Üridin ve üridin nükleotidlerinin nöroendokrin nöronlar üzerindeki aktive edici etkilerinin immünohistokimyasal olarak araştırılması
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Date
2023-12-26
Authors
Akbulut, Nursel Hasanoğlu
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Publisher
Bursa Uludağ Üniversitesi
Abstract
Tez çalışmasında intraserebroventriküler (i.s.v.) yolla uygulanan üridin ve üridin nükleotidleri olan üridin monofosfat (UMP), üridin difosfat (UDP), üridin trifosfat (UTP)'ın vazopressin, oksitosin ve nesfatin-1 nöronları üzerindeki aktive edici etkilerinin araştırılması ve bu etkide reseptör antagonistlerinin olası baskılayıcı rolünün değerlendirilmesi amaçlandı. Bu amaçla çalışmada 4-6 aylık Sprague Dawley cinsi erkek sıçanlar etken madde ve antagonist uygulamaları için 8 gruba ayrıldı (n=5/grup). Deney hayvanlarına salin (5 mikrolitre), üridin (1μmol), UMP (1μmol), UDP (1μmol), UTP (1μmol) i.s.v olarak uygulandı. Antagonist gruplarda UTP için P2Y₂ reseptör antagonist (AR-C118925XX-0,1μmol) UDP için hem P2Y₆ reseptör antagonisti (MRS2578-1μmol) ve hem P2Y₁₄ reseptör antagonisti (PPTN hidroklorür-1μmol) i.s.v. olarak etken maddelerin verilmesinden 15 dk önce uygulandı. Son enjeksiyonu takip eden 90. dakika sonunda perfüzyonla fikse edilen dokulardan alınan yüzen kesitlerde ikili indirekt immünoperoksidaz yöntemi kullanılarak vazopressin, oksitosin ve nesfatin-1 nöronlarında c-Fos işaretlemeleri yapıldı. Sonuçlar, ikili işaretlenen (aktive olan) nöron yüzdesi olarak değerlendirildi. Merkezi yolla uygulanan üridin, UMP ve UTP'nin kontrol grubu ile karşılaştırıldığında, supraoptik (SON), paraventriküler (PVN) ve periventriküler (PeV) çekirdeklerde istatistiki olarak anlamlı sayıda nesfatin-1 nöronunu aktive ettiği belirlendi. Benzer olarak SON ve PVN'de oksitosin nöronlarındaki aktivasyon artışı üridin, UMP ve UTP uygulanmasında anlamlı bulundu. Üridin ve nükleotidlerinin vazopressin nöronlarında aktive edici etkisi incelendiğinde hem SON hem de PVN'de UMP ve UTP'nin vazopressin nöronlarını salin grubuna göre anlamlı olarak aktive ettiği belirlendi. Tüm nöron gruplarında en yüksek aktivasyon artışı gösteren UTP'nin etkisi, P2Y₂ reseptör antagonist ön uygulanmasıyla baskılandığı gösterildi. İkili immünofloresans boyama ile tüm nöron tipinde P2Y₂ varlığı belirlendi. Sonuç olarak UTP'nin vazopressin, oksitosin ve nesfatin-1 nöronlarında lokalize P2Y₂ reseptörüne bağlanarak nöronları aktive ediyor olabileceğini düşündürdü.
The aim of the thesis study is to assess the effects of intracerebroventricularly (i.c.v.) adminestered uridine and uridine nucleotides including uridine monophosphate (UMP), uridine diphosphate (UDP) and uridine triphosphate (UTP) on the activation of vasopressin, oxytocin or nesfatin-1 neurons and to investigate the possible supressive effects of receptor antagonists on this activation. For this purpose, 4-6- month-old male Sprague Dawley rats were divided into 8 groups for active ingredient and antagonist applications (n=5/group). Saline (5 μl), uridine (1μmol), UMP (1μmol), UDP (1μmol), UTP (1μmol) were administered (i.c.v) to the experimental animals. In the antagonist groups, P2Y₂ receptor antagonist (AR-C118925XX, 0.1μmol) for UTP, P2Y₆ receptor antagonist (MRS2578, 1μmol) for UDP and P2Y₁₄ receptor antagonist (PPTN hydrochloride, 1μmol) were administered i.c.v. Antagonists were applied 15 minutes before the injection of active ingredients. At the end of the 90th minute following the last injection aimals were sacrified by perfusion-fixation and using the double indirect immunoperoxidase method on floating sections, c-Fos labeling was performed with vasopressin or oxytocin or nesfatin-1 labeling. The results were evaluated as the percentage of double labeled (activated) neurons. It was determined that centrally administered uridine, UMP and UTP caused activation in statistically significant number of nesfatin-1 neurons in the supraoptic (SON), paraventricular (PVN) and periventricular (PeV) nuclei when compared to the saline group. Similarly, the increase in activation of oxytocin neurons in the SON and PVN was found to be significant in the application of uridine, UMP and UTP. When the effects of uridine and its nucleotides on the activation of vasopressin neurons was examined, it was determined that UMP and UTP activated vasopressin neurons significantly in both SON and PVN when compared to the saline group. It was shown that the effect of UTP, which showed the highest increase in the number of activated neurons in all three neuron phenotypes, was suppressed by the pre-application of P2Y₂ receptor antagonist. The presence of P2Y₂ receptor expression was determined in all neuron types by double immunofluorescence staining. As a result, it was suggested that UTP may activate neurons by binding to the P2Y₂ receptor localized in vasopressin, oxytocin and nesfatin-1 neurons.
The aim of the thesis study is to assess the effects of intracerebroventricularly (i.c.v.) adminestered uridine and uridine nucleotides including uridine monophosphate (UMP), uridine diphosphate (UDP) and uridine triphosphate (UTP) on the activation of vasopressin, oxytocin or nesfatin-1 neurons and to investigate the possible supressive effects of receptor antagonists on this activation. For this purpose, 4-6- month-old male Sprague Dawley rats were divided into 8 groups for active ingredient and antagonist applications (n=5/group). Saline (5 μl), uridine (1μmol), UMP (1μmol), UDP (1μmol), UTP (1μmol) were administered (i.c.v) to the experimental animals. In the antagonist groups, P2Y₂ receptor antagonist (AR-C118925XX, 0.1μmol) for UTP, P2Y₆ receptor antagonist (MRS2578, 1μmol) for UDP and P2Y₁₄ receptor antagonist (PPTN hydrochloride, 1μmol) were administered i.c.v. Antagonists were applied 15 minutes before the injection of active ingredients. At the end of the 90th minute following the last injection aimals were sacrified by perfusion-fixation and using the double indirect immunoperoxidase method on floating sections, c-Fos labeling was performed with vasopressin or oxytocin or nesfatin-1 labeling. The results were evaluated as the percentage of double labeled (activated) neurons. It was determined that centrally administered uridine, UMP and UTP caused activation in statistically significant number of nesfatin-1 neurons in the supraoptic (SON), paraventricular (PVN) and periventricular (PeV) nuclei when compared to the saline group. Similarly, the increase in activation of oxytocin neurons in the SON and PVN was found to be significant in the application of uridine, UMP and UTP. When the effects of uridine and its nucleotides on the activation of vasopressin neurons was examined, it was determined that UMP and UTP activated vasopressin neurons significantly in both SON and PVN when compared to the saline group. It was shown that the effect of UTP, which showed the highest increase in the number of activated neurons in all three neuron phenotypes, was suppressed by the pre-application of P2Y₂ receptor antagonist. The presence of P2Y₂ receptor expression was determined in all neuron types by double immunofluorescence staining. As a result, it was suggested that UTP may activate neurons by binding to the P2Y₂ receptor localized in vasopressin, oxytocin and nesfatin-1 neurons.
Description
Keywords
Üridin, Üridin nükleotidleri, P2Y reseptörleri, İmmünohistokimya, Nöronal aktivasyon, Uridine, Uridine nucleotides, P2Y receptors, Immunohistochemistry, Neuronal activation
Citation
Akbulut, N. H. (2023). Üridin ve üridin nükleotidlerinin nöroendokrin nöronlar üzerindeki aktive edici etkilerinin immünohistokimyasal olarak araştırılması. Yayınlanmamış doktora tezi. Bursa Uludağ Üniversitesi Sağlık Bilimleri Enstitüsü.