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

PACAP is Protective Against Cellular Stress in Retinal Pigment Epithelial Cells

The integrity of the innermost, pigment epithelial layer of the retina is crucial for the photoreceptor survival and for maintaining the outer blood–retina barrier. In several ocular degenerations, such as diabetic retinopathy or macular edema, the stress caused by various harmful stimuli (hypoxia, oxidative stress, hyperosmosis) lead to severe molecular biological changes in this layer, promoting neovascularization of the retina. Pituitary adenylate cyclase activating polypeptide (PACAP) occurs throughout the whole body, including the eye. It has numerous functions in the retina, including the previously described anti-apoptotic and anti-angiogenic effects in retinal pigment epithelial cells. The aim of this present study was to investigate the influence of PACAP on different stress factors. In accordance with previous findings, PACAP significantly ameliorated the increased Hif1-α levels in hypoxic conditions. In H2O2-induced oxidative stress PACAP had an anti-apoptotic effect, it could decrease the expression of cytochrome-c and p53, while it upregulated the concentration of three antioxidants, namely SOD2, PON2 and thioredoxin. In conclusion, we provided new information on the molecular biological background of the retinoprotective effect of PACAP

Urocortin 2 treatment is protective in excitotoxic retinal degeneration

Urocortin 2 (Ucn 2) is a corticotrop releasing factor paralog peptide with many physiological functions and it has widespread distribution. There are some data on the cytoprotective effects of Ucn 2, but less is known about its neuro- and retinoprotective actions. We have previously shown that Ucn 2 is protective in ischemia-induced retinal degeneration. The aim of the present study was to examine the protective potential of Ucn 2 in monosodiumglutamate (MSG)-induced retinal degeneration by routine histology and to investigate cell-type specific effects by immunohistochemistry. Rat pups received MSG applied on postnatal days 1, 5 and 9 and Ucn 2 was injected intravitreally into one eye. Retinas were processed for histology and immunocytochemistry after 3 weeks. Immunolabeling was determined for glial fibrillary acidic protein, vesicular glutamate transporter 1, protein kinase Cα, calbindin, parvalbumin and calretinin. Retinal tissue from animals treated with MSG showed severe degeneration compared to normal retinas, but intravitreal Ucn 2 treatment resulted in a retained retinal structure both at histological and neurochemical levels: distinct inner retinal layers and rescued inner retinal cells (different types of amacrine and rod bipolar cells) could be observed. These findings support the neuroprotective function of Ucn 2 in MSG-induced retinal degeneration

MSG-indukálta retinális degeneráció és a PACAP neuroprotektív hatásának vizsgálata = MSG-induced retinal degeneration and the neuroprotective effect of PACAP

A monosodium-glutamát (MSG) egy ismert ízfokozó, amelyet a kínai konyha nagy mennyiségben adagol ételeihez. E vegyület retinakárosító hatását, valamint annak kivédésének lehetséges módszereit vizsgáltuk patkányban. Újszülött patkányokba subcutan MSG-t injektáltunk. Három hét elteltével vizsgáltuk a retinákat. Az kísérletek egy részeben a MSG injekciókkal együtt neuroprotektív szereket, főleg PACAP-ot, alkalmaztunk a degeneratív hatások kivédésére. Munkánk során a MSG-modellt optimalizáltuk és kifejlesztettünk egy ischemiás modellt is, amikor 2 hetes állatokon bilaterális carotis communis lekötést alkalmaztunk és 1 hét elteltével vizsgáltuk a retinákat. Ebben a modellben is lehetőség nyílt a protektív ágensek alkalmazására. Mindezen modellekben morfológiai, morfometriai, immuncitokémiai, és biokémiai módszerekkel vizsgáltuk a sejt- és molekuláris szintű eseményeket. Főbb eredményeink a következőkben foglalhatók össze: 1. A MSG modellben elsősorban a belső retinális rétegek károsodnak. 2. Az ischemiás modellben minden sejtrétegben találtunk károsodott sejteket. 3. A PACAP intravitreálisan adagolva mindkét modellben protektív hatású. 4. A PACAP protektív hatását a proapoptikus faktorok gátlásával és az anti-apoptotikus faktorok serkentésén keresztül éri el. 5. A védő hatás következtében a retina rétegei és sejttípusai megtartottak maradnak, a javulás funkcionális vizsgáló módszerrel (elektroretinogram) is igazolható. 6. Védő hatást gyakorol az ingergazdag környezet is. | Monosodium-glutamát (MSG) is a well-known aromatizer which is widely used in the Chinese cuisine. The retinotoxic effects of this substance and the possible neuroprotective strategies against this compound were examined in rats. Newborn rats were injected subcutaneously with MSG and their retinas were examined after three weeks. In some experiments neuroprotective agents were simulatneously applied with MSG. During the project we optimized the MSG model and also developed an ischemic retina model, in which the common carotid arteries of 2-week-old rats were ligated and the retinas were processed one week after the ligation. Also, a screen for neurorotective agents was executed. The experimental results were obtained with morphological, morphometric, immunocytochemical and biochemical methods. The main results are as follows: 1. After MSG treatment the inner retinal layers were damaged. 2. In the ischemic modell we found damaged cells in all cellular layers of the retina. 3. PACAP is neuroprotective in both models. 4. This effect of PACAP is mediated through inhibition of proapoptotic and stimulation of anti-apopototic factors. 5, Due to this protective effect, the histological layers and the cells of the retina remain relatively intact. These results are also supported by functional examinations (electroretinogram). 6. Enriched environments proved to be retinoprotective too

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