Investigation of PACAP38 and PAC1 Receptor Expression in Human Retinoblastoma and the Effect of PACAP38 Administration on Human Y-79 Retinoblastoma Cells.

Retinoblastoma represents the most prevalent malignant neoplasm affecting the eyes in childhood. The clear-cut origin of retinoblastoma has not yet been determined; however, based on experiments, it has been suggested that RB1 loss in cone photoreceptors causes retinoblastoma. Pituitary adenylate-cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide which has been shown to be affected in certain tumorous transformations, such as breast, lung, kidney, pancreatic, colon, and endocrine cancers. This study aimed to investigate potential changes in both PACAP38 and PAC1 receptor (PAC1R) expression in human retinoblastoma and the effect of PACAP38 administration on the survival of a human retinoblastoma cell line (Y-79). We analyzed human enucleation specimens removed because of retinoblastoma for PACAP38 and PAC1R immunostaining and the effect of PACAP38 on the survival of the Y-79 cell line. We described for the first time that human retinoblastoma cells from patients showed only perinuclear, dot-like immunopositivity for both PACAP38 and PAC1R, irrespective of laterality, genetic background, or histopathological features. Nanomolar (100 nM and 500 nM) PACAP38 concentrations had no effect on the viability of Y-79 cells, while micromolar (2 µM and 6 µM) PACAP38 significantly decreased tumor cell viability. These findings, along with general observations from animal studies showing that PACAP38 has strong anti-apoptotic, anti-inflammatory, and antioxidant effects on ocular tissues, together suggest that PACAP38 and its analogs are promising candidates in retinoblastoma therapy.

Protective Effects of Pituitary Adenylate-Cyclase-Activating Polypeptide on Retinal Vasculature and Molecular Responses in a Rat Model of Moderate Glaucoma.

Despite the high probability of glaucoma-related blindness, its cause is not fully understood and there is no efficient therapeutic strategy for neuroprotection. Vascular factors have been suggested to play an important role in glaucoma development and progression. Previously, we have proven the neuroprotective effects of pituitary adenylate-cyclase-activating polypeptide (PACAP) eye drops in an inducible, microbeads model in rats that is able to reproduce many clinically relevant features of human glaucoma. In the present study, we examined the potential protective effects of PACAP1-38 on the retinal vasculature and the molecular changes in hypoxia. Ocular hypertension was induced by injection of microbeads into the anterior chamber, while control rats received PBS. PACAP dissolved in vehicle (1 µg/drop) or vehicle treatment was started one day after the injections for four weeks three times a day. Retinal degeneration was assessed with optical coherence tomography (OCT), and vascular and molecular changes were assessed by immunofluorescence labeling. HIF1-α and VEGF-A protein levels were measured by Western blot. OCT images proved severe retinal degeneration in the glaucomatous group, while PACAP1-38 eye drops had a retinoprotective effect. Vascular parameters were deteriorated and molecular analysis suggested hypoxic conditions in glaucoma. PACAP treatment exerted a positive effect against these alterations. In summary, PACAP could prevent the severe damage to the retina and its vasculature induced by ocular hypertension in a microbeads model.

The Effects of a 12-Week-Long Sand Exercise Training Program on Neuromechanical and Functional Parameters in Type II Diabetic Patients with Neuropathy.

Studies have proven the effectiveness of different weight-bearing exercise interventions for diabetic patients with neuropathy; however, several adverse effects were reported using solid surfaces. Thus, in the present study, we investigated the effects of a novel sand exercise training intervention on biomechanical and functional parameters in seven diabetic patients (age = 62.7 ± 9.7 years) with neuropathy. Patients underwent a 12-week sand exercise training program, using strengthening, stretching, balance, and gait exercises. They were tested for ankle plantar- and dorsiflexion peak torque, active range of motion (ROM), timed up and go (TUG), and bilateral static balance. EMG activity of tibialis anterior (TA), gastrocnemius medialis (GM), and lateralis (GL) muscles were measured during unilateral isometric contraction in plantar- and dorsiflexion. In the intervention period, plantarflexion peak torque improved significantly (p = 0.033), while dorsiflexion torque remained unchanged. Plantar- and dorsiflexion ROM increased (p = 0.032) and (p = 0.021), respectively. EMG activity of GM (p = 0.005) and GL (p = 0.002) measured during dorsiflexion and postural sway in the balance test, as well as time to complete the TUG test, decreased significantly (p = 0.021) and (p = 0.002), respectively. No adverse effect was reported during the intervention period. We concluded that sand exercise training can be a safe and effective method to improve plantarflexion strength, ankle flexibility, and balance, which is reflected in better gait function in patients with diabetic peripheral neuropathy (DPN).

Aconitum Alkaloid Songorine Exerts Potent Gamma-Aminobutyric Acid-A Receptor Agonist Action In Vivo and Effectively Decreases Anxiety without Adverse Sedative or Psychomotor Effects in the Rat.

Songorine (SON) is a diterpenoid alkaloid from Aconitum plants. Preparations of Aconitum roots have been employed in traditional oriental herbal medicine, however, their mechanisms of action are still unclear. Since GABA-receptors are possible brain targets of SON, we investigated which subtypes of GABA-receptors contribute to the effects of SON, and how SON affects anxiety-like trait behavior and psychomotor cognitive performance of rats. First, we investigated the effects of microiontophoretically applied SON alone and combined with GABA-receptor agents picrotoxin and saclofen on neuronal firing activity in various brain areas. Next, putative anxiolytic effects of SON (1.0-3.0 mg/kg) were tested against the GABA-receptor positive allosteric modulator reference compound diazepam (1.0-5.0 mg/kg) in the elevated zero maze (EOM). Furthermore, basic cognitive effects were assessed in a rodent version of the psychomotor vigilance task (PVT). Local application of SON predominantly inhibited the firing activity of neurons. This inhibitory effect of SON was successfully blocked by GABA(A)-receptor antagonist picrotoxin but not by GABA(B)-receptor antagonist saclofen. Similar to GABA(A)-receptor positive allosteric modulator diazepam, SON increased the time spent by animals in the open quadrants of the EOM without any signs of adverse psychomotor and cognitive effects observed in the PVT. We showed that, under in vivo conditions, SON acts as a potent GABA(A)-receptor agonist and effectively decreases anxiety without observable side effects. The present findings facilitate the deeper understanding of the mechanism of action and the widespread pharmacological use of diterpene alkaloids in various CNS indications.

Well-Trained Elders Have Antioxidant Responses and an Equal Magnitude of EIMD as Young Adults.

Aim The aim of the study was to investigate acute and chronic effects of a two-week eccentric concentric, dynamometric training concerning the time-course changes of blood antioxidant parameters (total antioxidant capacity, catalase enzyme activity, thiol concentration), and to compare the adaptability of young and older muscle to this type of training. Methods Seventeen moderately trained young and older men participated in this research. Subjects performed six eccentric concentric exercise bouts during the training period and maximal voluntary isometric contraction torque, plasma CK and intensity of muscle soreness were determined before and 24 h after the first exercise. During five testing sessions (baseline, 24 h, 48 h, week 1, week 2) the level of blood antioxidants were measured. Results No significant changes were registered in total antioxidant capacity and catalase enzyme activity for any time points; furthermore, no difference was found between groups during the training period. However, total thiol concentrations measured two weeks after the first exercise bout significantly differed between the young and elderly groups. Plasma CK and the subjective intensity of soreness elevated significantly 24 h following the first training, while maximal voluntary isometric contraction torque decreased at the same time. Conclusions Our results do not support previous findings that chronic, short-term eccentric concentric training programs enhance the antioxidant defense of well-trained older and young men. This type and setting of exercise did not cause a different time course of changes in the markers of exercise-induced muscle damage (EIMD) in the studied population. Subjects may already have adapted to maintain constant levels of antioxidants and isometric torque due to their active lifestyle.

Distribution of PACAP and PAC1 Receptor in the Human Eye.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread distribution and diverse biological functions. Several studies show that PACAP has strong cytoprotective effects mediated mostly through its specific PAC1 receptor (PAC1-R) and it plays important roles in several pathological conditions. Its distribution and altered expression are known in various human tissues, but there is no descriptive data about PACAP and its receptors in the human eyebulb. Since PACAP38 is the dominant form of the naturally occurring PACAP, our aim was to investigate the distribution of PACAP38-like immunoreactivity in the human eye and to describe the presence of PAC1-R. Semiquantitative evaluation was performed after routine histology and immunohistochemical labeling on human eye sections. Our results showed high level of immunopositivity in the corneal epithelium and endothelium. Within the vascular layer, the iris and the ciliary body had strong immunopositivity for both PACAP and PAC1-R. Several layers of the retina showed immunoreactivity for PACAP and PAC1-R, but the ganglion cell layer had a special pattern in the immunolabeling. Labeling was observed in the neuropil within the optic nerve in both cases and glial cells displayed immunoreactivity for PAC1-R. In summary, our study indicates the widespread occurrence of PACAP and its specific receptor in the human eye, implying that the results from in vitro and animal studies have translational value and most probably are also present in the human eye.

Frontal Plane Neurokinematic Mechanisms Stabilizing the Knee and the Pelvis during Unilateral Countermovement Jump in Young Trained Males.

(1) The unilateral countermovement jump is commonly used to examine frontal plane kinetics during unilateral loading and to identify athletes with an increased risk of lower limb injuries. In the present study, we examined the biomechanical mechanisms of knee and pelvis stabilization during unilateral vertical jumps. (2) Healthy males performed jumps on a force plate with the dominant leg. Activity of the dominant-side gluteus medius and the contralateral-side quadratus lumborum and erector spinae muscles was recorded with surface EMG. The EMG data were normalized to the EMG activity recorded during maximal voluntary isometric hip abduction and lateral trunk flexion contractions. During jumps, the propulsive impulse was measured, and the pelvis and thigh segmental orientation angles in the frontal plane were recorded and synchronized with the EMG data. (3) The magnitude of knee valgus during the jump did not correlate with hip abduction force, but negatively correlated with gluteus medius activity. This correlation became stronger when gluteus medius activity was normalized to hip abduction force. Propulsive impulse did not correlate with any neuromechanical measurement. (4) We conclude that hip abduction force itself does not regulate the magnitude of knee valgus during unilateral jumps; rather, the gluteus medius should be highly activated to increase frontal-plane knee joint stability.

Retinoprotective Effects of PACAP Eye Drops in Microbead-Induced Glaucoma Model in Rats.

Glaucoma is associated with increased intraocular pressure (IOP), causing the apoptosis of retinal ganglion cells (RGCs) and the loss of their axons leading to blindness. Pituitary adenylate cyclase activating polypeptide (PACAP) is neuroprotective in several neural injuries, including retinopathies. The aim of this study was to investigate the effects of PACAP1-38 eye drops in a model of glaucoma. IOP was elevated bilaterally by injections of microbeads to block the aqueous humor outflow. The control groups received the same volume of saline. Animals were treated with PACAP1-38 (1 µg/drop, 3 × 1 drop/day) or vehicle for 4 weeks starting one day after the injections. Retinal morphology by histology and optical coherence tomography, function by electroretinography, and IOP changes were analyzed. Animals were sacrificed 8 weeks after the injections. Microbeads injections induced a significant increase in the IOP, while PACAP1-38 treatment lowered it to normal levels (~10 mmHg). Significant retinal degeneration and functional impairment were observed in the microbead-injected group without PACAP1-38 treatment. In the microbeads + PACAP1-38 group, the retinal morphology and functionality were close to the normal values. In summary, our results show that PACAP1-38, given in form of eye drops, is neuroprotective in glaucoma, providing the basis for potential future therapeutic administration.

Clinical Effect of End-range Maitland Mobilization in the Management of Knee Osteoarthritis - A Pilot Study.

BACKGROUND/AIM: Different manual therapy techniques and conservative therapy have been used separately for alleviation of pain and improvement of physical function in patients with knee osteoarthritis (KOA). However, no study has reported the effect of combination of these treatment modalities in the management of KOA. Our aim was to test the feasibility of the study design and to compare the effect of end-range Maitland mobilization to conservative therapy in KOA. PATIENTS AND METHODS: Fifteen patients (conservative therapy group: CG) received conservative therapy alone, fifteen patients (Maitland plus conservative therapy group: M+CG) received additionally end-range Maitland mobilization during the 3-week study period. Outcomes were pain intensity, measured with visual analogue scale (VAS) in general and during functional activities, passive range of motion (PROM) and peak muscle force during knee flexion and extension, Timed Up and Go test and 6-Minute Walk Test (6MWT). RESULTS: All outcomes improved significantly in both groups. Magnitude of changes was significantly greater in M+CG compared to CG regarding all VAS pain scores, flexion PROM of both knees, right hamstring peak muscle force and 6MWT. CONCLUSION: With few modifications, this study design seems feasible for the comparison of end-range Maitland mobilization with conservative therapy in KOA. Moreover, end-range Maitland mobilization in addition to conservative therapy appeared more effective in relief of pain and improvement of functional status than conservative therapy alone in KOA.

The Protective Effects of Endogenous PACAP in Oxygen-Induced Retinopathy.

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.

Stability Test of PACAP in Eye Drops.

PACAP is a neuropeptide with widespread distribution and diverse biological functions. It has strong cytoprotective effects mediated mainly through specific PAC1 receptors. Experimental data show protective effects of PACAP in the retina and cornea in several pathological conditions. Although intravitreal injections are a common practice in some ocular diseases, delivery of therapeutic agents in the form of eye drops would be more convenient and would lead to fewer side effects. We have previously shown that PACAP, in the form of eye drops, is able to pass through the ocular barriers and can exert retinoprotective effects. As eye drops represent a promising form of administration of PACAP in ocular diseases, it is important to investigate the stability of PACAP in solutions used in eye drops. In this study, the stability of PACAP1-27 and PACAP1-38 in eye drops was measured in four common media and a commercially available artificial tear solution at both room temperature and +4 °C. Mass spectrometry results show that the highest stability was gained with PACAP1-38 in water and 0.9% saline solution at +4 °C, representing 80-90% drug persistence after 2 weeks. PACAP1-38 in the artificial tear showed very fast degradation at room temperature, but was stable at +4 °C. In summary, PACAP1-38 has higher stability than PACAP1-27, with highest stability at +4 °C in water solution, but both peptides in each medium can be stored for relatively longer periods without significant degradation. These data can provide reference for future therapeutic use of PACAP in eye drops.

Association between Obesity and Overweight and Cardiorespiratory and Muscle Performance in Adolescents.

The high prevalence of obesity in childhood and adolescence has major public health consequences, since it is associated with various chronic diseases in the short- and long-term. The goal of our study was to examine the possible association between obesity and overweight and cardiorespiratory and muscle performance during a 4-year follow up period in adolescents. The body mass index (BMI) and physical performance of adolescents (360 girls and 348 boys) between 14-18 years of age was measured twice a year, and the possible correlation between overweight and obesity and cardiorespiratory and muscle performances were investigated. Our results revealed that cardiorespiratory performance increased significantly in boys during the 4 years (p < 0.001), but the aerobic performance of girls only showed seasonal fluctuation. Muscle performance significantly increased both in boys and girls (p < 0.001). Inverse association between obesity and cardiorespiratory and muscle performance was proved. Overweight was also inversely correlated with cardiorespiratory performance, but it demonstrated no correlation with muscle strength. Avoiding increased BMI and decreased physical fitness is essential for adolescents' health to prevent short- and long-term adverse effects.

Monitoring exercise-induced muscle damage indicators and myoelectric activity during two weeks of knee extensor exercise training in young and old men.

This study considered the effects of repeated bouts of short-term resistive exercise in old (age: 64.5±5.5 years; n = 10) and young men (age: 25.1±4.9 years; n = 10) who performed six knee extension exercise bouts over two weeks using various markers of exercise-induced muscle damage and electromyographic activity. We found that time-course changes in quadriceps isometric torque, creatine kinase activity, and muscle soreness in the two groups were similar. However, recovery in the acute torque deficit was mediated by more favourable electromyographic activity changes in the young group than in the older adults group. Muscle elastic energy storage and re-use assessed with dynamometry was selectively improved in the young group by the end of the protocol. Serum myoglobin concentration increased selectively in old group, and remained elevated with further bouts, suggesting higher sarcolemma vulnerability and less effective metabolic adaptation in the older adults, which, however, did not affect muscle contractility.

PARP Inhibitor Protects Against Chronic Hypoxia/Reoxygenation-Induced Retinal Injury by Regulation of MAPKs, HIF1α, Nrf2, and NFκB.

Purpose: In the eye, chronic hypoxia/reoxygenation (H/R) contributes to the development of a number of ocular disorders. H/R induces the production of reactive oxygen species (ROS), leading to poly(ADP-ribose) polymerase-1 (PARP1) activation that promotes inflammation, cell death, and disease progression. Here, we analyzed the protective effects of the PARP1 inhibitor olaparib in H/R-induced retina injury and investigated the signaling mechanisms involved. Methods: A rat retinal H/R model was used to detect histologic and biochemical changes in the retina. Results: H/R induced reductions in the thickness of most retinal layers, which were prevented by olaparib. Furthermore, H/R caused increased levels of Akt and glycogen synthase kinase-3ß phosphorylation, which were further increased by olaparib, contributing to retina protection. By contrast, H/R-induced c-Jun N-terminal kinase and p38 mitogen-activated protein kinases (MAPK) phosphorylation and activation were reduced by olaparib, via mitogen-activated protein kinase phosphatase 1 (MKP-1) expression. In addition, H/R-induced hypoxia-inducible factor 1α (HIF1α) levels were decreased by olaparib, which possibly contributed to reduced VEGF expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression was slightly increased by H/R and was further activated by olaparib. Nuclear factor-κB (NFκB) was also activated by H/R through phosphorylation (Ser536) and acetylation (Lys310) of the p65 subunit, although this was significantly reduced by olaparib. Conclusions: Olaparib reduced H/R-induced degenerative changes in retinal morphology. The protective mechanisms of olaparib most probably involved Nrf2 activation and ROS reduction, as well as normalization of HIF1α and related VEGF expression. In addition, olaparib reduced inflammation by NFκB dephosphorylation/inactivation, possibly via the PARP1 inhibition-MKP-1 activation-p38 MAPK inhibition pathway. PARP inhibitors represent potential therapeutics in H/R-induced retinal disease.

Retinoprotective Effects of TAT-Bound Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase Activating Polypeptide.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) belong to the same peptide family and exert a variety of biological functions. Both PACAP and VIP have protective effects in several tissues. While PACAP is known to be a stronger retinoprotective peptide, VIP has very potent anti-inflammatory effects. The need for a non-invasive therapeutic approach has emerged and PACAP has been shown to be retinoprotective when administered in the form of eye drops as well. The cell penetrating peptide TAT is composed of 11 amino acids and tagging of TAT at the C-terminus of neuropeptides PACAP/VIP can enhance the traversing ability of the peptides through the biological barriers. We hypothesized that TAT-bound PACAP and VIP could be more effective in exerting retinoprotective effects when given in eye drops, by increasing the traversing efficacy and enhancing the activation of the PAC1 receptor. Rats were subjected to bilateral carotid artery occlusion (BCCAO), and retinas were processed for histological analysis 14 days later. The efficiency of the TAT-bound peptides to reach the retina was assessed as well as their cAMP increasing ability. Our present study provides evidence, for the first time, that topically administered PACAP and VIP derivatives (PACAP-TAT and VIP-TAT) attenuate ischemic retinal degeneration via the PAC1 receptor presumably due to a multifactorial protective mechanism.

Alternative Routes of Administration of the Neuroprotective Pituitary Adenylate Cyclase Activating Polypeptide.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with diverse actions, including strong neurotrophic and neuroprotective effects. The aim of our present review is to provide a summary of the different approaches how in vivo neuroprotective effects can be achieved, emphasizing the potential translational values for future therapeutic applications. In the central nervous system, PACAP has been shown to have in vivo protective effects in models of cerebral ischemia, Parkinson's and Alzheimer's disease, Huntington chorea, traumatic brain and spinal cord injury and different retinal pathologies. PACAP passes through the bloodbrain barrier and therefore, systemic administration can affect the nervous system and lead to neuroprotective effects. This review summarizes results obtained in neuronal injury studies via local, such as intracerebral, intrathecal, intracerebroventricular, intravitreal and systemic treatments, such as intravenous, intraperitoneal and subcutaneous administration of PACAP. A few other options are summarized, like intranasal and eye drops treatments, as well as difficulties and side effects of different treatments are also discussed.

Protective Role of Endogenous PACAP in Inflammation-induced Retinal Degeneration.

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 PACAP-deficient (PACAP KO) mice provide evidence that endogenous PACAP has a 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-induced 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.

Passage through the Ocular Barriers and Beneficial Effects in Retinal Ischemia of Topical Application of PACAP1-38 in Rodents.

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.

Ocular Delivery of PACAP1-27 Protects the Retina From Ischemic Damage in Rodents.

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 µg/drop, 2 × 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.

PACAP Is Protective in a Rat Model of Retinopathy of Prematurity.

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.