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.     

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.

Recovery in hydrocephalic dementia after shunt operation.

Twenty-three patients with hydrocephalic dementia were studied before and after shunt operation, and improvement was found in 12. Before operation, the improved cases showed more symptoms of confabulation, gait disturbance, urinary incontinence, lack of insight, and constructional apraxia. The improvement was also most marked in these symptoms. The significance of general versus specific symptoms, duration, and aetiology is discussed from a differential diagnostic standpoint, and we conclude that the adequate and early diagnosis of hydrocephalic dementia is essential for good outcome after shunt operation, and that psychiatric and psychometric evaluation enable such a diagnosis to be made.     

Ornithine decarboxylase activity and immunohistochemical location in postischemic brain

Ornithine decarboxylase, rate-limiting in polyamine formation, has been found to be necessary for the development of vasogenic edema after cryogenic cerebral injury and is postulated to be of importance in late ischemic brain edema formation. Ornithine decarboxylase activity and accompanying edema was studied after transient cerebral ischemia in Mongolian gerbils. Bilateral carotid artery occlusion was utilized to produce dense forebrain ischemia. After 4 h of reperfusion a significant elevation in ornithine decarboxylase activity was present (72.5 +/- 24.7 vs 8.5 +/- 2 pmoles/mg protein/h, p less than 0.05). Immunohistochemical localization of ornithine decarboxylase indicated its presence in cortical neurons of ischemic gerbils. This was typically located in the perinuclear cytoplasm and extended into proximal dendrites. Nonischemic animals did not contain ornithine decarboxylase immunoreactivity. These studies show the presence and location of ornithine decarboxylase in cerebral tissue subjected to transient ischemia. The increase in this marker of polyamine activity paralleled previous studies in this model of cerebral edema formation and reperfusion deficit in blood flow and evoked potential, suggesting that ornithine decarboxylase is a marker for and may be associated with those late metabolic events leading to progressive functional deterioration after incomplete cerebral ischemia.     

Early ventriculoperitoneal shunt — effects on learning ability and synaptogenesis of the brain in congenitally hydrocephalic HTX rats

The learning ability of congenitally hydrocephalic HTX rats in which hydrocephalus had been arrested by the insertion of a V-P shunt 7 days after birth (early shunt) was assessed by means of the light-darkness discrimination test when the animals reached maturity. Early shunt placement resulted in marked reduction in size of abnormally enlarged ventricles and the prevention of both decreasing spine density and decay of synaptic vesicle protein (SVP-38) in the affected cerebral cortex. The learning ability of such animals was also found not to be impaired compared with that of animals in a sham-operation group. On the basis of these investigations, it is concluded that early shunt placement may have a beneficial role in preventing not only impairment of synaptogenesis of the brain by progressing hydrocephalus, but also learning disability. Recent biochemical investigation of the developing brains of hydrocephalic HTX rats revealed problems that cannot be resolved by early shunt insertion, and these are also discussed.Presented at the Consensus Conference: Hydrocephalus '92, Assisi, Italy, 26–30 April 1992     

Learning deficits in congenitally hydrocephalic rats and prevention by early shunt treatment

Shunt surgery is the usual treatment for infantile hydrocephalus; however, the extent to which it avoids subsequent neurological deficits is uncertain. The effect of early-onset hydrocephalus was tested in H-Tx rats using the Morris water maze. Spatial learning was assessed at 21 days after birth in control (n=18), hydrocephalic (n=18) and hydrocephalic rats shunt-treated at 4–5 (n=7) or at 10–12 days of life (n=13). The time taken to find a hidden platform was measured in five trials on 2 consecutive days and the data analyzed by one-and two-way ANOVA and t-tests. The latencies of the control rats decreased significantly between the first and second trial on the 1 st day, and learning was retained until the 2nd day. The hydrocephalic group had longer latencies than controls on both days, with no significant decrease between any trials. Performance was not significantly different between the two shunt groups. Overall, the shunted rats had latencies which were not significantly different from controls but were significantly lower than hydrocephalics. Despite this, the shunted rats did not perform as well as the controls. It is concluded that, although shunt treatment improved learning, some effects of early-onset hydrocephalus may not be reversible and/or a longer recovery time is required.     

Ornithine decarboxylase in motoneurons during regeneration

The activity of ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, was assayed in the isolated facial nucleus of the rat at various times after axotomy of the facial nerve. In addition, it was measured 24 h after the second of a series of two lesions (conditioning lesion design) with various times between the first and second operations. Ornithine decarboxylase activity was found to increase 8 h after nerve transection and was maximum after 24 h (300% of control). Thereafter the activity declined to subnormal levels where it remained for several weeks. Ornithine decarboxylase activity did not increase again when a second axotomy was made 2 weeks after the first lesion. However, ornithine decarboxylase did respond to the second axotomy if it was carried out 3 weeks after the first lesion. Histochemical localization of ornithine decarboxylase demonstrated that the increase in enzyme activity was mainly confined to the perikarya of the motoneurons. These data suggest that this enzyme is somehow involved in triggering the "regeneration program" and clearly indicate that at least some aspects of the neuronal response to axotomy are not further stimulated by a conditioning lesion.     

Immunohistochemical localization of L-ornithine decarboxylase in developing rat brain

L-Ornithine decarboxylase, the rate limiting enzyme of polyamine biosynthesis and a marker enzyme of tissue proliferation and maturation, was localized immunocytochemically in the developing rat central nervous system. It can be noted that the distribution of the enzyme protein underlies temporal alterations. Conclusions are drawn from the localization of the enzyme and possible functional roles played by ornithine decarboxylase in discrete brain areas.     

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.     

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.     

The effects of hydrocephalus upon the developing brain. Histological and quantitative studies of the ependyma and subependyma in hydrocephalic rats

A suspension of kaolin was injected into the cisterna magna of 44 rats at 2 weeks of age. Animals killed at intervals from 5--19 weeks of age showed varying degrees of hydrocephalus. Light microscopy, scanning and transmission electron microscopy revealed stretching and flattening of the ependymal cells but no significant loss of cilia. Histological evidence of periventricular tissue damage in these chronically hydrocephalic animals was only present when the ventricular dilatation was extensive. A quantitative assessment was made of the ependymal and subependymal cell reactions around the lateral ventricles of the hydrocephalic animals. Although the ependymal cells were clearly stretched around the ventricles, there was no apparent proliferation of these cells. An increase in the total number of subependymal cells was observed in hydrocephalic animals when compared with a series of 39 aged-matched controls. The greatest proliferation was in the dorsal and lateral walls of the ventricles which were the regions most severely stretched by the ventricular dilatation. There is evidence that subependymal cells differentiate into astrocytes and microglia so that proliferation of these cells may be interpreted as a response to continuing and progressive brain damage in chronic hydrocephalus. Such progressive tissue damage could adversely affect the developing brain.     

Free radical-induced elevation of ornithine decarboxylase activity in developing rat brain slices

Objective. In developing brain, we have previously shown both in vivo [L.D. Longo, S. Packianathan, J.A. McQueary, R.B. Stagg, C.V. Byus and C.D. Cain, Acute hypoxia increases ornithine decarboxylase activity and polyamine concentrations in fetal rat brain, Proc. Natl. Acad. Sci. USA, Vol. 90 (1993) 692–696] and in vitro [S. Packianathan, C.D. Cain, B.H. Liwnicz and L.D. Longo, Ornithine decarboxylase activity in vitro in response to acute hypoxia: a novel use of newborn rat brain slices, Brain Res., Vol. 688 (1995) 61–71] that acute hypoxia is associated with a significant increase in ornithine decarboxylase (ODC) activity and polyamine concentrations. We tested the hypothesis that oxygen free radicals induce an increase in ODC activity similar to that of hypoxia and that both this and the hypoxia-induced response are inhibited by free radical scavengers. Materials and methods. Slices of cerebrum, 300–500 μm thick, were made from P3 newborn Sprague-Dawley rat pups and equilibrated for 1 h in artificial cerebrospinal fluid continuously bubbled with 95% O₂/5% CO₂. Free radical-induced ODC activity response was measured beginning after a 1-h recovery period. Experiments were performed on slices treated with 5×10⁻⁷ M xanthine (X)+10 mU/ml xanthine oxidase (XO), with or without the free radical scavengers superoxide dismutase (SOD; 100 U/ml), catalase (CAT; 700 U/ml) or glutathione peroxidase (GPX; 3 U/ml). We also quantified slice malonaldehyde concentrations in response to hypoxia (21% O₂/5% CO₂/74% N₂). Results. Under control conditions, ODC activity was stable during the 2-h post-recovery period. In response to X/XO treatment, ODC activity increased 2.3-fold at 1.5 h post-recovery. In examining ODC activity as a function of xanthine dose, we noted that ODC activity increased in response to 2.5×10⁻⁷ M xanthine; however, it decreased in response to 7.5×10⁻⁷ M or higher concentrations. Free radical-induced ODC activity was significantly decreased by addition of the free radical scavengers, SOD, CAT or GPX. In addition, the hypoxic-induced increases in ODC activity and malonaldehyde concentration was also eliminated by the addition of SOD with CAT. Conclusions. (1) Oxygen free radicals, particularly hydroxyl radical (OH^.), appear to trigger an induction of ODC activity in newborn rat cerebrum slices. (2) Oxygen free radicals also appear to mediate the hypoxic-induced increase in ODC activity. (3) Any consequent increase in polyamine synthesis may have profound effects on neurogenesis and neurodifferentiation in the developing brain.     

Ornithine decarboxylase in the inner ear of the guinea pig

Ornithine decarboxylase, the rate limiting enzyme of polyamine synthesis and a possible marker enzyme for tissue proliferation and maturation, has been found in the developing guinea pig cochlea using the unlabelled horseradish-peroxidase-antiperoxidase technique. Ornithine decarboxylase-like immunoreactive material was detected in the neurons of the Ganglion spirale and in their axonal and/or dendritic fibers. The location of the enzyme and the possible functional role of ornithine decarboxylase plays in the development and maturation of the auditory organ and of the hearing process are discussed.     

Effects of malnutrition on cell genesis and migration in developing brain in rats

Rats were fed a diet containing either 5% (LP) or 25% (HP) casein throughout gestation. The animals, delivered by a cesarean section on the 21st day of gestation, were given a pulse of [³H]thymidine and killed 2 h later. No differences were observed in the mitotic index, but the labeling index of the ventricular zone cells of the lateral ventricle was higher in the case of litters of the LP dams. This we atribute to a disproportionate increase of the S phase in relation to the cell cycle in malnutrition. To study the effects of malnutrition on the programming of cell genesis, the dams were given [³H]thymidine on the 20th day of gestation, and the litters were killed on the 21st postnatal day. The labeled neurons reached the second layer of the cerebral cortex in both groups, indicating that the programming was not affected by gestational malnutrition. Postnatal undernutrition was induced by doubling the litter size. The cell generation cycle was prolonged in the undernourished animals. Not all phases, however, were evenly affected: S was prolonged, G₂ was not affected, but G₁ was shortened. The external granular layer maintained its proliferative activity in the undernourished animals beyond the stage it had ceased in the controls. The migration of the cerebellar granule neurons was slow. It is proposed that tardiness in the migration of the neurons may interfere with synaptogenesis.     

Energy metabolism in kaolin-induced hydrocephalic rat brain

Energy metabolism in kaolin-induced hydrocephalic rat brain was assessed by means of 31-phosphorus magnetic resonance spectroscopy and measurement of the activity of lactate dehydrogenase (LDH) as well as its isoenzyme patterns. Decreases in beta-adenosine triphosphate and phosphocreatine were observed in hydrocephalic rat brains. While the intracellular pH was decreased at 2 to 4 weeks, it showed some recovery 6 weeks after injection of kaolin. The activity of LDH increased in the hydrocephalic state, and its isoenzyme-pattern changes were as follows: the LDH5 fraction was predominant in 1-week to 4-week rats while the LDH4 fraction was predominant in 4-week rat brains, and at 6-weeks, the LDH4 and LDH5 fractions were decreased. These data from rat brains with kaolin-induced hydrocephalus indicate that anaerobic glycolysis is the primary pathway of energy metabolism in the acute hydrocephalic state, while in the chronic state the emphasis shifts to aerobic glycolysis.Presented at the 17th Annual Meeting of Pediatric Section of the American Association of Neurological Surgeons, Arizona 1988     

Ornithine decarboxylase in reversible cerebral ischemia: an immunohistochemical study

Summary Anesthetized 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 (P0.05 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.Supported by the Deutsche Forschungsgemeinschaft, Grant Pa 266/3-2, and by grants provided by the National Research Council for Natural Sciences, Academy of Finland and the Cancer Society of Finland.     

Evaluation of shunt function in hydrocephalic patients with the radionuclide 99mTc-pertechnetate

In most cases of shunted hydrocephalus, shunt malfunction is evaluated by clinical examination and neuro-imaging. However if there is a discrepancy between neurological examination and imaging, additional shuntography can be helpful in the evaluation of the shunt function. In our clinic, radionuclide-imaging shuntography using ^99mtechnetium-pertechnetate was performed in 85 children between 1992 and 1995. The results of shuntography were evaluated visually and from time-activity curves. Shuntography had a sensitivity of 96%, a specificity of 89%, and an accuracy of 93%, proved either by surgery or by clinical follow-up for 2–5 years. Corresponding to these results, we recommend the use of shuntography in cases with an uncertain shunt function before surgical revision. Received: 15 October 1998     

Evaluation of cerebrospinal fluid shunt function in hydrocephalic children using 99mTc-DTPA

The increasing numbers and survival of children with shunt-treated hydrocephalus make it mandatory to refine the methods for cerebrospinal fluid (CSF) shunt function evaluation. Radionuclide shuntography with ^99mTc-DTPA, which has proved to be a safe and effective method, was performed in eight children with suspected CSF-shunt dysfunction. Characteristic shuntography patterns were found for proximal and distal CSF-shunt catheter obstruction as well as for overdrainage and normal CSF-shunt function. Shuntography contributed to the explanation of suspected CSF-shunt dysfunction in all children investigated.     

Indicators of hypoxia in cerebrospinal fluid of hydrocephalic children with suspected shunt malfunction

We used high performance liquid chromatography to determine the concentration of purine metabolites in the cerebrospinal fluid of three hydrocephalic children with a history of shunt malfunction. Hypoxanthine and xanthine levels were high in comparison with controls. We consider these purines to be valuable indicators of disturbance of neuronal metabolism following the sustained rise in intracranial pressure caused by shunt valve malfunction.