint, reserpine, and splanchnicotomy on the ornithine decarboxylase activity of rat adrenal medulla and cortex

The activity of ornithine decarboxylase (EC 4.1.1.17, ODC) of the adrenal medulla and cortex was studied after subjecting rats to three different forms of stress: administration of reserpine, exposure to short periods of cold, or bodily restraint. Reserpine, cold exposure, and immobilization significantly increased ODC activity in both adrenal tissues. For medullary ODC this effect was prevented by denervation of the adrenal gland. In the cortex, splanchnicotomy reduced or prevented the stimulation of ODC activity caused by reserpine and cold exposure. When animals pretreated with reserpine were subjected to restraint, a potentiation was observed for the medullary enzyme; this effect was prevented by denervation. The cortical enzymic activity attained levels of activity similar to those observed with the individual stimuli. Exposure of rats treated with reserpine to cold led to a reduction of adrenal ODC activity, a reduction that was statistically significant only for the cortical enzyme. Denervation had no effect. Cortical ODC responses appear to be strongly influenced by hypothalamopituitary factors which, in turn, are known to be modified by stress, whereas medullary ODC activity seems to be more susceptible to changes in sympathetic activity associated with stress.     

Increased ornithine decarboxylase activity and protein level in the cortex following traumatic brain injury in rats

There is increasing evidence that the elevated levels of polyamines play an important role in the secondary injury following traumatic brain injury (TBI). Ornithine decarboxylase (ODC) is the rate-limiting enzyme of polyamine biosynthesis. Presently, we measured the ODC protein levels by Western blot analysis in the cerebral cortex of rats sacrificed at 2 h, 6 h, 24 h, 72 h and 168 h after controlled cortical impact injury. TBI resulted in a significant increase in ODC protein levels (2.5 to 5.5 fold, P<0.05) and enzyme activity (13 to 21 fold, p<0.01) between 2 and 6 h after the injury. ODC protein levels and enzyme activity returned to normal, control levels by 72 h after the injury. Increased ODC protein and enzyme activity could contribute to vasogenic edema and the pathogenesis of neuronal dysfunction after TBI by stimulating the formation of polyamines.     

Ornithine decarboxylase in developing brain of normal and hydrocephalic rats Biochemical and immunohistochemical studies

Polyamines have various roles in cortical development. We examined the ontogenic changes in ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, in cerebral cortices of normal and hydrocephalic rats. Both biochemical and immunohistochemical examinations revealed increased ODC protein and enzyme activity during the perinatal period. Apical dendrites of developing neuroblasts migrating from the superficial layer of cortical plate showed intense ODC immunoreactivity. Once they had settled at their final destination, ODC immunoreactivity weakened. Both ODC immunoreactivity and enzyme activity reached very low levels after completion of layer formation of cortex. The enzyme activity of ODC in hydrocephalic cortices exceeded that in normal cortex during the perinatal periods. ODC was rather overexpressed, but no characteristic distribution was observed in the hydrocephalic cortex. These findings indicate the participation of polyamines in the cortical development, especially in the layer formation. The overexpression of ODC in hydrocephalus appears to promote development despite increased hydrostatic pressure.     

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

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 alpha-difluoromethylornithine (DFMO; injected intraperitoneally) immediately before recirculation. At the end of the experiments brains were frozen and 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 most 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.