Decrease of immunoreactive catalase protein in specific areas of ageing rat brain.

The activity of catalase, the main enzyme responsible for detoxification against hydrogen peroxide, decreases in specific brain areas of aged rats. The reduction of enzyme activity appears to be the consequence of a decreased protein expression rather than an impaired function of the native enzyme. In fact, diminution of the immunoreactive protein parallels enzyme activity decrease. Since the extent of decrease of both activity and protein content was observed to be area dependent, we hypothesise that this phenomenon may underlie, at least in part, the increased susceptibility of specific brain regions to oxidative insults observed in pathological situations related to ageing.

N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors mediate seizures and CA1 hippocampal damage induced by dendrotoxin-K in rats.

The epileptogenic and neurodegenerative effects of dendrotoxin K, from Dendroaspis polylepis, a specific blocker of a non-inactivating, voltage-sensitive K+ channel, were studied after focal injection into one dorsal hippocampus in rats. Administration of 35 pmol dendrotoxin K elicited motor seizures and bilateral electrocortical discharges after a latent period (5.3 +/- 2.1 min), in all of the treated animals (n = 6). At 24 h, histological examination of brain (n = 5) coronal sections (10 microns; n = 6 per brain) detected bilateral damage to the hippocampal formation which extended 300 microns rostral and caudal to the injection tract. Quantitation of the damage revealed significant bilateral neuronal cell loss in the CA1 and CA4 pyramidal cell layer relative to the corresponding brain regions of rats (n = 3) injected with bovine serum albumin (105 pmol), which per se was ineffective in all respects. Dendrotoxin K (35 pmol) also caused a significant loss of CA3 pyramidal neurons and dentate gyrus granule cells ipsilateral to the site of toxin injection. In one out of six rats, a lower dose (3.5 pmol) of dendrotoxin K produced convulsive behaviour and electrocortical seizures but after a longer latency and these were accompanied by significant neuronal loss in the CA1, CA3 and CA4 pyramidal cell layer ipsilateral to the injected side. The lowest dose (0.35 pmol; n = 6 rats) of dendrotoxin K used failed to induce seizures and did not cause hippocampal damage (n = 6 rats). Systemic (i.p.) treatment with dizocilpine maleate (3 mg/kg) or LY 274614 (5 mg/kg i.p.), two N-methyl-D-aspartate receptor antagonists (given 15 min beforehand), prevented dendrotoxin K (35 pmol)-induced motor seizures and electrocortical epileptogenic discharges in 100% of the animals (n = 6 per group) treated. Similarly, these antagonists minimized the damage typically produced in the rat hippocampus, with no significant neuronal loss being observed. By contrast, NBQX (30 mg/kg, i.p. given 15 min previously), a non-N-methyl-D-aspartate antagonist, failed to prevent seizures normally evoked by dendrotoxin K (35 pmol; n = 6 rats); also, this treatment was unable to abolish CA1 pyramidal cell loss but minimized the loss in hippocampal sectors distant to the site of dendrotoxin K injection. However, complete protection against motor and electrocortical seizures and hippocampal damage was afforded by GYKI 52466 (10 mg/kg i.p.; n = 6 rats), a more effective non-N-methyl-D-aspartate receptor antagonist. These findings differ from the reported lack of protection by N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor antagonists to rats receiving intra-hippocampal injection of alpha-dendrotoxin; this difference may stem from the ability of alpha-dendrotoxin to block predominantly a slowly inactivating K+ current whereas dendrotoxin K inhibits a non inactivating variant. In conclusion, the present data on dendrotoxin K, together with the previously described pattern of neurotoxicity for alpha-dendrotoxin, show that these homologues act via different mechanisms and, thus, can be used effectively as complementary tools to study seizures and neuronal cell death.

Age-dependent changes of NO synthase activity in the rat brain.

We report that NO synthase activity, as expressed by citrulline and nitrite formation in brain homogenates, is decreased in 24-month old in comparison to 3-month old rats. In particular, a Ca(++)-dependent NO synthase activity was detected in homogenates obtained from cortical, hippocampal, cerebellar and lower brain stem slices from both 3- and 24 month-old rats. The amount of citrulline generated from L-arginine was significantly decreased in the hippocampus and lower brain stem by 40 and 48%, respectively. No changes were observed in NO synthase activity in cortical and cerebellar homogenates. Thus, the L-arginine-NO pathway seems to be impaired in selected areas of rat brain and this may contribute to the understanding of pathophysiological mechanisms underlying age-related cerebral disorders.

Potent protective effect of melatonin on in vivo paraquat-induced oxidative damage in rats.

The in vivo effect of melatonin on paraquat-induced oxidative damage in rat lung and liver was studied using two parameters: the concentration of malonaldehyde and 4-hydroxyalkenals as indices of lipid peroxidation; changes in total and oxidized glutathione. Melatonin (10 mg/kg) or an equal volume of saline were administered intraperitoneally (ip) to rats 30 min prior to an ip injection of paraquat (20 mg/kg or 70 mg/kg). After paraquat treatment, the animals received melatonin or saline ip injections every six hours for 24 hours. Rats were killed 24 hours after paraquat injection. In lung, both the low and high dose of paraquat, when administered with saline, augmented lipid peroxidation (100% and 18%, respectively) above levels found in control animals. Treatment with melatonin completely reversed this effect. In liver, paraquat (70 mg/kg) increased lipid peroxidation by 40% over the levels of control animals. The increase was completely abolished by treatment with melatonin. Paraquat at 20 mg/kg did not induce any significant change in liver lipid peroxidation. Paraquat treatment resulted in a significant decrease of total glutathione concentration and increased oxidized glutathione in both lung and liver. These effects were abolished by treatment with melatonin. The results suggest that melatonin confers marked protection against paraquat-induced oxidative toxicity in both the lung and liver.

Melatonin restores immunodepression in aged and cyclophosphamide-treated mice.

Melatonin, the main hormone of the pineal gland, when chronically injected into young mice or mice immunodepressed by aging or by cyclophosphamide treatment, was able to enhance the antibody response to a T-dependent antigen. The enhancement of antibody response was associated with increased induction of T helper cell activity and IL-2 production as evidenced in mice immunodepressed by aging or by cyclophosphamide treatment. These observations suggest that melatonin may be successfully used in the therapy of immunodepressive conditions.

Effect of L-arginine on thymic function. Possible role of L-arginine: Nitric oxide (no) pathway.

The thymus and in particular its epithelial component produces hormonal peptides which are required for differentiation of stem cells into mature T-cells. With advancing age, there occurs a progressive reduction of the plasma level of one of the best known thymic peptides, i.e. thymulin. In old mice, oral supplementation with arginine (9x10(-4) gr/day/mouse) for 1 month is able to induce a regrowth of the thymus and recovery of the reduced thymulin plasma level to the values observed in young animals. The direct immunological target of arginine seems to be the thymus gland. In fact, the transplantation of thymus from old arginine treated mice into young thymectomized recipients is able to restore thymulin plasma level in thymectomized recipients to nearly the same level as do thymuses from young mice. Furthermore, arginine supplementation young thymectomized recipients is unable to induce the reappearance of thymulin activity in the blood. With regard to the mechanism of action of arginine, two pathways may be suggested. The first one may be indirect and mediated by the secretagogue action of arginine on growth hormone. The second one, may rely on a direct action through the L-arginine: NO pathway. Lymphocyte-depleted thymic explants from young mice, when incubated in vitro with the NO-synthetase inhibitor L-NAME (6 mM), are, in fact, incapable of producing and realising thymulin in the supernatant. The in vitro addition of L-arg (60 mM) is able to recover such a production to the values observed in supernatants of control thymic cultures. The present findings offer the first evidence that also the thymic endocrine activity is modulated by L-arginine:NO pathway.

Inhibition by N omega-nitro-L-arginine methyl ester of the electrocortical arousal response in rats.

In rats chronically implanted with cannulae into one lateral cerebral ventricle and recording electrodes onto the fronto-parietal cortex, the effects of systemic or intraventricular administration of the nitric oxide (NO) synthesis inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), on electrocortical (ECoG) arousal response evoked by sound stimulation were studied. In control animals, a single acoustic stimulation (80 dB for 15 s) produced a significant decrease in ECoG total voltage power lasting approximately 25 s. No tolerance developed after repeating the same sound stimulation at 15, 30, 60 min and 24 h intervals. Under these experimental conditions, pretreatment with L-NAME, given systemically (10 mg kg-1, i.p.) or intracerebroventricularly (300 micrograms), significantly reduced the sound-evoked arousal response 1 h and 15 min later, respectively. In conclusion, the present data are in favour of a physiological role of NO in the control of arousal mechanisms.

Melatonin as immunomodulator in immunodeficient mice.

Melatonin, the main hormone of the pineal gland, when chronically injected into young mice or mice immunodepressed by aging or by cyclophosphamide treatment, is able to enhance the antibody response to a T-dependent antigen. The enhancement of the antibody response is associated with increased induction of T helper cell activity and IL-2 production, as evidenced in mice immunodepressed by aging or by cyclophosphamide treatment. These observations suggest that melatonin treatment may be successfully used in the therapy of immunodepressive conditions.

NGF restores decrease in catalase activity and increases superoxide dismutase and glutathione peroxidase activity in the brain of aged rats.

The effects of ageing on the activity of copper-zinc superoxide dismutase (SOD), selenium-dependent and independent glutathione peroxidase (GSH-Px) and catalase in several areas of the brain in 3-, 12-, and 24-month-old rats were studied. In addition, the effects of a subacute intracerebroventricular treatment of NGF (1 microgram daily for 28 consecutive days) on SOD, GSH-Px, and catalase activity in the same areas of the brain were assessed. The effects of ageing on the activities of antioxidant enzymes varied considerably in the different brain areas studied. Copper-zinc SOD was alone in being unaffected by ageing. Intraventricular infusion of NGF significantly increased SOD activity in the prefrontal cortex, hypothalamus, caudate nucleus, and mesencephalon of 24-month-old rats. Selenium-dependent GSH-Px activity did not significantly change in 12-month-old rats but it increased in the lower brain stem of 24-month-old animals. In comparison to vehicle-treated rats, NGF significantly increased selenium-dependent GSH-Px activity in all brain areas studied in 12- and 24-month-old rats. Catalase activity decreased significantly in the majority of the brain areas studied in 12- and 24-month-old rats. NGF completely restored the fall in catalase activity in 12- and 24-month-old animals to levels similar to those occurring in young rats. In conclusion, the present experiments show, for the first time, that long-term intraventricular administration of NGF significantly increases in old animals the activity of key enzymes involved in the metabolic degradation of superoxide radicals and hydrogen peroxide.

Age-related changes in Cu,Zn superoxide dismutase, Se-dependent and -independent glutathione peroxidase and catalase activities in specific areas of rat brain.

Oxidative injury of tissues involves both accumulation of damage due to persistent oxidative stress and loss of the proper balance of antioxidative enzymes. These events may produce a faster rate of tissue senescence. In this regard, we have assayed the antioxidative enzyme activities (Cu,Zn superoxide dismutase, glutathione peroxidase and catalase), in various areas of rat brain (prefrontal cortex, parietal cortex, hippocampus, hypothalamus, caudate nucleus, mesencephalon and lower brain stem) for the age groups of 3, 6, 12, 24 months. The results obtained show that the levels of antioxidant enzyme activities differed considerably in the various brain parts studied. Furthermore, changes in the specific activities of superoxide dismutase, catalase, and glutathione peroxidase did not follow the same pattern as a function of aging. In particular, in prefrontal cortex and caudate nucleus, superoxide dismutase and glutathione peroxidase activities did not change, while catalase activity decreased. In parietal cortex and mesencephalon, superoxide dismutase and glutathione peroxidase activities increased, but the catalase activity decreased in parietal cortex and did not change in mesencephalon. In lower brain stem, the activities of glutathione peroxidase and catalase decreased in 3-12-month-old rats. The activity of glutathione peroxidase was increased in the hippocampus and was decreased in hypothalamus during aging. In this area the catalase activity was also significantly diminished.

Behavioural, electrocortical spectrum power and body temperature changes after microinfusion of some lymphokines in the rat brain.

Interleukin-1, interleukin-2 and interleukin-3 injected into the third cerebral ventricle produce typical behavioural sedation and/or sleep and ECoG synchronization in rats. Much smaller doses were required to produce similar behavioural and ECoG spectrum power effects after infusion of interleukins into the locus coeruleus. Interleukin-1 and interleukin-3 induced pyrogenic effects while interleukin-2 produced no pyrogenic actions. These effects were blocked by specific anti-interleukin receptors monoclonal antibodies. In addition, naloxone was able to antagonize the central effects of IL-2 but it did not affect the typical changes induced by IL-1. These results are consistent with the hypothesis that the behavioural, body temperature and ECoG changes of these lymphokines are mediated by specific receptors.

NGF restores decrease in catalase and increases glutathione peroxidase activity in the brain of aged rats.

The effects of subchronic administration of nerve growth factor (NGF) into the lateral ventricle on catalase and selenium-dependent glutathione-peroxidase (GSH-Px) activity in several areas of the brain in 3-, 12- and 24-month-old rats were studied. NGF given daily (1 microgram for 28 consecutive days) produced in all brain areas studied a significant increase in catalase activity in 12- and 24-month-old rats. The most important finding was a complete restoration in 12- and 24-month-old rats of catalase activity to levels similar to those occurring in young (3-month-old) rats. In addition, NGF produced in comparison to 3-month-old rats and to same age vehicle-treated rats a significant increase in selenium-dependent GSH-Px in all the brain areas studied in 12- and 24-month-old animals, whereas selenium-independent GSH-Px was unaffected. In conclusion, the present results show that long-term administration of NGF into the lateral ventricle significantly increases in old animals the activity of key enzymes involved in the metabolic degradation of hydrogen peroxide.

Behavioural and electrocortical effects after injection of two ornithine decarboxylase inhibitors into several areas of the brain in the rat.

The behavioural and electrocortical effects of alpha-methylornithine and alpha-difluoromethylornithine, two ornithine decarboxylase inhibitors, were evaluated after their infusion into several areas of the brain in the rat. Both compounds induced, in dose-dependent manner, similar epileptogenic effects, stereotyped behaviour and postural asymmetry, depending upon the site of injection. Unilateral injection of DFMO into the entopeduncular nucleus or the substantia nigra pars reticulata, caused an increase in locomotor activity. Unilateral injection of DFMO into the caudate nucleus or the substantia nigra pars compacta had no effect on locomotor activity. Among the areas of the brain studied, the most sensitive site from which electrocortical epileptogenic disorders were evoked, was the hippocampus. The GABA agonist muscimol, infused prior to DFMO, resulted in an anticonvulsant action, while DFMO, infused prior to putrescine, increased the convulsant properties of putrescine. In conclusion, these results indicate that DFMO and alpha-methylornithine produced epileptogenic effects after their intracerebral infusion and suggest that may act as analogues of putrescine.

Anticonvulsant properties of flunarizine on reflex and generalized models of epilepsy.

The anticonvulsant activity of 1-bis(4-fluorophenyl)methyl-4-(3-phenyl-2-propenyl)-piperazine, flunarizine, was studied after intraperitoneal administration in DBA/2 mice (seizures induced by sound), intravenous administration in Papio papio (myoclonus induced by photic stimulation) and oral administration in Wistar rats (seizures induced by cefazolin). Protection against sound-induced seizures was observed after intraperitoneal administration of flunarizine (5-40 mg/kg). The ED50 for suppression of tonic, clonic and wild running phases of sound-induced seizures was 3.3, 9.8 and 17.5 mg/kg, respectively. This protective action was significantly reduced by pretreatment with aminophylline (50 mg/kg, i.p.). In photosensitive baboons flunarizine (0.5-1.0 mg/kg, i.v.) provided partial protection against myoclonic responses to stroboscopic stimulation. After flunarizine (2 mg/kg, i.v.) this protection lasted for more than 5 hr (and was complete at 2-3 hr). Cefazolin-induced seizures in rats were prevented by administration of flunarizine (20-40 mg/kg, orally). The ED50 for the suppression of tonic and clonic seizures evoked by subsequent intravenous administration of cefazolin was 25 mg/kg. The protective effects of flunarizine (40 mg/kg, orally) were maximal after 3-6 hr and were maintained for 16-24 hr. Behavioural effects of flunarizine included signs of sedation in both mice and rats. Tolerance to the antiepileptic effects of flunarizine was not seen after chronic treatment in rats. The role of purinergic receptors and of calcium entry blockade in the anticonvulsant action of flunarizine requires further study.

Comparative effects of indoprofen and indomethacin on rat hypothalamic prostaglandin-synthetase activity.

The inhibiting effects of indoprofen were compared with those of indomethacin on prostaglandin-synthetase activity in rat hypothalamus. A dose-dependent inhibition of PG-synthetase activity was obtained after intramuscular administration of both antiphlogistic agents; however, indoprofen was found more powerful. In conclusion, the present experiments provide additional evidence against the idea that pyrogen fever is due to the release of PG's of the E type within the hypothalamus since when comparing results obtained with the two antiphlogistic drugs used, no correlation exists between potency in inhibiting PG's synthesis and antipyretic activity.

Behavioral and electrocortical effects of beta-endorphin after intracerebral infusion in rats.

In rats with cannulae chronically implanted into specific areas of the brain, the effects of beta-endorphin were studied on behavior and electrocortical activity. It has been shown that analgesia and catatonia were evoked after infusing beta-endorphin into the III cerebral ventricle or into the hypothalamus. On the contrary, beta-endorphin microinjected into the caudate nucleus or into the substantia nigra produced an intense pattern of stereotyped movements, occasional contralateral circling, contralateral myoclonic jerks and asymmetric posture. Stereotyped gnawing, grooming and "wet-dog" syndrome preceded catatonia after intrahypothalamic administration of beta-endorphin. Bilateral (III ventricle) or ipsilateral (hypothalamus, caudate nucleus and substantia nigra) high voltage electrocortical spikes and other ECoG pathological changes accompanied by motor disorders (stereotypies, myoclonic jerks) or without any overt behavioral change were constantly observed. Behavioral and electrocortical changes evoked by beta-endorphin were long-lasting and rapidly reversed by the specific opiate antagonist, naloxone.