Dóra Reglődi

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.

Peripheral and central alterations of pituitary adenylate cyclase activating polypeptide-like immunoreactivity in the rat in response to activation of the trigeminovascular system

Pituitary adenylate cyclase activating polypeptide (PACAP) is present in the cranial arteries and trigeminal sensory neurons. We therefore examined the alterations in PACAP-like immunoreactivity (PACAP-LI) in a time-dependent manner in two rat models of trigeminovascular system (TS) activation. In one group chemical stimulation (CS) was performed with i.p. nitroglycerol (NTG), and in the other one the trigeminal ganglia (TRG) were subjected to electrical stimulation (ES). The two biologically active forms, PACAP-38 and PACAP-27, were determined by means of radioimmunoassay (RIA) and mass spectrometry (MS) in the plasma, the cerebrospinal fluid (CSF), the trigeminal nucleus caudalis (TNC), the spinal cord (SC) and the TRG. The tissue concentrations of PACAP-27 were 10 times lower than those of PACAP-38 in the TNC and SC, but about half in the TRG. PACAP-38, but not PACAP-27, was present in the plasma. Neither form could be identified in the CSF. PACAP-38-LI in the plasma, SC and TRG remained unchanged after CS, but it was increased significantly in the TNC 90 and 180 min after NTG injection. In response to ES of the TRG, the level of PACAP-38 in the plasma and the TNC was significantly elevated 90 and 180 min later, but not in the SC or the TRG. The alterations in the levels of PACAP-27 in the tissue homogenates in response to both forms of stimulation were identical to those of PACAP-38. The selective increases in both forms of PACAP in the TNC suggest its important role in the central sensitization involved in migraine-like headache.

Divergent peripheral effects of pituitary adenylate cyclase-activating polypeptide-38 on nociception in rats and mice

Abstract Pituitary adenylate cyclase‐activating polypeptide‐38 (PACAP‐38) and its receptors have been shown in the spinal dorsal horn, on capsaicin‐sensitive sensory neurons and inflammatory cells. The role of PACAP in central pain transmission is controversial, and no data are available on its function in peripheral nociception. Therefore, the aim of the present study was to analyze the effects of locally or systemically administered PACAP‐38 on nocifensive behaviors, inflammatory/neuropathic hyperalgesia and afferent firing. Intraplantar PACAP‐38 (0.2 nmol) injection inhibited carrageenan‐evoked inflammatory mechanical allodynia, mild heat injury‐induced thermal hyperalgesia, as well as nocifensive behaviors in the early and late phases of the formalin test in rats. However, the above dose did not alter basal mechanical or heat thresholds. In mice, PACAP‐38 (0.2 nmol/kg s.c.) significantly diminished acetic acid‐induced abdominal contractions, but exerted no effect on sciatic nerve ligation‐induced neuropathic mechanical hyperalgesia. In contrast, local PACAP‐38 injection markedly increased rotation‐induced afferent firing in the inflamed rat knee joint clearly demonstrating a peripheral sensitization in this organ. These actions were blocked by VPAC1/VPAC2 receptor antagonist pretreatment, but were not altered by PAC1 receptor antagonism. This paper presents the first data for the peripheral actions of PACAP‐38 on nociceptive transmission mediated by VPAC receptors. These effects seem to be divergent depending on the mechanisms of nociceptor activation and the targets of PACAP actions. In acute somatic and visceral inflammatory pain models, PACAP exerts anti‐nociceptive, anti‐hyperalgesic and anti‐allodynic effects. It has no significant peripheral role in traumatic mononeuropathy, but induces mechanical sensitization of knee joint primary afferents.

Effects of maternal separation on dynamics of urocortin 1 and brain-derived neurotrophic factor in the rat non-preganglionic Edinger-Westphal nucleus.

Although mood disorders are frequently genetically determined and to some degree gender‐dependent, the concept of early life ‘programming’, implying a relation between perinatal environmental events and adult mood disorders, has recently gained considerable attention. In particular, maternal separation (MS) markedly affects various stress‐sensitive brain centers. Therefore, MS is considered as a suitable experimental paradigm to study how early life events affect brain plasticity and, hence, cause psychopathologies like major depression. In adult mammals, the classical hypothalamo‐pituitary‐adrenal (HPA‐) axis and the urocortin 1 (Ucn1)‐containing non‐preganglionic Edinger‐Westphal nucleus (npEW) respond in opposite ways to chronic stressors. This raises the hypothesis that MS, which is known to increase vulnerability for adult mood disorders via the dysregulation of the HPA‐axis, will affect npEW dynamics as well. We have tested this hypothesis and, moreover, studied a possible role of brain‐derived neurotrophic factor (BDNF) in such npEW plasticity. By triple immunocytochemistry we show that BDNF and Ucn1 coexist in rat npEW‐neurons that are c‐Fos‐positive upon acute stress. Quantitative immunocytochemistry revealed that MS increases the contents of Ucn1 and BDNF in these cells. Furthermore, in males and females, the c‐Fos response of npEW‐Ucn1 neurons upon restraint stress was blunted in animals with MS history, a phenomenon that was concomitant with dampening of the HPA corticosterone response in females but not in males. Based on these data we suggest that the BDNF‐containing npEW‐Ucn1 system might be affected by MS in a sex‐specific manner. This supports the idea that the npEW would play a role in the appearance of sex differences in the pathogenesis of stress‐induced mood disorders.

Differential regulatory role of pituitary adenylate cyclase-activating polypeptide in the serum-transfer arthritis model.

Pituitary adenylate cyclase–activating polypeptide (PACAP) expressed in capsaicin‐sensitive sensory neurons and immune cells has divergent functions in inflammatory and pain processes. This study was undertaken to investigate the involvement of PACAP in a mouse model of rheumatoid arthritis.

Effects of pituitary adenylate cyclase activating polypeptide and its fragments on retinal degeneration induced by neonatal monosodium glutamate treatment

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the growing family of neurotrophic and neuroprotective factors playing important roles during neuronal development and protection against different types of injuries, such as Parkinsons disease, excitotoxicity, and ischemia. As shown with other neuronal tissues, we provide evidence that PACAP is protective in the retina against toxic injury induced by monosodium glutamate (MSG) in vivo. The need for characterization of its fragments and analogues has recently been emphasized. The aim of the present study was to compare the effects of the physiologically occurring fragments PACAP1–38 and 1–27 and the widely used antagonists (PACAP6–38 and 6–27) in retinal degeneration induced by MSG in neonatal pups. Histological analysis showed that MSG treatment caused the degeneration of the entire inner plexiform layer and the inner nuclear and ganglion cell layers seemed fused. The total thickness of the retina was significantly reduced. Similar and substantial protective effects could be observed after three treatments with PACAP1–38 and 1–27, while MSG toxicity was further aggravated by the PACAP antagonists PACAP6–38 and 6–27. Glutamate‐induced toxicity is known to play a role in several retinal pathologies. Our results provide further evidence for the effectiveness of the endogenously present PACAP forms in counteracting retinotoxicity and call for further studies leading to the discovery of potent analogues that could be used in human ophthalmic diseases.

Early postnatal enriched environment decreases retinal degeneration induced by monosodium glutamate treatment in rats

Environmental enrichment is known to influence the development of the nervous system and it provides beneficial effects in various kinds of brain lesions. Enriched housing conditions also influence the development and functioning of the visual system. The aim of the present study was to investigate whether retinal degeneration induced by neonatal monosodium glutamate (MSG) in rats can be ameliorated by expanded cage size or enriched environment. Control rats were kept in a regular cage, another group of pups was kept in an expanded cage and a third group was kept under complex environmental enriched conditions from the first postnatal day. Half of the rats received MSG treatment on postnatal days 1, 5, and 9, while the other half of the groups received only saline treatment. Retinas were removed at 5 weeks of age and processed for histological analysis. Microscopical analysis revealed a substantial inner retinal degeneration in MSG-treated animals: the retinal thickness was less than 25% of the normal retinas, and the inner layers were completely fused. Expanded cage environment had a significant protective effect: the layers of the retina were well visible, and the thickness of the entire retina had a nearly 50% amelioration. Best results were obtained in retinas from rats living in enriched environment: the retina suffered only an approximately 25% reduction in thickness, and all layers were significantly thicker than in MSG-treated retinas. In summary, the present study showed that expanded field provided some degree of neuroprotection, while a complex environmental enrichment led to a manifest protection against retinal degeneration induced by neonatal MSG treatment in rats.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) signalling exerts chondrogenesis promoting and protecting effects: Implication of calcineurin as a downstream target

Pituitary adenylate cyclase activating polypeptide (PACAP) is an important neurotrophic factor influencing differentiation of neuronal elements and exerting protecting role during traumatic injuries or inflammatory processes of the central nervous system. Although increasing evidence is available on its presence and protecting function in various peripheral tissues, little is known about the role of PACAP in formation of skeletal components. To this end, we aimed to map elements of PACAP signalling in developing cartilage under physiological conditions and during oxidative stress. mRNAs of PACAP and its receptors (PAC1,VPAC1, VPAC2) were detectable during differentiation of chicken limb bud-derived chondrogenic cells in micromass cell cultures. Expression of PAC1 protein showed a peak on days of final commitment of chondrogenic cells. Administration of either the PAC1 receptor agonist PACAP 1-38, or PACAP 6-38 that is generally used as a PAC1 antagonist, augmented cartilage formation, stimulated cell proliferation and enhanced PAC1 and Sox9 protein expression. Both variants of PACAP elevated the protein expression and activity of the Ca-calmodulin dependent Ser/Thr protein phosphatase calcineurin. Application of PACAPs failed to rescue cartilage formation when the activity of calcineurin was pharmacologically inhibited with cyclosporine A. Moreover, exogenous PACAPs prevented diminishing of cartilage formation and decrease of calcineurin activity during oxidative stress. As an unexpected phenomenon, PACAP 6-38 elicited similar effects to those of PACAP 1-38, although to a different extent. On the basis of the above results, we propose calcineurin as a downstream target of PACAP signalling in differentiating chondrocytes either in normal or pathophysiological conditions. Our observations imply the therapeutical perspective that PACAP can be applied as a natural agent that may have protecting effect during joint inflammation and/or may promote cartilage regeneration during degenerative diseases of articular cartilage.

PACAP and VIP signaling in chondrogenesis and osteogenesis

Skeletal development is a complex process regulated by multifactorial signaling cascades that govern proper tissue specific cell differentiation and matrix production. The influence of certain regulatory peptides on cartilage or bone development can be predicted but are not widely studied. In this review, we aimed to assemble and overview those signaling pathways which are modulated by PACAP and VIP neuropeptides and are involved in cartilage and bone formation. We discuss recent experimental data suggesting broad spectrum functions of these neuropeptides in osteogenic and chondrogenic differentiation, including the canonical downstream targets of PACAP and VIP receptors, PKA or MAPK pathways, which are key regulators of chondro- and osteogenesis. Recent experimental data support the hypothesis that PACAP is a positive regulator of chondrogenesis, while VIP has been reported playing an important role in the inflammatory reactions of surrounding joint tissues. Regulatory function of PACAP and VIP in bone development has also been proved, although the source of the peptides is not obvious. Crosstalk and collateral connections of the discussed signaling mechanisms make the system complicated and may obscure the pure effects of VIP and PACAP. Chondro-protective properties of PACAP during oxidative stress observed in our experiments indicate a possible therapeutic application of this neuropeptide.

Role of Pituitary Adenylate-Cyclase Activating Polypeptide and Tac1 gene derived tachykinins in sensory, motor and vascular functions under normal and neuropathic conditions

Pituitary Adenylate-Cyclase Activating Polypeptide (PACAP) and Tac1 gene-encoded tachykinins (substance P: SP, neurokinin A: NKA) are expressed in capsaicin-sensitive nerves, but their role in nociception, inflammation and vasoregulation is unclear. Therefore, we investigated the function of these neuropeptides and the NK1 tachykinin receptor (from Tacr1 gene) in the partial sciatic nerve ligation-induced traumatic mononeuropathy model using gene deficient (PACAP(-/-), Tac1(-/-), and Tacr1(-/-)) mice. Mechanonociceptive threshold of the paw was measured with dynamic plantar aesthesiometry, motor coordination with Rota-Rod and cutaneous microcirculation with laser Doppler imaging. Neurogenic vasodilation was evoked by mustard oil stimulating sensory nerves. In wildtype mice 30-40% mechanical hyperalgesia developed one week after nerve ligation, which was not altered in Tac1(-/-) and Tacr1(-/-) mice, but was absent in PACAP(-/-) animals. Motor coordination of the PACAP(-/-) and Tac1(-/-) groups was significantly worse both before and after nerve ligation compared to their wildtypes, but it did not change in Tacr1(-/-) mice. Basal postoperative microcirculation on the plantar skin of PACAP(-/-) mice did not differ from the wildtypes, but was significantly lower in Tac1(-/-) and Tacr1(-/-) ones. In contrast, mustard oil-induced neurogenic vasodilation was significantly smaller in PACAP(-/-) mice, but not in Tacr1(-/-) and Tac1(-/-) animals. Both PACAP and SP/NKA, but not NK1 receptors participate in normal motor coordination. Tachykinins maintain basal cutaneous microcirculation. PACAP is a crucial mediator of neuropathic mechanical hyperalgesia and neurogenic vasodilation. Therefore identifying its target and developing selective, potent antagonists, might open promising new perspectives for the treatment of neuropathic pain and vascular complications.