Citation: Werling D, Reglodi D, Banks WA, et al. Ocular delivery of PACAP1-27 protects the retina from ischemic damage in rodents. Invest Ophthalmol Vis Sci. 2016;57:6683-6691. DOI: 10.1167/iovs.16-20630 PURPOSE. Pituitary adenylate cyclase activating polypeptide (PACAP) is neuroprotective in neuronal injuries. Bilateral common carotid artery occlusion (BCCAO) causes chronic hypoperfusion-induced degeneration in the rat retina, where we proved the retinoprotective effect of intravitreal PACAP. Although this route of administration is a common clinical practice in several diseases, easier routes are clinically important. Our aim was to investigate the potential retinoprotective effects of PACAP eye drops in BCCAO-induced ischemic retinopathy.METHODS. After performing BCCAO in rats, the right eyes were treated with PACAP1-27 eye drops (1 lg/drop, 2 3 1 drops/day for 5 days), containing different vehicles: saline, water for injections, thiomersal or benzalkonium solution for ophthalmic use (SOCB). Histology and immunohistochemistry were performed 2 weeks after surgery, while molecular analysis was performed 24 hours after BCCAO. Passage of PACAP1-27 through the ocular layers was tested with radioactive PACAP-SOCB in mice.RESULTS. Bilateral common carotid artery occlusion led to a severe degeneration of all retinal layers. Solution for ophthalmic use was the most effective vehicle for delivering PACAP (PACAP-SOCB), significantly ameliorating BCCAO-induced damage. The massive upregulation of GFAP was not observed in retinas treated with PACAP-SOCB eye drops. PACAP-SOCB treatment also increased activation of the protective Akt and ERK1/2 in hypoperfused retinas. The cytokine profile showing upregulation in different cytokines was attenuated by PACAP-SOCB. Radioactive PACAP reached the retina when delivered in SOCB-containing eye drops.CONCLUSIONS. PACAP1-27, delivered in the SOCB vehicle as eye drops, was retinoprotective in ischemic retinopathy, providing the basis for future therapeutic administration.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a widespread neuropeptide that is well known for its general cytoprotective effects in different neuronal injuries, such as traumatic brain and spinal cord injury, models of neurodegenerative diseases, and cerebral ischemia. PACAP and its receptors also occur in the retina. In this review, we summarize the retinoprotective effects of PACAP. In vitro, PACAP is protective against glutamate, thapsigargin, anisomycin, oxidative stress, UV light, high glucose, infl ammation, and anoxia. Both the neural retina and the pigment epithelial cells can be protected by PACAP in various experimental paradigms. In vivo, the protective effects of intravitreal PACAP treatment have been shown in the following models in rats and mice: excitotoxic injury induced by glutamate, N -methyl-D -aspartate (NMDA) or kainate, ischemic injury induced by carotid artery ligation and high intraocular pressure, degeneration caused by UV-A light, optic nerve transection, and streptozotocin-induced diabetic retinopathy as well as retinopathy of prematurity. Molecular biological methods have revealed that PACAP activates anti-apoptotic, while inhibits pro-apoptotic signaling pathways, and it also stimulates an anti-infl ammatory environment in the retina. Altogether, PACAP is suggested to be a potential therapeutic retinoprotective agent in various retinal diseases.

Purpose. Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuroprotective peptide that has been shown to exert protective effects in different models of neurodegenerative diseases, including retinal degenerations. Data obtained from PACAPdeficient (PACAP KO) mice provide evidence that endogenous PACAP has neuroprotective role in different pathologies. PACAP KO mice show enhanced sensitivity to different insults, such as oxidative stress, hypoxia and inflammation. The aim of the present study was to investigate the protective effects of endogenous PACAP in retinal inflammation. Methods. Endotoxin-caused eye inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS) in PACAP KO and wild type (Wt) mice. After LPS treatment, retinas were processed for histological examination. To detect the alterations of different proteins and cytokines, immunohistochemical, western blot and cytokine array were used. We also performed dark-adapted electroretinography (ERG) to detect the functional differences. Results. The thickness of nearly all layers was significantly less in LPS-injected PACAP KO mice compared to Wt animals. Increased expression of glial fibrillary acidic protein (GFAP) was induced in Müller glial cells after LPS treatment, which was more intense in PACAP KO mice. The levels of pAkt and pGSK were decreased in PACAP KO group during inflammation. LPS treatment significantly increased cytokines (sICAM-1, JE, TIMP-1) in both treated groups, but it was more expressed in PACAP KO animals. Furthermore, ERG responses were disturbed after LPS injection in PACAP KO mice. Conclusion. Our results showed that endogenous PACAP has a protective role in LPS-caused retinal inflammation.

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has two active forms, PACAP1-27 and PACAP1-38. Among the well-established actions are PACAP’s neurotrophic and neuroprotective effects, which have also been proven in models of different retinopathies. The route of delivery is usually intravitreal in studies proving PACAP’s retinoprotective effects. Recently, we have shown that PACAP1-27 delivered as eye drops in benzalkonium-chloride was able to cross the ocular barriers and exert retinoprotection in ischemia. Since PACAP1-38 is the dominant form of the naturally occurring PACAP, our aim was to investigate whether the longer form is also able to cross the barriers and exert protective effects in permanent bilateral common carotid artery occlusion (BCCAO), a model of retinal hypoperfusion. Our results show that radioactive PACAP1-38 eye drops could effectively pass through the ocular barriers to reach the retina. Routine histological analysis and immunohistochemical evaluation of the Müller glial cells revealed that PACAP1-38 exerted retinoprotective effects. PACAP1-38 attenuated the damage caused by hypoperfusion, apparent in almost all retinal layers, and it decreased the glial cell overactivation. Overall, our results confirm that PACAP1-38 given in the form of eye drops is a novel protective therapeutic approach to treat retinal diseases.

The oxygen-induced retinopathy (OIR) is a well-established rodent model of retinopathy of prematurity (ROP), which is one of the most common causes of childhood visual impairment affecting preterm babies. Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to have neuroprotective effects. Several studies have revealed the presence of PACAP and its receptors in the retina and reported its protective effects in ischemic and diabetic retinopathy. In this study, we investigated whether PACAP administration can influence the vascular changes in the rat OIR model. OIR was generated by placing the animals in daily alternating 10/50 oxygen concentrations from postnatal day (PD) 0 to PD14 then returned them to room air. Meanwhile, animals received PACAP or saline intraperitoneally or intravitreally from PD1 to PD8 or on PD11, PD14, and PD17, respectively. On PD19 ± 1, the retinas were isolated and the vessels were visualized by isolectin staining. The percentage of avascular to whole retinal areas and the number of branching points were measured. Change in cytokine expression was also determined. Intravitreal treatment with PACAP remarkably reduced the extent of avascular area compared to the non- and saline-treated OIR groups. Intraperitoneal PACAP injection did not influence the vascular extent. Retinal images of room-air controls did not show vascular alterations. No changes in the number of vessel branching were observed after treatments. Alterations in cytokine profile after local PACAP injection further supported the protective role of the peptide. This is the first study to examine the effects of PACAP in ROP. Although the exact mechanism is still not revealed, the present results show that PACAP treatment can ameliorate the vascular changes in the animal model of ROP.

Early life events are critical in the development of the central nervous system. Injuries in this period can cause severe damage with permanent disabilities. The early changes following a perinatal lesion have prognostic significance. The nervous system in young age has a potential for plasticity and regeneration, which can prevent the negative effects of neuronal damage, and the most important objective of rehabilitation is to enhance this inner potential of the developing brain. Experimental examination of the environmental factors affecting this regeneration and remodeling process is very important. Endogenous factors, such as neurotrophic factors, which play a role in neurogenesis, migration, and differentiation of neurons, and development of neuronal circuits, are also in the center of interest. Most studies concerning the effect of positive or negative perinatal treatments focus mainly on long-term effects, and most examinations are carried out on adult animals following perinatal injuries. Less data are available on short-term effects and early neurobehavioral changes. In the past several years, we have shown how different (positive or negative) perinatal events affect the early neuronal development. Applying different tests widely used for behavioral testing, we have established a standardized testing method. This includes measuring parameters of somatic growth and facial development, appearance of basic neurological reflexes and also reflex performance, more complex motor coordination tests, and open-field and novelty-seeking tests. In the present chapter, we summarize data on early neurobehavioral development of newborn rats subjected to negative (perinatal asphyxia, hypoxia, excitotoxic injury, stress) and positive (enriched environment, neurotrophic factor treatment) stimuli during early postnatal life.

Pituitary adenylate cyclase–activating polypeptide (PACAP) is a neuropeptide having trophic and protective functions in neural tissues, including the retina. Previously, we have shown that intravitreal PACAP administration can maintain retinal structure in the animal model of retinopathy of prematurity (ROP). The purpose of this study is to examine the development of ROP in PACAP-deficient and wild-type mice to reveal the function of endogenous PACAP. Wild-type and PACAP-knockout (KO) mouse pups at postnatal day (PD) 7 were maintained at 75% oxygen for 5 consecutive days then returned to room air on PD12 to develop oxygen-induced retinopathy (OIR). On PD15, animals underwent electroretinography (ERG) to assess visual function. On PD16, eyes were harvested for either immunohistochemistry to determine the percentage of the central avascular retinal area or molecular analysis to assess angiogenesis proteins by array kit and anti-apoptotic protein kinase B (Akt) change by western blot. Retinas of PACAP-deficient OIR mice showed a greater central avascular area than that of the wild types. ERG revealed significantly decreased b-wave amplitude in PACAP KO compared to their controls. Several angiogenic proteins were upregulated due to OIR, and 11 different proteins markedly increased in PACAP-deficient mice, whereas western blot analysis revealed a reduction in Akt phosphorylation, suggesting an advanced cell death in the lack of PACAP. This is the first study to examine the endogenous effect of PACAP in the OIR model. Previously, we have shown the beneficial effect of exogenous local PACAP treatment in the rat OIR model. Together with the present findings, we suggest that PACAP could be a novel retinoprotective agent in ROP.

Background: Several studies focus on the effects of prenatal stress in adulthood. Relatively little is known about the early neurodevelopmental consequences of such experiences and their predictive value. Thus we examined the early neurobehavioral responses of offspring whose mothers were exposed to restraint stress.