István Lengvári

Neurological reflexes and early motor behavior in rats subjected to neonatal hypoxic-ischemic injury.

Severe perinatal hypoxia-ischemia is an important cause of brain injury in both full-term and premature newborns, with a high risk of future behavioral and neurological deficits. The most commonly used animal model of neonatal hypoxia-ischemia is the unilateral ligation of the common carotid artery followed by exposure to hypoxia in 7-day-old rats. In spite of the wide use of this model, lot of contradictions and discrepancies exist between the results obtained by different laboratories regarding behavioral deficits and there are no data regarding the possible delay of the appearance of neurological reflexes and the time-course of reflex performances following neonatal hypoxic-ischemic injury in rats. In the present study we showed that neonatal hypoxia-ischemia retarded the development of somatic growth and several neurological reflexes (ear twitch, grasping, gait and negative geotaxis). Hypoxic animals also displayed retarded performance in righting, geotaxis and gait reflexes. Although hypoxic pups performed worse in most tests for motor coordination, they reached normal levels by 5 weeks of age except in the footfault test. In the open-field, hypoxic animals were generally more active, except at 3 weeks, when activity of normal pups increased enormously as well. Brain areas were significantly reduced in hypoxic animals, but no close correlation was found with behavioral deficits.

Pituitary adenylate cyclase activating polypeptide protects dopaminergic neurons and improves behavioral deficits in a rat model of Parkinson's disease.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide, exerting different actions in the central and peripheral nervous systems. Among others, it has neurotrophic and neuroprotective effects. In the present study, we investigated the effects of PACAP in a rat model of Parkinsons disease. Rats were given unilateral injections of 6-hydroxydopamine (6-OHDA) into the substantia nigra. PACAP-treated animals received 0.1 microg PACAP as a pretreatment. Control animals without PACAP treatment displayed severe hypokinesia at 1 and 10 days postlesion when compared to animals receiving saline only. In only 1 day postlesion, by contrast, PACAP-treated rats showed no hypokinesia. Asymmetrical signs, such as turning, rearing and biased thigmotaxic scanning were observed in all lesioned animals 1 day postlesion. PACAP-treated animals, however, showed better recovery as they ceased to display asymmetrical signs 10 days later and showed markedly less apomorphine-induced rotations. Tyrosine-hydroxylase immunohistochemistry revealed that control animals had more than 95% loss of the dopaminergic cells in the ipsilateral substantia nigra, while PACAP-treated animals had only approximately 50% loss of dopaminergic cells. In summary, the present results show the neuroprotective effect of PACAP in 6-OHDA-induced lesion of substantia nigra, with less severe acute neurological symptoms and a more rapid amelioration of behavioral deficits.

Pituitary adenylate cyclase activating polypeptide protects cardiomyocytes against oxidative stress-induced apoptosis

Pituitary adenylate cyclase activating polypeptide (PACAP) has well-known neuroprotective effects, and one of the main factors leading to neuroprotection seems to be its anti-apoptotic effects. The peptide and its receptors are present also in the heart, but whether PACAP can be protective in cardiomyocytes, is not known. Therefore, the aim of the present study was to investigate the effects of PACAP on oxidative stress-induced apoptosis in cardiomyocytes. Our results show that PACAP increased cell viability by attenuating H2O2-induced apoptosis in a cardiac myocyte culture. PACAP also decreased caspase-3 activity and increased the expression of the anti-apoptotic markers Bcl-2 and phospho-Bad. These effects of PACAP were counteracted by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP is able to attenuate oxidative stress-induced cardiomyocyte apoptosis.

Effects of pituitary adenylate cyclase activating polypeptide in retinal degeneration induced by monosodium-glutamate.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide with a wide range of effects in the central and peripheral nervous systems. PACAP has well-documented neurotrophic and neuroprotective actions in both in vitro and in vivo models of different neuronal injuries. The aim of the present study was to investigate the possible neuroprotective effect of PACAP in retinal degeneration induced by monosodium-glutamate (MSG) in neonatal rats. Preceding the MSG treatment, PACAP (1 or 100pmol/5mul) was injected unilaterally into the vitreous body on postnatal days 1, 5 and 9. Immediately after the PACAP treatment, pups were treated with 2mg/g body weight MSG subcutaneously. At 3 weeks of age, rats were sacrificed and retinas were removed and processed for histological examination. Our results show that MSG treatment caused severe degeneration, primarily of the inner retinal layers. The thickness of the entire retina was only approximately half of that of the normal retinas, and the inner nuclear layer seemed to be fused with the ganglionic cell layer, with no discernible inner plexiform layer. Retinas of animals treated with 1pmol PACAP showed a similar degree of degeneration. However, retinas of rats treated with 100pmol PACAP showed significantly less damage, with clearly distinguishable inner retinal layers. In summary, our present study shows that local PACAP treatment could attenuate the retinal degeneration induced by the excitotoxic effects of glutamate.

The neuroprotective effects of PACAP in monosodium glutamate-induced retinal lesion involve inhibition of proapoptotic signaling pathways

Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are present in the retina and exert several distinct functions. PACAP has well-known neuroprotective effects in neuronal cultures in vitro and against different insults in vivo. Recently we have shown that PACAP is neuroprotective against monosodium glutamate (MSG)-induced retinal degeneration. In the present study we investigated the possible signal transduction pathways involved in the protective effect of intravitreal PACAP administration against apoptotic retinal degeneration induced by neonatal MSG treatment. MSG induced activation of proapoptotic signaling proteins and reduced the levels of antiapoptotic molecules in neonatal retinas. Co-treatment with PACAP attenuated the MSG-induced activation of caspase-3 and JNK, inhibited the MSG-induced cytosolic translocation of apoptosis inducing factor (AIF) and cytochrome c, and increased the level of phospho-Bad. Furthermore, PACAP treatment alone decreased cytosolic AIF and cytochrome c levels, while PACAP6-38 increased cytochrome c release, caspase-3 and JNK activity and decreased phospho-Bad activity. In summary, our results show that PACAP treatment attenuated the MSG-induced changes in apoptotic signaling molecules in vivo and suggest that also endogenously present PACAP has neuroprotective effects. These results may have further clinical implications in reducing glutamate-induced excitotoxicity in several ophthalmic diseases.

Effects of pituitary adenylate cyclase activating polypeptide (PACAP) on the PKA-Bad-14-3-3 signaling pathway in glutamate-induced retinal injury in neonatal rats.

The neuropeptide PACAP (pituitary adenylate cyclase activating polypeptide) and its receptors are widely expressed in the nervous system including the retina. PACAP has well-known neuroprotective effects in neuronal culturesin vitro and against different insultsin vivo. Recently, we have shown that PACAP1-38 is neuroprotective against monosodium glutamate (MSG)-induced retinal degeneration. Studying the molecular mechanisms of this protection has revealed that PACAP1-38 stimulates antiapoptotic mechanisms such as phosphorylation of ERK1/2 and inhibits pro-apoptotic signaling molecules such as JNK1/2, p38MAPK, caspase-3 and the translocation of mitochondrial cytochromec and apoptosis inducing factor in glutamate-treated retinasin vivo. In the present study we investigated the effects of PACAP1-38 on a further signal transduction pathway possibly involved in the protective effect of intravitreal PACAP1-38 administration against apoptotic retinal degeneration induced by neonatal MSG treatment. The focus of the present study was the protein kinase A (PKA)-Bad-14-3-3 transduction pathway.In vivo MSG treatment led to a reduction in the levels of anti-apoptotic molecules (phospho-PKA phospho-Bad, Bcl-xL and 14-3-3 proteins) in the retina. Co-treatment with PACAP1-38 counteracted these effects: the level of phospho-PKA, phospho-Bad, Bcl-xL and 14-3-3 were increased. All effects of PACAP1-38 were inhibited by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP1-38 activates the PKA-Bad-14-3-3 pathway which is inhibited by MSG treatment. Our results also provide new insights into the signaling mechanisms possibly involved in the PACAP-mediated anti-apoptotic effects.

Ultrastructural characteristics of immunolabelled, corticotropin releasing factor (CRF)-synthesizing neurons in the rat brain

SummaryThe corticotropin releasing factor (CRF)-synthesizing perikarya and neural processes were detected at ultrastructural level in the hypothalamic paraventricular nucleus and in the median eminence of control and colchicine-pretreated rats. The unlabelled antibody peroxidase-antiperoxidase complex (PAP) immunohistochemical method was used in a pre-embedding manner, on thick, non-frozen sections. In CRF-perikarya, neurosecretory granules (80–120 nm in diameter), free ribosomes, and the rough endoplasmic reticulum were labelled. Unlabelled axon terminals formed asymmetric synapses on CRF-containing perikarya and dendrites. Immunolabelled axons terminated in the palisadic zone of the median eminence.

Age and gender differences in behavioral and morphological outcome after 6-hydroxydopamine-induced lesion of the substantia nigra in rats

It is well known that Parkinsons disease occurs more commonly in older people and men are more frequently affected than women. Animal studies in models of the disease mainly use young male animals. The effects of aging on the neurochemical changes in the dopaminergic system have been extensively studied, however, data on comparative behavioral consequences of lesions of the dopaminergic system in aging and in female animals are limited. The aim of the present study was to give a detailed comparative behavioral analysis of young and aging male and female rats following 6-hydroxydopamine (6-OHDA) lesion of the substantia nigra. Young males and females, as well as aging males and females underwent 6-OHDA-induced lesion of the substantia nigra. Behavioral analysis in an open-field was performed before the injury, and 1 and 10 days after the operation. Tyrosine-hydroxylase (TH) immunohistochemistry was done in order to assess the dopaminergic cell loss in the substantia nigra. It was found that both young and aging male animals were more susceptible to 6-OHDA than females: female rats had a significantly less dopaminergic cell loss and responded to 6-OHDA with a significantly higher degree of behavioral recovery after the injury. Although the dopaminergic cell loss was not significantly different between young and aging animals of the same sex, aging rats showed more severe behavioral deficits. In summary, our present results showed clear age- and gender differences in the behavioral and histological outcome following 6-OHDA lesion of the substantia nigra.

Immunohistological detection of degenerating CRF-immunoreactive nerve fibers in the median eminence after lesion of paraventricular nucleus of the rat. A light and electron microscopic study

Corticotropin releasing factor (CRF)-immunoreactive neurons were detected in the paraventricular nuclei (PVN) of the rat brain, using both the traditional and the recently developed silver-gold intensified PAP methods at light and electron microscopic levels. The latter technique was more sensitive, compared to the classical PAP method, and proved to be highly specific at the ultrastructural level. The immunolabeled perikarya showed smooth or rough contoured fusiform or multipolar shape. Bilateral surgical destruction of PVN caused a gradual decrease in the number of CRF-immunopositive fibers of the median eminence. Following the second post-operative week, CRF-immunoreactivity practically disappeared from this area. In the case of unilateral lesion of PVN, the diminution of immunoreactivity was restricted to the ipsilateral side of the median eminence-pituitary stalk region. Applying the silver-gold intensified PAP method to electron microscopy, the detection of immuno-labeled degenerating fibers became possible, among morphologically similar, densely degenerating, but unlabeled, profiles. This study reports that CRF fibers to the capillary system of the median eminence of the rat originate principally from PVN.

Morphological and functional effects of PACAP in 6-hydroxydopamine-induced lesion of the substantia nigra in rats

Pituitary adenylate cyclase activating polypeptide (PACAP) has several different actions in the nervous system, including neuroprotective effects. In the present study, we investigated the effects of different doses of PACAP on the functional and morphological outcome in a rat model of Parkinsons disease. Rats were given unilateral injections of 6-hydroxydopamine (6-OHDA) into the substantia nigra. PACAP-treated animals received 1, 0.1 or 0.01 microg PACAP as a pretreatment. Control animals without PACAP treatment displayed severe hypokinesia at 1 and 10 days post-lesion when compared to normal animals or those receiving saline only. PACAP treatment resulted in less severe acute hypokinesia, and complete recovery by 10 days. Asymmetrical signs were observed in all lesioned animals 1 day post-lesion. PACAP-treated animals, however, showed better recovery as they ceased to display asymmetrical signs 10 days later and showed markedly less apomorphine-induced rotations. Best behavioral outcome was observed in animals treated with 0.1 microg PACAP. Tyrosine-hydroxylase (TH) immunohistochemistry revealed increased number of dopaminergic neurons in the substantia nigra pars compacta and in the ventral tegmental area in all PACAP-treated rats in contrast to the severe cell loss in control animals. These results indicate that PACAP may be a promising therapeutic agent in Parkinsons disease.