Christine Rauca

Formation of free hydroxyl radicals after pentylenetetrazol-induced seizure and kindling

The present study indicates that free radicals have been implicated in pentylenetetrazol (PTZ)-induced seizure and kindling. In our experiments we used a method in which free hydroxyl radicals (* OH) are trapped by systemically applied salicylate in vivo, resulting in the stable and quantifiable products, 2, 3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA. We investigated the formation of both 2,3- and 2,5-DHBA in the whole brain (without cerebellum) of rats treated with an acute seizure-inducing dose of 48 mg/kg PTZ and in PTZ-kindled animals which were injected with the kindling dose of 37.5 mg/kg PTZ at different times after the last PTZ injection. An increase of * OH was observed in kindled rats compared with acutely convulsing animals when the PTZ application was performed 1 min after the salicylate treatment. The amount of DHBAs increased significantly in both convulsed groups 30 min after the application of PTZ compared with animals treated with NaCl instead of PTZ. Sixty minutes after PTZ the DHBA levels normalized to NaCl control values.

The role of superoxide dismutase and α-tocopherol in the development of seizures and kindling induced by pentylenetetrazol - influence of the radical scavenger α-phenyl-N-tert-butyl nitrone

Abstract Previous experiments have shown that the generation of free hydroxyl radicals in rat brain homogenates is increased following pentylenetetrazol (PTZ) kindling. The present study was performed in order to evaluate the involvement of endogeneous radical defence systems as the superoxide dismutase (SOD) and the level of α-tocopherol, an important lipid-soluble and membrane-bound antioxidant in brain homogenate of rats after acute seizure and kindling induced by PTZ. The activities of the total SOD were significantly reduced after acute seizure and tend towards an enhancement in kindled animals. Western blot analysis shows an upregulation of Mn-SOD in rat brain homogenates after kindling. The level of the chain-breaking antioxidant α-tocopherol was reduced in acutely convulsing rats and was not modified in kindled rats. Second, we studied the influence of exogeneously supplied radical scavenger α-phenyl-N-tert-butyl-nitrone (PBN) on seizure and kindling following PTZ treatment. After a single injection of PTZ at a dose evoking clonic–tonic seizures, PBN did not modify either the formation of free hydroxyl radicals measured by the levels of 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA or the susceptibility to PTZ. In the kindling group, subchronic treatment with PBN (over a period of 4 weeks) prevented the increase in the formation of free hydroxyl radicals, and the susceptibility to PTZ was transiently decreased during the development of kindling, but PBN did not influence the susceptibility to PTZ in fully kindled rats. Pretreatment with PBN increased the activities of total SOD and the protein content of Mn-SOD and decreased the level of α-tocopherol in comparison to saline controls. The results suggest that the formation of free hydroxyl radicals is not reflected by an enhanced susceptibility to PTZ classified according to the modified RACINE scale. Additionally, it may be assumed that the increased generation of hydroxyl radicals in kindled animals is not primary caused by an exhaustion of both the defence systems measured. Adaptive mechanisms, as the induction of Mn-SOD, may be taken into consideration to counteract oxidative stress-mediated free radical formation.

The importance of free hydroxyl radicals to hypoxia preconditioning.

Hypoxia preconditioning states that a sublethal hypoxia period will afford neuroprotection against a second harmful event. In our experiments, we carried out a procedure for the development of hypoxia preconditioning in adult male Wistar rats using hypoxic exposure (9% O(2); 91% N(2)) for 1 h. The protection against pentylenetetrazol (PTZ)-induced seizures was studied. For this, rats were tested by a single injection of PTZ (55 mg/kg i.p.) on days 1-21 after hypoxia exposure. The hypoxia exposure significantly prevented the development of acute PTZ convulsion at different times after hypoxia. The present study was designed to determine the effect of N-t-butyl-alpha-phenylnitrone (PBN), an electron-trapping agent and free radical scavenger, on hypoxia preconditioning against PTZ seizures 7 days after hypoxia exposure. PBN abolished the protective action of hypoxia exposure. The generation of free hydroxyl radicals in the brains of animals exposed to hypoxia was determined in a second experiment. For this purpose, the rats were i. p. pretreated with 30 mg/kg PBN and NaCl, respectively, 20 min before the start of hypoxia exposure. Forty-five minutes later the rats were i.p. injected with 300 mg/kg sodium salicylate and once again exposed to hypoxia for 15 min. Immediately after that the animals were decapitated and the free hydroxyl radicals and the salicylate content were estimated in the whole brain without cerebellum. Hypoxia preconditioned animals pretreated with NaCl showed a significantly higher extent of free hydroxyl radicals in the brain compared with PBN-injected preconditioned animals and with naive and sham exposed controls. The results pointed out that the generation of free reactive oxygen species under hypoxic conditions in the brain is involved in the development of the hypoxic preconditioning phenomenon.

Hypoxia protects against the neurotoxicity of kainic acid

A normobar hypoxia (9% oxygen) of 8 h reduces the neurotoxicity of a subcutaneous injection of 10 mg/kg kainic acid given one week later. Both seizures and degenerative changes, including cell death of hippocampal and cortical neurons are markedly decreased by hypoxia. It is also shown that hypoxia also markedly reduced the extensive depletion of zinc from mossy fiber terminals normally induced by kainic acid. This suggests that a protective mechanism induced by hypoxia may affect the glutamatergic transmission in these synapses and prevent excessive synaptic excitation. The possible involvement of adenosine and/or GABA in this protective mechanism is discussed.

Effect of age on pentylenetetrazol-kindling and kindling-induced impairments of learning performance.

Epileptogenesis during ontogeny may not be linearly related to time. It is known that the behavioral manifestations of seizures are age-dependent, but more research is needed to clarify ontogenetic aspects of epilepsies and related alterations, including cognitive deficits. Kindling is an accepted animal model for the study of the convulsive component of epilepsy and its consequences on behavior. Recently, we demonstrated an impairment in acquisition of a conditioned reaction in young adult kindled rats, using pentylenetetrazol (PTZ) as the kindling stimulus. The present study was undertaken to investigate the dependence on age of alterations in the induction of PTZ kindling in rats. We started the kindling protocol in 4-, 6-, and 8-week- and 6-, 12-, 18-, and 24-month-old rats. The PTZ kindling showed an age-dependent decrease in expression of convulsions. The diminished kindling capacity was already seen in 6-month-old rats. In contrast, kindling-related impairment effects on cognitive functions increased with age. Thus, the correlation between learning impairment and occurrence of tonic-clonic seizures that we had demonstrated in 8-week-old rats was abolished in older rats. On the other hand, when the kindling procedure was started in 6-week-old rats, no impairment was found in fully kindled rats.

Hypothermia Inhibits Pentylenetetrazol Kindling and Prevents Kindling-Induced Deficit in Shuttle-Box Avoidance

In this study, we evaluated the effects of hypothermic exposure on pentylenetetrazol (PTZ) kindling and the resulting deficit of shuttle-box avoidance learning in rats. Additionally, to acknowledge neuronal cell loss, we estimated the number of toluidine blue-positive cells in different brain regions after PTZ kindling and hypothermia exposure in comparison to different normothermic and hypothermic controls. To obtain hypothermic conditions over a period of up to about 3 h, 30 min after PTZ application the animals were treated with 5 mg/kg chlorpromazine (CP) and 25 min later exposed to 15 degrees C cold water for 5 min. Under these conditions the rectal and the striatal temperature were reduced up to a maximum of 5 degrees C. The additional injection of CP did not influence the development of PTZ kindling. Animals treated with PTZ/CP and exposed to hypothermia did not reach the criterion for kindling. Furthermore, this group of animals did not demonstrate any learning deficit. Forty-eight hours after the last kindling application the number of toluidine blue-stained cells was decreased in the investigated brain regions (hippocampal CA1 and CA3 sector, hilus, and cingular cortex) of kindled rats. Hypothermia protected from cell damage in the hippocampal CA3 sector and in the hilus. Results suggest that the inhibiting effect of hypothermia on the development of kindling and the following learning deficit possibly resulted from the suppression of cell damage in distinct brain structures on PTZ-kindled rats.

Neuropeptide Y and somatostatin immunoreactivity in the rat hippocampus after moderate hypoxia

Transient moderate hypoxia has been previously shown to exert a potent protective role to subsequently applied convulsant drugs. We now investigated neuropeptide Y and somatostatin immunoreactivities seven days after moderate hypoxia (9% O2 in N2 for two times 8 h) in the hippocampus of the rat. A slight reduction of somatostatin immunoreactive cells was observed in the hilus of the dorsal and ventral hippocampus. At the same time, the total number of neuropeptide Y immunoreactive neurons was increased in this area due to a pronounced increase in staining of presumable basket cells. There was also increased staining of neuropeptide Y positive fibers in the outer molecular layer. Our data suggest activation of neuropeptide Y containing interneurons after a moderate or a mild transient hypoxia. Activation of these inhibitory neurons may contribute to the protective effect of this treatment.

Does a moderate hypoxia induce manganese-superoxide dismutase or heat shock proteins to act as possible protective factors?

Male Wistar rats were kept in a hypoxia chamber (9% O2) for eight hours. Control animals breathed room air in the same chamber for a similar period of time. One week later the brains of all rats were prepared for the immunohistochemical demonstration of Mn-superoxide dismutase (Mn-SOD). In comparison with the sham-exposed controls, the hypoxia-treated animals showed an increase in the number of Mn-SOD-immunoreactive neurons in several hippocampal structures. The 72 kD heat shock protein was not found to be induced one week after a moderate hypoxia.

Does fucose or piracetam modify the effect of hypoxia preconditioning against pentylenetetrazol-induced seizures?

To clarify the question whether the duration of hypoxia exposure has an influence on the point in time or the strength of hypoxic preconditioning, hypoxia exposure of rats lasting 1 and 8 h was tested regarding the modification of susceptibility to acute pentylenetetrazol-induced seizures. Following the short-lasting (1 h) hypoxia, the maximum level of preconditioning action was observed 7 days after hypoxia, whereas the longer-lasting hypoxia (8 h) produced the maximum level of protection 14 days after hypoxia. We investigated the influence of fucose and piracetam on the effect of hypoxia preconditioning by the application of the substances 20 min before the beginning of hypoxia exposure. Fucose did not modify the result of hypoxia preconditioning. But after the treatment with piracetam, the preconditioning effect was prevented following hypoxia lasting 1 and 8 h. We suggest that the radical scavenger properties of piracetam are responsible for the absence of protection against pentylenetetrazol-evoked seizures.