Tamas Atlasz

PACAP is an endogenous protective factor-insights from PACAP-deficient mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a widespread neuropeptide with a diverse array of biological functions. Not surprisingly, the lack of endogenous PACAP therefore results in a variety of abnormalities. One of the important effects of PACAP is its neuroprotective and general cytoprotective role. PACAP protects neurons and other tissues against ischemic, toxic, and traumatic lesions. Data obtained from PACAP-deficient mice provide evidence that endogenous PACAP also has protective functions. Mice lacking PACAP are more vulnerable to different in vitro and in vivo insults. The present review summarizes data on the increased sensitivity of PACAP-deficient mice against harmful stimuli. Mice lacking PACAP respond with a higher degree of injury in cerebral ischemia, autoimmune encephalomyelitis, and axonal lesion. Retinal ischemic and excitotoxic injuries also produce increased cell loss in PACAP-deficient mice. In peripheral organs, kidney cell cultures from PACAP-deficient mice are more sensitive to oxidative stress and in vitro hypoxia. In vivo, PACAP-deficient mice have a negative histological outcome and altered cytokine response in kidney and small intestine ischemia/reperfusion injury. Large intestinal inflammation, toxic lesion of the pancreas, and doxorubicin-induced cardiomyopathy are also more severe with a lack of endogenous PACAP. Finally, an increased inflammatory response has been described in subacute endotoxin-induced airway inflammation and in an oxazolone-induced allergic contact dermatitis model. In summary, lack of endogenous PACAP leads to higher vulnerability in a number of injuries in the nervous system and peripheral organs, supporting the hypothesis that PACAP is part of the endogenous cytoprotective machinery.

Pituitary adenylate cyclase activating polypeptide in the retina: focus on the retinoprotective effects.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide that has been shown to exert protective effects against different neuronal injuries, such as traumatic brain and spinal cord injury, models of neurodegenerative diseases, and cerebral ischemia. PACAP and its receptors are present in the retina. In this study, we summarize the current knowledge on retinal PACAP with focus on the retinoprotective effects. Results of histological, immunohistochemical, and molecular biological analysis are reviewed. In vitro, PACAP shows protection against glutamate, thapsigargin, anisomycin, and anoxia. In vivo, the protective effects of intravitreal PACAP treatment have been shown in the following models of retinal degeneration in rats: excitotoxic injury induced by glutamate and kainate, ischemic injury, degeneration caused by UV‐A light, optic nerve transection, and streptozotocin‐induced diabetic retinopathy. Studying the molecular mechanism has revealed that PACAP acts by activating antiapoptotic and inhibiting proapoptotic signaling pathways in the retina in vivo. These studies strongly suggest that PACAP is an excellent candidate retinoprotective agent that could be a potential therapeutic substance in various retinal diseases.

Evaluation of the protective effects of PACAP with cell-specific markers in ischemia-induced retinal degeneration

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide that has been shown to exert protective effects in different neuronal injuries, such as traumatic brain injury, models of neurodegenerative diseases and cerebral ischemia. We have provided evidence that PACAP is neuroprotective in several models of retinal degeneration in vivo. In our previous studies we showed that PACAP treatment significantly ameliorated the damaging effects of permanent bilateral common carotid artery occlusion (BCCAO). In the present study cell-type-specific markers were used in the same models in order to further specify the protective effects of PACAP. In rats BCCAO led to severe degeneration of all retinal layers that was attenuated by PACAP (100 pmol) administered unilaterally immediately following BCCAO into the vitreous body of one eye. Retinas were processed for immunohistochemistry after 3 weeks. Immunolabeling was executed for vesicular glutamate transporter 1 (VGLUT 1), vesicular gamma-aminobutyric acid transporter (VGAT), protein kinase Calpha (PKCalpha), glial fibrillary acidic protein (GFAP) and calcium-binding proteins, such as calbindin, calretinin, parvalbumin. In BCCAO retinas, intensity of immunopositivity for all antisera was dramatically decreased, except in the case of GFAP. In PACAP-treated retinas, immunostaining was similar to that of the control animals. In summary, our study presented immunohistochemical identification of cell types sensitive to chronic retinal hypoperfusion and the protective effects of PACAP. This analysis revealed that the retinoprotective effects of PACAP are not phenotype-specific, but it rather influences general cytoprotective pathways irrespective of the neuronal subtypes in the retina subjected to chronic hypoperfusion.

Degree of damage compensation by various PACAP treatments in monosodium glutamate-induced retinal degeneration.

Pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to be neuroprotective in retinal ischemia and monosodium L-glutamate (MSG)-induced retinal degeneration. Here we describe how different MSG treatments (1x and 3x application) cause retinal damage and finally lead to the destruction of the entire inner retina and how PACAP attenuates this effect. Newborn rats from both sexes were injected subcutaneously with 2 mg/g bodyweight MSG on postnatal days 1, 5 and 9. The left eye was left intact while we injected 5 µl PACAP38 solution (100 pmol) into the vitreous of the right eye with a Hamilton syringe at the time of (i) the first, (ii) the first two or (iii) all three MSG injections. Histological analysis has shown that the above described MSG treatment caused the entire inner plexiform layer (IPL) to degenerate, and the inner nuclear (INL) and ganglion cell layers (GCL) seemed fused. One time PACAP38 treatment at the first MSG application did not change the degenerative capacity of MSG. However, if animals received PACAP38 into the vitreous of the eye at the first 2 or all 3 times, a substantial protective effect could be observed. The IPL remained well discernible, the INL retained 2–3 cell rows and the number of cells in the GCL was substantially higher than in the MSG-treated retinas, and was not significantly different from that observed in the control tissue. We conclude that (i) 2 or 3 times PACAP treatment attenuates retinal degeneration; (ii) one PACAP treatment does not provide protection against repeated excitotoxic insults, and (iii) repeated application of PACAP under these experimental conditions may lead to a primed state in which further neurotoxic insults are ineffective.

Protective effects of the neuropeptide PACAP in diabetic retinopathy

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with highly potent neurotrophic and neuroprotective effects. PACAP and its receptors occur in the retina and PACAP has been applied in animal models of metabolic retinal disorders to reduce structural and functional damage. Furthermore, PACAP has been implicated as a potential anti-diabetic peptide. Our aim has been to investigate, by using a complex morphological, immunochemical and molecular biological approach, whether PACAP attenuates diabetic retinopathy. Diabetes was induced in rats with a single streptozotocin injection. PACAP was injected intravitreally into one eye (100 pmol) three times during the last week of a 3-week survival period. Retinas were processed for the following procedures: routine histology, immunohistochemistry (single and double labeling, whole-mount), quantitative reverse transcription with the polymerase chain reaction and Western blotting. Cone photoreceptors and dopaminergic amacrine and ganglion cells degenerated in diabetic retinas and glial fibrillary acidic protein were upregulated in Müller glial cells. The number of cones, the length of their outer segments and the cell number in the ganglion cell layer were decreased. PACAP ameliorated these structural changes. Moreover, PACAP increased the levels of PAC1-receptor and tyrosine-hydroxylase as detected by molecular biological methods. Thus, PACAP has significant protective effects in the diabetic retina. PACAP treatment attenuates neuronal cell loss in diabetic retinopathy, the protective effects of PACAP probably being mediated through the activation of PAC1-receptor. These results suggest that PACAP has a therapeutic potential in diabetic retinopathy.

Search for the optimal monosodium glutamate treatment schedule to study the neuroprotective effects of PACAP in the retina

Abstract:  We have previously shown the protective effects of pituitary adenylate cyclaseactivating polypeptide (PACAP) in monosodium glutamate (MSG)‐induced retinal degeneration. In the present article, we have investigated the optimal model for examining this neuroprotective effect. One MSG treatment on postnatal (P) days 1 or 5, in spite of leading to same ultrastructural changes, does not cause enough damage to study neuroprotection. When retinas were treated three times with MSG, the entire inner retina degenerated. Neuroprotection with PACAP was achieved with at least two treatments. Evidence suggests that PACAP provides protection against excitotoxicity, therefore, it may be a useful agent in reducing excitotoxic damage in the retina.

Novel Neuroprotective Strategies in Ischemic Retinal Lesions

Retinal ischemia can be effectively modeled by permanent bilateral common carotid artery occlusion, which leads to chronic hypoperfusion-induced degeneration in the entire rat retina. The complex pathways leading to retinal cell death offer a complex approach of neuroprotective strategies. In the present review we summarize recent findings with different neuroprotective candidate molecules. We describe the protective effects of intravitreal treatment with: (i) urocortin 2; (ii) a mitochondrial ATP-sensitive K+ channel opener, diazoxide; (iii) a neurotrophic factor, pituitary adenylate cyclase activating polypeptide; and (iv) a novel poly(ADP-ribose) polymerase inhibitor (HO3089). The retinoprotective effects are demonstrated with morphological description and effects on apoptotic pathways using molecular biological techniques.

Effect of PACAP on MAP kinases, Akt and cytokine expressions in rat retinal hypoperfusion

Pituitary adenylate cyclase activating polypeptide (PACAP) is known for its potent neuroprotective effects, including the retinoprotective actions in several types of retinal injuries. We have shown earlier that PACAP treatment causes activation of protective pathways and inhibition of pro-apoptotic signaling in excitotoxic retinal lesions. The aim of the present study was to gain insight into the in vivo protective mechanism of PACAP in retinal hypoperfusion injury induced by bilateral common carotid artery occlusion (BCCAO). Rats underwent BCCAO and received intravitreal PACAP (PACAP38) treatment. We investigated the activation level of the protective Akt pathway as well as the different mitogen activated protein kinases (MAPKs) by Western blot analysis and the expression of cytokines using a cytokine array kit. We found that PACAP treatment alone did not influence the phosphorylation of Akt or the MAPKs, but decreased the hypoperfusion-induced activation of both p38MAPK and JNK and increased the activation of the protective Akt and ERK1/2 in hypoperfused retinas. The cytokine profile was dramatically changed after BCCAO, with most cytokines and chemokines showing an increase, which was attenuated by PACAP (such as CINC, CNTF, fractalkine, sICAM, IL-1, LIX, Selectin, MIP-1, RANTES and TIMP-1). In addition, PACAP increased the expression of VEGF and thymus chemokine. The present results provide further insight into the neuroprotective mechanism induced by PACAP in ischemic retinal injuries, showing that PACAP ameliorates hypoperfusion injury involving Akt, MAPK pathways and anti-inflammatory actions.

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.

Effects of perinatal asphyxia on the neurobehavioral and retinal development of newborn rats.

Perinatal asphyxia during delivery produces long-term deficits and represents a major problem in both neonatal and pediatric care. Several morphological, biochemical and behavioral changes have been described in rats exposed to perinatal asphyxia. The aim of the present study was to evaluate how perinatal asphyxia affects the complex early neurobehavioral development and retinal structure of newborn rats. Asphyxia was induced in ready-to-deliver mothers by removing the pups by cesarian section after 15 min of asphyxia. Somatic and neurobehavioral development was tested daily during the first 3 weeks, and motor coordination tests were performed on postnatal weeks 3-5. After completion of the testing procedure, retinas were removed for histological analysis. We found that in spite of the fast catch-up-growth of asphyctic pups, nearly all examined reflexes were delayed by 1-4 days: negative geotaxis, sensory reflexes, righting reflexes, development of fore- and hindlimb grasp and placing, gait and auditory startle reflexes. Time to perform negative geotaxis, surface righting and gait reflexes was significantly longer during the first few weeks in asphyctic pups. Among the motor coordination tests, a markedly weaker performance was observed in the grid walking and footfault test and in the walk initiation test. Retinal structure showed severe degeneration in the layer of the photoreceptor and bipolar cell bodies. In summary, our present study provided a detailed description of reflex and motor development following perinatal asphyxia, showing that asphyxia led to a marked delay in neurobehavioral development and a severe retinal degeneration.