Revista Cienfica, FCV-LUZ / Vol. XXXV Recibido: 08/10/2025 Aceptado: 19/01/2026 Publicado: 11/02/2026 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 6 Revista Cienfica, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Demonstraon of ovarian and brain ssue interacons as a result of smulaon of oogenesis in Migraine Modeled rats. Demostración de las interacciones del tejido Ovárico y Cerebral como resultado de la esmulación de la Ovogénesis en ratas con Migraña modelada. Berna Özdenoğlu-Kutlu* , Serpil Ünver-Saraydin ¹Sivas Cumhuriyet University, Medical Faculty, Histology&Embriyology Department, Sivas, Türkiye *Corresponding author: bernaozdenoglu@gmail.com ABSTRACT Migraine, is a primary neurological disease. Although there is no gender difference in the prepubertal period, migraine is more common in adult women than in adult men. This situaon decreases with menopause, but sll its dominance in women connues. In our study, it is aimed to determine whether follitropin alfa could have a developmental benefit on the ovarian follicles of animals in migraine model aſter creang experimental migraine model with nitroglycerin on rats. In this study, the animals were divided into a total of four groups: a control group and three experimental groups. The first experimental group animals were injected with nitroglycerin, the other groups were injected with follitropin alfa and follitropin alfa together with nitroglycerin. At the end of 21 days, euthanasia was provided with pentothal sodium. Immunohistochemical methods were applied to post-mortem brain and ovarian ssues of rats. c-Fos was used as a migraine marker, Zona Pellucida 3 was used to show changes in the zona pellucida on the ovarian surface, MMP-9 and transient receptor potenal vanilloid 1 were used to elucidate the pathogenesis of migraine. In our study, samples were evaluated by comparison with immunohistochemical staining. In animals, high c-Fos localizaon in the brain stem, high expression of Matrix metalloproteinase-9 in granular neurons, transient receptor potenal vanilloid 1 in theca layer and Zona Pellucida 3 in the zona pellucida region were detected. The expression relaonships of c-Fos, Matrix metalloproteinase-9, transient receptor potenal vanilloid 1 and Zona Pellucida 3 in the brain stem, brain frontal cortex and ovary ssues where migraine and follitropin alfa were studied together have been shown for the first me in this study. Key words: Brain; migraine; ovary; rat; c-Fos. RESUMEN La migraña es una enfermedad neurológica primaria. Si bien no existen diferencias de género en el período prepuberal, es más común en mujeres adultas que en hombres. Esta situación disminuye con la menopausia, pero persiste su prevalencia en mujeres. Nuestro estudio busca determinar si la folitropina alfa podría tener un efecto beneficioso sobre el desarrollo de los folículos ováricos de animales en un modelo de migraña, tras crear un modelo experimental de migraña con nitroglicerina en ratas. En este estudio, los animales se dividieron en un total de cuatro grupos: un grupo de control y tres grupos experimentales. Los animales del primer grupo experimental recibieron nitroglicerina; los demás grupos recibieron folitropina alfa y folitropina alfa junto con nitroglicerina. Al cabo de 21 días, se les praccó la eutanasia con pentotal sódico. Se aplicaron métodos inmunohistoquímicos a tejidos cerebrales y ováricos post mortem de ratas. Se ulizó c-Fos como marcador de migraña, zona pelúcida 3 para mostrar cambios en la zona pelúcida en la superficie ovárica, metaloproteinasa de matriz-9 y receptor de potencial transitorio vaniloide 1 para dilucidar la patogénesis de la migraña. En nuestro estudio, las muestras se evaluaron comparándolas con nción inmunohistoquímica. En animales, se detectó una alta localización de c-Fos en el tronco encefálico, una alta expresión de metaloproteinasa de matriz-9 en neuronas granulares, receptor de potencial transitorio vaniloide 1 en la capa de la teca y zona pelúcida 3 en la región de la zona pelúcida. Las relaciones de expresión de c-Fos, metaloproteinasa de matriz-9, receptor de potencial transitorio vaniloide 1 y zona pelúcida 3 en el tronco encefálico, la corteza frontal cerebral y los tejidos ováricos donde se estudiaron conjuntamente la migraña y la folitropina alfa se han demostrado por primera vez en este estudio. Palabras clave: Cerebro; migraña; ovario; rata; c-Fos https://doi.org/10.52973/rcfcv-e361819

Ovarian And Brain Tissue İnteracon İn Migraine Rats / Özdenoğlu-Kutlu et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Migraine is a disease that is triggered by various factors with not yet fully known pathology and progresses with different clinical pictures. Although there is no gender difference in the prepubertal period, migraine is more common in women than in adult men. This decreases with menopause, but its predominance in women connues [1]. Since the pathophysiology of migraine, which causes significant loss of labor force in the society, has not yet been fully explained, the drugs used for the treatment of migraine today are prophylacc and symptomac and a treatment parameter that provides complete recovery has not been developed [2]. There are available many studies showing that nitric oxide plays a very important role in the pathophysiology of migraine disease. Systemic administraon of nitroglycerin (NG), a nitric oxide donor, acvates the brain nuclei involved in nocicepve transmission. NG infusion is an approved migraine model in humans [3]. However, the experimental migraine model induced by NG infusion in free and to the rats that roam awake is shown as the closest model to the human model of migraine accepted by everyone [4]. The c-Fos protein, one of the proto-oncogenes whose synthesis increasing with pain or a noxious smulus, is a good marker for neuronal acvaon. Neuronal acvaon areas are widely used to show pain [5]. In this respect, an increase in c-Fos expression in the caudalis of the trigeminal nucleus is expected in the headache phase of migraine [6]. In this study, it was invesgated whether follitropin alpha (FA), a follicle smulang hormone, could have a developmental benefit on ovarian follicles in migraine modeled animals by inducing an experimental migraine model with NG in rats. FA belongs to the gonadotropins hormone family [7]. Gonadotropins are associated with reproducon and ferlity. In adult women, Gonal-f is used to induce ovulaon in women who are unable to ovulate and have not responded to treatment with clomiphene citrate, and to promote the development of mulple follicles in assisted reproducve technology (IVF) such as in vitro ferlizaon. Gonal-f contains a drug called ‘follitropin alfa’ [7]. Gonal-f was used in this study to determine whether migraine has any role in the relaonship between migraine and ferlity. c-Fos acvity is used as a neuronal marker in terms of showing neuronal acvaon [8]. It has been emphasized that MMP-9 is very important in the physiology and pathology of the brain. In fact, its amount is quite low in healthy brain ssue. In physiological smulaon and pathological condions, protein synthesis, gene expression and enzymac there is an increase in the level of acvity [9]. In the study conducted by Feng et al. [10], have demonstrated that transient receptor potenal vanilloid 1 (TRPV1) smulates sensory neurons involved in the transport and determinaon of pain sensaon and these channels have been shown to be highly important regulators of nocicepve and inflammatory pain. Zona pellucida glycoproteins have important roles in ferlizaon. Therefore, any deficiency or defect in zona pellucida glycoproteins may adversely affect ferlizaon [11]. It was aimed to determine the expressions of c-Fos, Matrix metalloproteinase-9 (MMP-9), TRPV1 and Zona Pellucida 3 (ZP3) in the brainstem, brain frontal cortex and ovarian ssues where migraine and FA were studied together and to show the interacons between ovarian and brain ssue as a result of oogenesis smulaon in rats with a migraine model. The objecves set forth set this study apart from similar studies. MATERIAL AND METHODS In this study, 32 8-week-old adult female Wistar albino rats (Raus norvegicus) with a body weight of 200-250 grams were used in the Experimental Animal Laboratory. The animals were fed with standard pellet feed and tap water during the experiment and then the animals were divided into a total of four groups: a control group and three experimental groups. The first experimental group animals were injected with NG, the second experimental group animals were injected with FA, and the third experimental group animals were injected with both NG and FA (Gonal-f 900 IU/1.5 mL). For immunohistochemical staining, c-Fos, ZP3, MMP-9 and TRPV1 were used, the ssues obtained were compared and evaluated and photographs were taken from the appropriate areas. For semiquantave evaluaon of ssue samples from the brain, brainstem and ovary, 5 preparaons per group were scoring was done by selecng. A semi-quantave scoring method was applied to evaluate staining intensity and distribuon. Specifically, three independent observers visually assessed the histochemical intensity in each structure, and the average of their scores was used to describe the final expression levels. Immunohistochemical staining Serial 3 μm secons from ssue samples taken from paraffin blocks were incubated in an oven overnight, and the following steps were applied. Deparaffinizaon was performed in xylol. Subsequently, the samples were incubated in 100 %, 95 %, 80 %, and 70 % alcohol series for 2 minutes (min) each, followed by a 5-min rinse in dislled water. Hydrogen peroxide was added and allowed to stand at room temperature for 10 min. Sodium citrate buffer (pH = 6) was used for the boiling soluon, and the samples were boiled in the microwave for a total of 10 min. The edges of the ssue samples were outlined with a Pap pen (Daido Sangyo Co., Ltd. Tokyo, Japan). They were then washed in dislled water. To prevent nonspecific transfer of immunoglobulin, the secons were incubated in SuperBlock (Sky Tech Lab, USA) soluon at room temperature for approximately 30 min. Tissue samples were incubated with primary anbodies at 4 °C in a humid, dark environment overnight. 2 of 6

Revista Cienfica, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico The primary anbody used in this study was MMP-7, and the anbody diluon rao was 1:100. The primary anbody (ab5706, Abcam) was diluted with anbody diluent reagent (Invitrogen, USA) at pH 7.4. They were washed with dislled water. Aſter 20 min of Streptavidin/Bion protein binding, the samples were finally washed with dislled water. Counterstaining was performed with hematoxylin for 1 min and then washed. Coverslip medium was used to cover the ssues, and the ssues were then covered. Semiquantave Assessment All secons were examined by two independent observers, and the ovary ssues were evaluated according to the degree of anbody staining: no staining was considered negave (-), mild staining was considered (+), moderate staining was considered (++), strong staining was considered (+++), and very strong staining was considered (++++). RESULTS AND DISCUSSION In nitroglycerin group animals, very intense c-Fos expression was observed in granular neurons in the Trigeminal Nucleus Caudalis (TNC) region of the brain stem, while localizaon was not observed in control group animals (FIG. 1). FIGURE 1. C-Fos immunolocalizaon of the (A, B) control group and (C, D) nitroglycerin group in the brain stem. A) x40 scale bar: 20 µm, B) x100 scale bar: 10 µm, C) x40 scale bar: 20 µm D) x100 scale bar: 10 µm. Granular neurons (black arrow). In the control group, non-localized MMP-9 was expressed in the cerebral cortex region, but mostly in granular and occasionally motor neurons in the lamina granularis interna and externa regions in the experimental groups. MMP-9 was localized especially in the peripheral parts of granular neurons (FIG. 2). FIGURE 2. MMP-9 immunolocalizaon of (A, B) control group and (C) nitroglycerin group and (D) nitroglycerin + follitropin alpha group in the brain. A) x40 scale bar: 20 µm, B) x100 scale bar: 10 µm, C) x100 scale bar: 10 µm D) x100 scale bar: 10 µm. Granular neurons (black arrow). Transient receptor potenal vanilloid 1, showed intense expression, especially in the NG group. Although TRPV1 was strongly observed in the cytoplasm of pyramidal neurons, its density was quite evident in both large and small motor neurons, but TRPV1 localizaon was not observed in granular neurons. TRPV1 localizaon was moderate in granular neurons in the FA + NG group. TRPV1 expression was not observed in the FA group (FIG. 3). FIGURE 3. MMP-9 immunolocalizaon of (A, B) nitroglycerin group, (C) nitroglycerin + follitropin alpha group, and (D) follitropin alpha group in the brain. A) x40 scale bar: 20 µm, B) x100 scale bar: 10 µm, C) x100 scale bar: 10 µm D) x40 scale bar: 20 µm. Motor and granular neurons (black arrow). In the ovary, MMP-9 was observed in the most prominent corpus luteum, cytoplasmic areas of granulosa lutein and theca lutein cells in the control group, while MMP-9 localizaon was observed in the ovarian epithelium and primordial follicle epithelium. When the ovarian samples taken from the NG 3 of 6

Ovarian And Brain Tissue İnteracon İn Migraine Rats / Özdenoğlu-Kutlu et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico injecon group were examined, MMP-9 staining was generally weak. MMP-9 expression was significantly higher in the FA group compared to the control and NG groups. In the FA + NG group, a very severe MMP-9 staining was observed in the medulla region and granulosa lutein and theca lutein cells (FIG. 4). FIGURE 4. MMP-9 immunolocalizaon of (A) control group, (B) nitroglycerin group, (C) follitropin alpha group and (D) nitroglycerin + follitropin alpha group in the ovary. A) x40 scale bar: 20 µm, B) x40 scale bar: 20 µm, C) x40 scale bar: 20 µm D) x100 scale bar: 10 µm. Theca lutein cells (black arrow), primordial follicle (curved black arrow), germinal epithelium (yellow arrow with black stripes), granulosa lutein cells (yellow, blue arrow). Transient receptor potenal vanilloid 1 immunolocalizaon appeared more prominent in the ovaries of the FA and FA + NG groups compared to the control and NG groups. High level of TRPV1 expression was observed in the medulla layer, corpus luteum cells, granulosa lutein and theca lutein cells, and intermediate connecve ssue cells (FIG. 5). FIGURE 5. TRPV1 immunolocalizaon in the ovary (A) control group, (B) nitroglycerin group, (C) follitropin alpha group and (D) nitroglycerin + follitropin alpha group. A) x40 scale bar: 20 µm, B) x40 scale bar: 20 µm, C) x40 scale bar: 20 µm D) x40 scale bar: 20 µm. Theca lutein cells (black arrow), granulosa lutein cells (red arrow with black stripes), antrum (black star), secondary follicle (red star), theca interna (black arrow with green stripe), theca externa (black arrow with orange stripe), connecve ssue areas (ring). The zona pellucida 3 was intensely expressed in the zona pellucida regions of the ovarian follicles, especially in the secondary follicles (FIG. 6). FIGURE 6. ZP3 immunolocalizaon in the ovary, (A) control group, (B) nitroglycerin group, (C) follitropin alpha group and (D) nitroglycerin + follitropin alpha group. A) x40 scale bar: 20 µm, B) x40 scale bar: 20 µm, C) x40 scale bar: 20 µm D) x40 scale bar: 20 µm. Zona pellucida (black arrow). Many studies have been conducted to elucidate the pathophysiology of migraine, however the exact mechanism remains unclear due to the complex nature of migraine pain. Systemic administraon of NG, a nitric oxide donor, brain nuclei are acvated involved in nocicepve transmission [12 , 13 , 14]. In this study, we think that the high localizaon of c-Fos in the TNC region in animals in which we created an experimental migraine model compared to normal ssues may be due to the increased expression of Fos proteins by growth factors and c-Fos gene, mitogens and many extracellular factors and the relaonship between this increase and very important biological processes such as proliferaon, differenaon, growth and apoptosis occurring in the cell. In addion, since c-Fos acvity is used as a neuronal marker in terms of showing neuronal acvaon [13], it is possible to say that the significant difference in expression and its higher intensity in the experimental group may be due to NG. FAis a follicle-smulang hormone belonging to the gonadotropins hormone family. In adult women who are unable to ovulate and have not responded to treatment with clomiphene citrate, Gonal-f is used to promote the development of mulple follicles in IVF such as in vitro ferlizaon, which is used to induce ovulaon [14]. In this study, c-Fos localizaon decreased in the brainstem samples of the FA group, especially in the NG group, suggesng that c-Fos gene is one of the first genes may be due to the fact that the expression and accumulaon of this core protein in the cell can be seen without any effect [15], and it can also accept it as an indicaon that FA that it does not exert an effect externally on pain. It has been emphasized that MMP-9 is very important in the physiology and pathology of the brain. In fact, its amount is quite low in healthy brain ssue. 4 of 6

Revista Cienfica, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico In physiological smulaon and pathological condions, protein synthesis, gene expression and enzymac acvity there is an increase in the level [9]. The absence of MMP-9 localizaon in the brain ssue of the control group in this study may be due to the very low amount of MMP-9 in the healthy brain ssue. Studies have shown that cell death occurs in the extracellular matrix area of neurons as a result of MMP-9 disrupon [16], and that MMP-9 expression may occur in ischemic neurons due to apoptosis, one of the important mechanisms of programmed cell death [17]. In this study, MMP-9 expression occurred in the periphery of the cytoplasmic areas of granular neurons and in certain accompanying extracellular matrix areas in the experimental group in the NG-induced migraine model. It is conceivable that the intense MMP-9 expression in granular neurons may be triggered by migraine, in line with the possibility that apoptosis may have occurred in granular neurons, especially in cells close to their peripheral regions, since MMP-9 can be expressed in cells undergoing apoptosis. In the ovary, MMP-9 was observed intensely among the staining paerns, especially in the granulosa lutein and teka lutein cells of the control group corpus luetum. We think that NG may have affected Follicle-smulang hormone Nitric oxide synthase levels and suppressed folliculogenesis. Therefore, MMP-9, whose expression increases with the progression of follicular development, may be one of the proteins that can be used to determine follicular development in migraine ssues. In addion, the fact that MMP-9 acvity was predominantly observed in intersal glandular cells and luteal ssue due to the known reproducve effect of FA, which we used in this study, and the fact that hormonal differences may cause changes in localizaon levels, in light of the studies menoned above, clarifies that the difference in MMP-9 staining is seen in the intersal cells of the follicle, which are open to hormonal interacons. However, the fact that localizaon was predominantly observed in the corpus luteum in the FA + NG group may be due to the fact that female sex hormones are effecve in migraine pathophysiology [17] and FA had an increasing effect on MMP-9 expression in the NG-induced migraine model. It is also known that TRPV1 is an effecve control mechanism in the iniaon of various pathological phenomena such as pain sensaon [18], 19] and is effecve in the development of pathological condions such as inflammaon, excessive pain sensivity and visceral hyperreflexia [20 , 21]. In the experimental group with migraine, TRPV1 localizaon in pyramidal neurons was observed intensely. This is similar to the informaon that TRPV1 increases in migraine disease states and is localized in neurons. Since it is also known that TRPV1 triggers pain and inflammaon in the central nervous system, it is an expected result that V1 shows a different course from the control group with the effect of NG. The localizaon of TRPV1 in pyramidal neurons in the brain ssue of migraine group animals and the fact that the study showed a difference compared to the control group supports that the study is in the right direcon. Therefore, in line with the findings obtained, the idea that TRPV1 can be used in studies to determine pain is supported and brings to mind the idea that it may be one of the determinant proteins of migraine disease. FA group samples did not show TRPV1 expression in brain ssue. When animals given FA together with NG were compared with animals that received only FA injecon, a significant difference was found in terms of c-Fos expression in granular neurons Although Transient Receptor Potenal channels are not selecve against caons, they are known to be highly sensive to calcium (Ca+2). In this study, there is no localizaon due to the fact that FA does not have a NG-like pain-inducing effect and possibly interferes with Ca+2 influx or FA suppresses the excitatory pathway of TRPV1. It is noteworthy that the expression of TRPV1 is more intense in the teca interna and teca externa layer of terary follicles, intermediate connecve ssue, granulosa cells and cytoplasmic areas of cells. In the realizaon of TRPV1 expression, it can be said that funconal informaon may have been received through neurons and sent to hypothalamic centers via receptors located around the follicle, or TRPV1 acvity may have occurred through sensory nerves [22]. In the absence of ZP3, one of the major components of the zona pellucida, folliculogenesis connues in mice, but these mice are inferle because oocytes adhere to the oviducts aſter ovulaon. Zona pellucida glycoproteins have important roles in ferlizaon. ZP3 glycoprotein acts as the primary receptor in the acrosome reacon. Therefore, any deficiency or defect in zona pellucida glycoproteins negavely affects ferlizaon [23]. The observaon of ZP3 expression in mullayered primary, secondary and terary follicles in ovarian control group samples indicates the presence of ZP3 protein in the zona pellucida areas of follicles. ZP3 protein is a protein capable of recognizing carbohydrate domains [24]. The reason for the intense localizaon of ZP3 in the NG group in this study is that NG also recognizes carbohydrate binding sites like ZP3, and the fact that no localizaon was observed in the terary follicle in the FA + NG group suggests that NG may have disrupted the funconal aspect of ZP3 together with FA. Several differences were found between the expressions. The results of this study support the differenal immunolocalizaon of c-Fos, MMP-9, TRPV1 and ZP3 proteins in rat ovarian and brain ssues, their widespread presence in these ssues and their important roles in migraine and FA. c-Fos, MMP-9 and TRPV1 may have important roles in the pathogenesis of migraine, since the physiopathology of pain is quite complex, determining the cause of pain, It is thought that TRPV1 and MMP-9 may have roles in finding the right treatment approaches and maintaining the treatments effecvely, MMP-9 and ZP3 may have roles in the results that can be obtained by comparing migraine with hormonal level differences. CONCLUSION This study, demonstrated for the first me the relaonship between c-Fos, MMP-9, TRPV1 and ZP3 expression in brainstem, brain frontal cortex and ovarian ssues in which migraine and FA were studied together. MMP-9, TRPV1 and ZP3 proteins can be used to show the relaonship between female sex hormones and migraine. 5 of 6

Ovarian And Brain Tissue İnteracon İn Migraine Rats / Özdenoğlu-Kutlu et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico In addion, it has been shown that experimental migraine can be induced by showing c-Fos expression in the brain stem as a result of administraon of NG (10 mg/kg) to rats, and in brain and ovarian ssue samples taken from animals as a result of administraon of Gonal-F to rats with a total of 4 IU. ACKNOWLEDGEMENT This work was supported by Sivas-Cumhuriyet University Scienfic Research Project Unit (No: T-2021-799) Conflict of Interest There is no conflict of interest between the authors BIBLIOGRAPHIC REFERENCES [1] Lisicki M, Schoenen J. Metabolic treatments of migraine. Expert. Rev. Neurother. [Internet]. 2020; 20(3):295-302 doi: hps://doi.org/gk7wrw [2] Goadsby P, Holland P, Marns-Oliveira M, Hoffmann J, Schankin C, Akerman S. 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