Synergistic neuroprotective action of prolactin and 17β-estradiol on kainic acid-induced hippocampal injury and long-term memory deficit in ovariectomized rats.

Synergistic neuroprotective action of prolactin and 17β-estradiol on kainic acid-induced hippocampal injury and long-term memory deficit in ovariectomized rats. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synergistic neuroprotective action of prolactin and 17β-estradiol on kainic acid-induced hippocampal injury and long-term memory deficit in ovariectomized rats. Karen De la Torre, Marco Antonio Cerbón, Gladys Molina-Salinas, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3409192/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Purpose The neuroprotective actions of the ovarian hormone 17β-estradiol (E2) against different brain lesions have been continuously confirmed in a variety of models including kainic acid (KA) lesions. In the same line, the pituitary hormone prolactin (PRL), traditionally associated to lactogenesis, has recently been linked to a great diversity of functions, including neurogenesis, neuroprotection, and cognitive processes. While the mechanisms of actions of E2 regarding its neuroprotective and behavioral effects have been extensively explored, the molecular mechanisms of PRL related to these roles remain under investigation. The aim of the current study was to explore if the simultaneous administration of PRL and a low dose of E2 prevents the KA-induced cognitive deficit and if this action is associated to changes in hippocampal neuronal density. Methods Ovariectomized (OVX) rats were treated with saline, PRL and/or E2 in the presence or absence of KA. Neuroprotection was assessed by Nissl staining and neuron counting. Evaluation of memory was carried out by means of the novel object recognition test (NOR). Results These findings indicate that both PRL and E2 prevent short- and long-term memory deficits in lesioned animals. In addition, both hormones exert neuroprotection against KA-induced excitotoxicity in the hippocampus. Interestingly, the combined hormonal treatment was superior at improving the behavioral performance of rats in the NOR and neuronal survival than either treatment administered separately. Conclusion Taken together, these results suggest that these hormones act in different ways at the hippocampus to produce their behavioral, proliferative, and neuroprotective effects. estradiol prolactin kainic acid recognition memory hippocampus Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The neuroprotective actions of the ovarian hormone 17β-estradiol (E2), also synthesized in the brain, against diverse injuries has been consistently confirmed in a variety of models including: β-amyloid toxicity, kainic acid (KA) and quinolinic acid lesions, among others [ 1 , 2 ]. These preclinical results have been found in brain regions closely related to cognitive functions including the hippocampus, amygdala, and the prefrontal cortex. In this line some experiments, mainly carried out in rodents, have shown that estrogens enhance cognitive functions [ 4 , 5 ] mostly through their effects in the hippocampus. These E2-procognitive actions have been observed in female rodents at different ages and hormonal conditions: adult and middle-aged female rats, ovariectomized (OVX) and reproductively senescent female rats [ 3 , 6 ] The pituitary hormone prolactin (PRL) has traditionally been associated to lactogenesis but over the last years it has been related to more than three hundred functions in the organism including neurogenesis, neuroprotection, and cognition [ 7 ]. While the literature about the pro-cognitive effects of E2 is extensive (see Taxier et al., [ 8 ] for a review), reports regarding the actions of PRL on learning and memory are scarce and the results of these reports are controversial. For instance, hyperprolactinemic male rats show impaired object recognition without modifications in spatial learning [ 9 ], while the lack of PRL receptors in both female and male mice results in learning and memory deficits [ 10 ]. One more recent study reported that this hormone can prevent the KA-induced cognitive deficits in OVX rats evaluated in the novel object recognition test (NOR) [ 11 ]. Furthermore, from clinical experiments, it has been observed that older men having high PRL levels show low cognitive performance in verbal and working memory [ 12 ], which is in line with the observation that cabergoline, a dopaminergic agonist able to block the PRL secretion, produces an improvement in cognitive abilities in hyperprolactinemic people [ 13 ]. As known, the insult produced by KA on hippocampal neurons results in a serious impairment of different types of memory [ 14 ]. Since PRL seems to exert its neuroprotective actions independently of the ovarian hormones [ 15 ], we used KA-lesioned OVX rats to assess whether combining PRL and E2 administration produce a synergic effect on short- and long-term memory (STM and LTM, respectively). Thus, a low dose of E2 in combination with a standard dose of PRL was essayed in KA-lesioned animals tested in the NOR. We also sought to evaluate the effect of these treatments on the neuronal density in CA1 and CA3 hippocampal subregions and whether they were correlated to our behavioral data. Material and methods A total of 70 adult female Wistar rats (200–220 g) were obtained from the animal facility of the Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico. All experimental procedures were performed in accordance with the guidelines and standards established by the Ethics Committee of the Faculty of Medicine, UNAM, Mexico City, project registration number FM/DI/046/2018 and by the Internal Committee for Use and Care of Laboratory Animals No. 006/CIC/2018. Animal care was carried out according to the “International Guiding Principles for Biomedical Research Involving Animals”, Council for International Organizations of Medical Sciences, 2012. Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines were also followed. Efforts were taken to minimize animals’ suffering throughout all experimental procedures. Rats were housed in a vivarium under controlled temperature (21 ± 1°C) and relative humidity (50 ± 10%) under a 12 h light/dark cycle (lights-on at 8:00 h). Water and food were available ad libitum . Three weeks before the start of experimental procedures, animals from all groups were OVX under 2,2,2-tribromoethanol anesthesia (0.2 g/kg, i.p.). Immediately after surgery, rats were placed on a warm platform until their complete recovery and, after checking for their well-being, they were housed in their standard home cages. Three weeks later, all animals were randomly assigned to seven groups: VEH, E2, PRL, KA, KA + E2, KA + PRL and KA + E2 + PRL (see below). The first 3 groups of rats were treated for 3 days with a daily dose of either vehicles (0.1 M PBS, i.p. w/o 0.1 mL oil, s.c.; VEH group), ovine prolactin (100 µg/rat, i.p.; PRL group), or 17β-estradiol (10 µg/rat s.c.; E2 group). A fourth group received a single dose of kainic acid (7 mg/kg i.p.) on the second day (KA group). The last 3 groups were treated with either E2, PRL or both, as described above, and, in addition, received KA on the second day. All reagents were purchased from Sigma-Aldrich, MO, USA. One hour after the end of the treatments, animals were evaluated in the NOR; this paradigm is a cortical and hippocampus-dependent memory test, which is useful for evaluating the ability of rodents to recognize previously encountered objects after short (1 h) or longer times (24 h) following initial exposure [ 16 , 17 ]. This initial exposure session (sample phase) consists of an open-field arena (30×30×15 cm) with two identical objects (F + F ́) located in opposite and symmetrical corners which rats are made to explore for 5 min. After this first session, animals were tested again 1 h (STM) and 24 h (LTM) in the same arena, with the exception that one of the now familiar objects (F) was replaced by a novel one (N). All sessions were videotaped for later scoring by a single observer, who was blind to the treatment conditions. Data are presented as the discrimination index (DI), which expresses the difference between the time spent exploring the F and N objects [time spent with N/(time spent with N + time spent with F) X 100] [18]. After completing the behavioral test, 3 females from each experimental group were randomly chosen, deeply anesthetized with an overdose of pentobarbital (200 mg/kg, i.p) and perfused transcardially with 250 ml of 0.9% saline followed by 250 ml of 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in sodium phosphate buffer (PBS) (pH 7.4, 10°C). Brains were removed and immediately cryoprotected in 30% sucrose-PBS solution for 2–3 weeks at 4°C. Coronal sections (20 µm) were cut through the dorsal hippocampus with a cryostat (Leica Microsystems Inc. IL, USA). A total of six 20µM sections, spanning the whole anteroposterior length of the hippocampi were obtained for each animal. To have a better representation of the regions analyzed, one series of six cuts were eliminated, while six cuts were collected and mounted on slides. After Nissl staining, microscopic images within the CA1 or CA3 subfields of the dorsal hippocampus were obtained with a digital camera attached to a Nikon microscope (Nikon, Tokyo, Japan), and images were analysed using IMAGEJ, version 1.41 (NIH, Bethesda MD, USA). To avoid false positives on the identification of neurons, the presence of a distinguishable nucleus was considered the main criterion for identification at x20 magnification. For both CA1 and CA3, counts were restricted to the region delimited by the anteroposterior coordinates − 2.5 to -4.5 mm relative to bregma [ 19 ] and were performed blind as to the experimental condition. All image analysis was carried out using ImageJ software (ImageJ, NIH. MD, USA). Data from all groups were tested for normal distribution by the Shapiro-Wilk test; homoscedasticity was also assessed. Behavioral data and neuronal density were analyzed by one-way ANOVAs (ordinary or Brown-Forsythe, accordingly) followed, when indicated, by multiple comparisons with post hoc Tukey tests. In all cases, a p value < 0.05 was considered significant. Results Figure 1 shows the average DIs from the sample phase (exploration of two identical objects) of the OVX rats treated with E2 and/or PR previously lesioned with KA. As expected at this acquisition phase of the NOR, performance was similar in all groups (F (6,26.5) = 0.79; p = 0.59). One hour after the acquisition phase, these same rats were evaluated for STM. A One-Way ANOVA comparing the DIs of rats from every group unveiled a statistically significant effect, (F (6,55) = 5.9; p <0.0001) with Tukey post-hoc tests showing that only the KA group differed from our control VEH group (p < 0.001). Noteworthy, KA-lesioned animals treated with either E2, PRL or both did not differ from controls, suggesting a protecting effect of any of these hormonal treatments on STM. Rats were next tested 24 hours later to assess LTM. Here again, a One-Way ANOVA unveiled significant differences among groups (F (6,55) = 5.42, p < 0.001). Like STM, the LTM of the KA group appeared impaired when compared to the VEH, although in this case it did not reach significance (p = 0.07). However, both KA-lesioned E2-treated and PRL-treated rats showed better LTM performance compared to their untreated KA-lesioned counterparts (DIs = 0.69 ± 0.04 and 0.72 ± 0.03 for E2 + KA and PRL + KA groups, respectively versus 0.54 ± 0.05 for the KA group, p < 0.05 in both cases). Furthermore, lesioned rats treated with the hormonal combination showed a much more robust rescuing effect on their LTM performance with average DIs of 0.82 ± 0.03 versus 0.54 ± 0.05 for the KA + E2 + PRL and KA groups, respectively (p < 0.0001). We next explored whether our results showing rescuing effects of our hormonal treatments on the NOR could be linked to neuroprotective effects in the hippocampus, a region involved in object recognition. Representative images of neurons from CA1 and CA3 hippocampal regions (Figs. 2 and 3 ) reveal marked differences in neuronal density among the different experimental conditions. Data from morphometric analysis showed significant differences among groups in both CA1 and CA3 regions (CA1: F (6,14) = 89.47, p <0.0001; CA3: F (6,14) = 252.1, p <0.0001). Post hoc Tukey tests revealed a significant loss of neurons after kainic treatment in both CA1 and CA3 regions (p < 0.0001 in both cases), which was prevented by the independent injection of E2 or PRL. Surprisingly, and only observed in lesioned animals, the combination of these hormones resulted in an increase of neuronal density over the control (VEH) group in both regions (p < 0.0001, Fig. 4 ). Discussion The main findings derived from this work are: 1) E2 or PRL administration prevented the KA-induced cognitive deficit in the NOR; 2) the combination of E2 plus PRL in KA-lesioned animals resulted in an improvement of long-term memory over those groups treated only with E2 or PRL, which was accompanied by 3) an increase of neurons density at CA1 and CA3 regions. As mentioned previously, the pro-cognitive and neuroprotective actions of E2 have been deeply explored in a variety of animal models. For instance, it has been established that it promotes synaptic plasticity of hippocampal neurons [ 23 ], attenuates apoptosis and KA-induced excitotoxicity [ 24 , 25 ], increases spinogenesis [ 26 ] and improves several types of memory, specifically long-term memory [ 3 , 4 , 27 ]. Similarly, although research on the role of PRL in cognition and neuroplasticity is rather scarce, it has been recently reported that PRL, like E2, facilitates axogenesis, synaptic plasticity, dendrite growth and neurotrophic factors synthesis in the hippocampus [ 28 , 29 ]. Although the present findings appear to point to a pro-cognitive effects of PRL, its role in this regard remains controversial. Some authors have found that high levels of PRL are associated to poor performance in several memory tasks. For instance, in male rats, hyperprolactinemia was shown to impair object recognition and declarative memory but not spatial learning [ 9 ]. By contrast, the low levels of this hormone observed in PRL null mice, were related to spatial learning deficit, which could be rescued by chronic (28 days) infusion of PRL into the hippocampus [ 30 ]. On a different level, lactation seems to be associated to neuroprotection and cognitive improvement in rats [ 31 , 32 ]. In this sense, recent research from our group has shown that the number of pregnancies in these animals was related to an increase in dendritic spines of neurons from prefrontal cortex and CA1 hippocampal subfield [ 33 ], along with improved performance in the Morris Water-Maze task [ 34 ]. Additionally, other authors have found a close correlation between poor recognition memory and low spine density in both PFC and hippocampus from ovariectomized rats [ 35 ]. These studies suggest that the lack of PRL may be correlated with impaired learning and memory and supports the role for this hormone as both a neuroprotective and pro-cognitive agent. Our results showing that PRL prevents excitotoxic damage and improves object-recognition memory are in line with the beneficial effects previously reported for this hormone (for a review see Duc Nguyen et al., [ 36 ]). The neuroprotective and proliferative actions of E2 have been explained considering its antioxidant activity, its stimulation of intracellular and membranal E2 receptors and its effects on different growth factors and neuronal signaling pathways, among others [ 37 – 39 ]. Therefore, E2 has been shown to reduce inflammation, apopotosis, gliosis, and induces neurogenesis. In this regard, E2-induced cell proliferation in the hippocampus seems to be enhanced by ischemic processes, epilepsy, stress, kainic acid, etc. [ 40 , 41 ]. However, it is known that neuronal damage itself triggers cell proliferation processes. For instance, Chamma et al., [ 42 ] showed that low doses of KA into the anterior thalamic nucleus boost proliferation in the dentate gyrus, an effect mediated by thalamo-hippocampal connections. Furthermore, it has been reported that KA-induced epileptic seizures are followed by enhanced neurogenesis [ 43 ], while KA lesions in neonatal rats induce cell proliferation in CA1 and CA3 hippocampal subfields [ 44 ]. One of the most intriguing findings was the fact that a low dose of E2 administered daily for three days, attenuated KA-induced lesion at the hippocampus. Previously we reported a similar effect injecting a high dose of this hormone (100 ug/rat) [ 20 ]. The data reported herein suggest that low doses of E2 are sufficient to promote neuroprotection and proliferative effects when combined with PRL. In line with this, there are reports where even physiological doses of E2 protect neurons against several harmful agents [ 21 ] and increase neurogenesis in the dentate gyrus [ 22 ]. On the other hand, the pro-cognitive and proliferative actions of PRL could be linked to the fact that it facilitates axogenesis, dendrite growth and neurotrophic factors synthesis, which seem to be associated with hippocampal dependent memory [ 28 ]. Previously we reported that in OVX rats, this hormone prevents the PRL receptors reduction induced by KA and increases the vesicular glutamate transporter 1 (VGLUT1) as a long-term bounce response to the lesion by KA in CA1 subfield [ 3 ]. Accordingly, subchronic (7 days) administration of PRL increases the number of hippocampal neurosphere cells in the adult mouse, both in vitro and in vivo [ 10 ]. E2 and PRL activate different biochemical pathways to mitigate neural damage and induce cell proliferation. Thus, it is possible that the long-term memory recovery observed in lesioned rats after the combined treatment, together with the cell density increase observed in CA1 and CA3 hippocampal subfields, are due to synergistic effects of both hormones. The fact that an effect was observed only in lesioned animals is in line with Turner et al., [ 45 ] who reported that a high dose of PRL rescues adult hippocampal neurogenesis during chronic stress. These findings open the possibility of implementing new substitution therapies based not only on the combination of E2 and progesterone but also including PRL. To our knowledge, this is the first report about the improvement at long-term of DI together with cellular increase, over the control group, produced by the combined E2-PRL treatment. We propose that this effect is due to different mechanisms of action for E2 and PRL. However, further experiments are needed to test this hypothesis. Conclusions The present study shows, for the first time, the improvement of long-term memory along with the increase of hippocampal cell density produced by the combined treatment of E2-PRL. We propose that this effect is due to a synergistic action of these hormones through different mechanisms. However, further experiments are needed to test this hypothesis. Declarations Acknowledgments This study was partially supported by CONACYT (286446), COFAA, SIP-IPN and DGAPA PAPIIT UNAM grant numbers IN224019 to GRR and IN227123 to JPM. Disclosure of potential conflicts of interest. The authors have no conflicts of interest to declare. Research involving Human Participants and/or Animals. All experimental procedures were performed in accordance with the guidelines and standards established by the Ethics Committee of the Faculty of Medicine, UNAM, Mexico City, project registration number FM/DI/046/2018 and by the Internal Committee for Use and Care of Laboratory Animals No. 006/CIC/2018. 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The Journal of Neuroscience 23:1742–1749. https://doi.org/10.1523/JNEUROSCI.23-05-01742.2003 Torner L, Karg S, Blume A, et al (2009) Prolactin Prevents Chronic Stress-Induced Decrease of Adult Hippocampal Neurogenesis and Promotes Neuronal Fate. The Journal of Neuroscience 29:1826–1833. https://doi.org/10.1523/JNEUROSCI.3178-08.2009 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Minor revisions 17 Jan, 2024 Reviewers agreed at journal 22 Nov, 2023 Reviewers invited by journal 17 Oct, 2023 Editor assigned by journal 13 Oct, 2023 First submitted to journal 12 Oct, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3409192","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":240810682,"identity":"eee41faa-32db-4d82-bf8c-a0ef66a317c2","order_by":0,"name":"Karen De la Torre","email":"","orcid":"","institution":"Instituto Politecnico Nacional","correspondingAuthor":false,"prefix":"","firstName":"Karen","middleName":"De la","lastName":"Torre","suffix":""},{"id":240810683,"identity":"5be91341-ae8e-498e-a3f6-674918c9f07d","order_by":1,"name":"Marco Antonio Cerbón","email":"","orcid":"","institution":"Universidad Nacional Autonoma de Mexico Facultad de Quimica","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"Antonio","lastName":"Cerbón","suffix":""},{"id":240810684,"identity":"c35271c0-7027-4452-85ec-227a3058fb60","order_by":2,"name":"Gladys Molina-Salinas","email":"","orcid":"","institution":"Universidad Nacional Autonoma de Mexico","correspondingAuthor":false,"prefix":"","firstName":"Gladys","middleName":"","lastName":"Molina-Salinas","suffix":""},{"id":240810685,"identity":"2b1959a9-244e-4d1e-a949-86869f63deed","order_by":3,"name":"José Eduardo Suárez-Santiago","email":"","orcid":"","institution":"Universidad Autonoma de Chiapas","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Eduardo","lastName":"Suárez-Santiago","suffix":""},{"id":240810686,"identity":"da6053ce-b866-48e1-8ad1-808a821ed130","order_by":4,"name":"Jean Pascal Morin","email":"","orcid":"","institution":"Universidad Nacional Autonoma de Mexico","correspondingAuthor":false,"prefix":"","firstName":"Jean","middleName":"Pascal","lastName":"Morin","suffix":""},{"id":240810687,"identity":"aa842cb4-0a08-4797-9049-d941b6f75e90","order_by":5,"name":"Gabriel Roldán-Roldán","email":"","orcid":"","institution":"Universidad Nacional Autonoma de Mexico","correspondingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"","lastName":"Roldán-Roldán","suffix":""},{"id":240810688,"identity":"e7cdfb3d-506e-45e2-b7b7-372489f6460f","order_by":6,"name":"Ofir Picazo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYDACZsYHDB8qbBLAnIQCorQwGzDOOJOWwMAG0mJAnDUGzLxthyFaGIjRYt7OzPiYt+18Hr98d+KHBwYM8vxiB/BrkTnMzGw459ztYsk23s0SQIcZzpydgF+LBDP/MYk3ZbcTNxzj3QDSkmBwm6AWZjYJHrZzIC2bfxCtRZKn7QBIyzaibWE2nHEmOXFmW+42iwQDCSL8wn+Y8cGHCrvEfuazm2/+qLCR55cmoAXDCNKUj4JRMApGwSjADgB4Aj54nH/xsAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4320-4997","institution":"Instituto Politecnico Nacional","correspondingAuthor":true,"prefix":"","firstName":"Ofir","middleName":"","lastName":"Picazo","suffix":""}],"badges":[],"createdAt":"2023-10-04 03:54:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3409192/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3409192/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":44940823,"identity":"a4b2ac5a-89e4-461b-8f6e-632a736f3030","added_by":"auto","created_at":"2023-10-19 17:51:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":57840,"visible":true,"origin":"","legend":"\u003cp\u003eValues (mean ± SEM) for the discrimination index (DI) in the NOR during the initial acquisition period (A), short-term memory (B) and long-term memory (C). Ovariectomized females (n = 10 in each group) were tested after different treatments: VEH = saline solution; KA= kainic acid; E2 = 17β-estradiol; PRL= prolactin. * p\u0026lt; 0.05; ** p\u0026lt;0.01; *** p\u0026lt;0.001; **** p\u0026lt;0.0001, Tukey test.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-3409192/v1/d6a1c48d308eef78edbb99cb.png"},{"id":44940824,"identity":"29843928-2952-4eb5-b7f6-b8631394687f","added_by":"auto","created_at":"2023-10-19 17:51:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3292486,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative Nissl staining sections from CA1 region of ovariectomized rats treated with A) saline solution B) kainic acid, C)17ß-estradiol, D) prolactin, E) kainic acid+17ß-estradiol, F) kainic acid+prolactin and G) kainic acid+17ß-estradiol+prolactin. Scale = 100 uM.\u003c/p\u003e","description":"","filename":"Fig.2CA1.png","url":"https://assets-eu.researchsquare.com/files/rs-3409192/v1/a1ab7a2f962d4496a2c9b85f.png"},{"id":44940826,"identity":"dfede591-0bbe-4897-b831-502752c1e2d6","added_by":"auto","created_at":"2023-10-19 17:51:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3642919,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative Nissl staining sections from CA3 region of ovariectomized rats treated with A) saline solution B) kainic acid, C)17ß-estradiol, D) prolactin, E) kainic acid+17ß-estradiol, F) kainic acid+prolactin and G) kainic acid+17ß-estradiol+prolactin. Scale = 100 uM.\u003c/p\u003e","description":"","filename":"Fig.3CA3.png","url":"https://assets-eu.researchsquare.com/files/rs-3409192/v1/e4020b8c629ebdc1df25a694.png"},{"id":44940825,"identity":"ef1e8f21-7ef3-449d-8f23-787aaa544e98","added_by":"auto","created_at":"2023-10-19 17:51:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":80247,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of hippocampal neurons from ovariectomized rats (three weeks before) treated with VEH = saline solution; KA= kainic acid; E2 = 17β-estradiol and PRL= prolactin. Hormones were administered alone or in combination on lesioned rats injected with KA. Data are cell number ± SD, n=3/group for both CA1 and CA3 regions, * p\u0026lt;0.0001 \u003cem\u003evs\u003c/em\u003e. VEH; # p\u0026lt;0.0001 \u003cem\u003evs\u003c/em\u003e. all other groups.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3409192/v1/039ee100094a4c6d7006f26c.png"},{"id":44941675,"identity":"4c380535-5f32-4484-9498-17885b74d02d","added_by":"auto","created_at":"2023-10-19 17:59:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1936670,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3409192/v1/62bf6984-edc0-4a7d-8008-1b86712768f8.pdf"}],"financialInterests":"","formattedTitle":"Synergistic neuroprotective action of prolactin and 17β-estradiol on kainic acid-induced hippocampal injury and long-term memory deficit in ovariectomized rats.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe neuroprotective actions of the ovarian hormone 17β-estradiol (E2), also synthesized in the brain, against diverse injuries has been consistently confirmed in a variety of models including: β-amyloid toxicity, kainic acid (KA) and quinolinic acid lesions, among others [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These preclinical results have been found in brain regions closely related to cognitive functions including the hippocampus, amygdala, and the prefrontal cortex. In this line some experiments, mainly carried out in rodents, have shown that estrogens enhance cognitive functions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] mostly through their effects in the hippocampus. These E2-procognitive actions have been observed in female rodents at different ages and hormonal conditions: adult and middle-aged female rats, ovariectomized (OVX) and reproductively senescent female rats [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe pituitary hormone prolactin (PRL) has traditionally been associated to lactogenesis but over the last years it has been related to more than three hundred functions in the organism including neurogenesis, neuroprotection, and cognition [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. While the literature about the pro-cognitive effects of E2 is extensive (see Taxier et al., [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] for a review), reports regarding the actions of PRL on learning and memory are scarce and the results of these reports are controversial. For instance, hyperprolactinemic male rats show impaired object recognition without modifications in spatial learning [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], while the lack of PRL receptors in both female and male mice results in learning and memory deficits [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. One more recent study reported that this hormone can prevent the KA-induced cognitive deficits in OVX rats evaluated in the novel object recognition test (NOR) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Furthermore, from clinical experiments, it has been observed that older men having high PRL levels show low cognitive performance in verbal and working memory [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], which is in line with the observation that cabergoline, a dopaminergic agonist able to block the PRL secretion, produces an improvement in cognitive abilities in hyperprolactinemic people [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs known, the insult produced by KA on hippocampal neurons results in a serious impairment of different types of memory [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Since PRL seems to exert its neuroprotective actions independently of the ovarian hormones [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], we used KA-lesioned OVX rats to assess whether combining PRL and E2 administration produce a synergic effect on short- and long-term memory (STM and LTM, respectively). Thus, a low dose of E2 in combination with a standard dose of PRL was essayed in KA-lesioned animals tested in the NOR. We also sought to evaluate the effect of these treatments on the neuronal density in CA1 and CA3 hippocampal subregions and whether they were correlated to our behavioral data.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eA total of 70 adult female Wistar rats (200\u0026ndash;220 g) were obtained from the animal facility of the Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico. All experimental procedures were performed in accordance with the guidelines and standards established by the Ethics Committee of the Faculty of Medicine, UNAM, Mexico City, project registration number FM/DI/046/2018 and by the Internal Committee for Use and Care of Laboratory Animals No. 006/CIC/2018. Animal care was carried out according to the \u0026ldquo;International Guiding Principles for Biomedical Research Involving Animals\u0026rdquo;, Council for International Organizations of Medical Sciences, 2012. Animal Research: Reporting of \u003cem\u003eIn Vivo\u003c/em\u003e Experiments (ARRIVE) guidelines were also followed. Efforts were taken to minimize animals\u0026rsquo; suffering throughout all experimental procedures.\u003c/p\u003e \u003cp\u003eRats were housed in a \u003cem\u003evivarium\u003c/em\u003e under controlled temperature (21\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) and relative humidity (50\u0026thinsp;\u0026plusmn;\u0026thinsp;10%) under a 12 h light/dark cycle (lights-on at 8:00 h). Water and food were available \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThree weeks before the start of experimental procedures, animals from all groups were OVX under 2,2,2-tribromoethanol anesthesia (0.2 g/kg, i.p.). Immediately after surgery, rats were placed on a warm platform until their complete recovery and, after checking for their well-being, they were housed in their standard home cages. Three weeks later, all animals were randomly assigned to seven groups: VEH, E2, PRL, KA, KA\u0026thinsp;+\u0026thinsp;E2, KA\u0026thinsp;+\u0026thinsp;PRL and KA\u0026thinsp;+\u0026thinsp;E2\u0026thinsp;+\u0026thinsp;PRL (see below).\u003c/p\u003e \u003cp\u003eThe first 3 groups of rats were treated for 3 days with a daily dose of either vehicles (0.1 M PBS, i.p. w/o 0.1 mL oil, s.c.; VEH group), ovine prolactin (100 \u0026micro;g/rat, i.p.; PRL group), or 17β-estradiol (10 \u0026micro;g/rat s.c.; E2 group). A fourth group received a single dose of kainic acid (7 mg/kg i.p.) on the second day (KA group). The last 3 groups were treated with either E2, PRL or both, as described above, and, in addition, received KA on the second day. All reagents were purchased from Sigma-Aldrich, MO, USA.\u003c/p\u003e \u003cp\u003eOne hour after the end of the treatments, animals were evaluated in the NOR; this paradigm is a cortical and hippocampus-dependent memory test, which is useful for evaluating the ability of rodents to recognize previously encountered objects after short (1 h) or longer times (24 h) following initial exposure [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This initial exposure session (sample phase) consists of an open-field arena (30\u0026times;30\u0026times;15 cm) with two identical objects (F\u0026thinsp;+\u0026thinsp;F ́) located in opposite and symmetrical corners which rats are made to explore for 5 min. After this first session, animals were tested again 1 h (STM) and 24 h (LTM) in the same arena, with the exception that one of the now familiar objects (F) was replaced by a novel one (N). All sessions were videotaped for later scoring by a single observer, who was blind to the treatment conditions. Data are presented as the discrimination index (DI), which expresses the difference between the time spent exploring the F and N objects [time spent with N/(time spent with N\u0026thinsp;+\u0026thinsp;time spent with F) X 100] [18].\u003c/p\u003e \u003cp\u003eAfter completing the behavioral test, 3 females from each experimental group were randomly chosen, deeply anesthetized with an overdose of pentobarbital (200 mg/kg, i.p) and perfused transcardially with 250 ml of 0.9% saline followed by 250 ml of 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in sodium phosphate buffer (PBS) (pH 7.4, 10\u0026deg;C). Brains were removed and immediately cryoprotected in 30% sucrose-PBS solution for 2\u0026ndash;3 weeks at 4\u0026deg;C. Coronal sections (20 \u0026micro;m) were cut through the dorsal hippocampus with a cryostat (Leica Microsystems Inc. IL, USA). A total of six 20\u0026micro;M sections, spanning the whole anteroposterior length of the hippocampi were obtained for each animal. To have a better representation of the regions analyzed, one series of six cuts were eliminated, while six cuts were collected and mounted on slides. After Nissl staining, microscopic images within the CA1 or CA3 subfields of the dorsal hippocampus were obtained with a digital camera attached to a Nikon microscope (Nikon, Tokyo, Japan), and images were analysed using IMAGEJ, version 1.41 (NIH, Bethesda MD, USA).\u003c/p\u003e \u003cp\u003eTo avoid false positives on the identification of neurons, the presence of a distinguishable nucleus was considered the main criterion for identification at x20 magnification. For both CA1 and CA3, counts were restricted to the region delimited by the anteroposterior coordinates \u0026minus;\u0026thinsp;2.5 to -4.5 mm relative to bregma [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and were performed blind as to the experimental condition. All image analysis was carried out using ImageJ software (ImageJ, NIH. MD, USA).\u003c/p\u003e \u003cp\u003eData from all groups were tested for normal distribution by the Shapiro-Wilk test; homoscedasticity was also assessed. Behavioral data and neuronal density were analyzed by one-way ANOVAs (ordinary or Brown-Forsythe, accordingly) followed, when indicated, by multiple comparisons with \u003cem\u003epost hoc\u003c/em\u003e Tukey tests. In all cases, a \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the average DIs from the sample phase (exploration of two identical objects) of the OVX rats treated with E2 and/or PR previously lesioned with KA. As expected at this acquisition phase of the NOR, performance was similar in all groups (F\u003csub\u003e(6,26.5)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.79; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.59). One hour after the acquisition phase, these same rats were evaluated for STM. A One-Way ANOVA comparing the DIs of rats from every group unveiled a statistically significant effect, (F\u003csub\u003e(6,55)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.9; \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001) with Tukey post-hoc tests showing that only the KA group differed from our control VEH group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Noteworthy, KA-lesioned animals treated with either E2, PRL or both did not differ from controls, suggesting a protecting effect of any of these hormonal treatments on STM. Rats were next tested 24 hours later to assess LTM. Here again, a One-Way ANOVA unveiled significant differences among groups (F\u003csub\u003e(6,55)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.42, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Like STM, the LTM of the KA group appeared impaired when compared to the VEH, although in this case it did not reach significance (p\u0026thinsp;=\u0026thinsp;0.07). However, both KA-lesioned E2-treated and PRL-treated rats showed better LTM performance compared to their untreated KA-lesioned counterparts (DIs\u0026thinsp;=\u0026thinsp;0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 and 0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 for E2\u0026thinsp;+\u0026thinsp;KA and PRL\u0026thinsp;+\u0026thinsp;KA groups, respectively \u003cem\u003eversus\u003c/em\u003e 0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 for the KA group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 in both cases). Furthermore, lesioned rats treated with the hormonal combination showed a much more robust rescuing effect on their LTM performance with average DIs of 0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u003cem\u003eversus\u003c/em\u003e 0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 for the KA\u0026thinsp;+\u0026thinsp;E2\u0026thinsp;+\u0026thinsp;PRL and KA groups, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next explored whether our results showing rescuing effects of our hormonal treatments on the NOR could be linked to neuroprotective effects in the hippocampus, a region involved in object recognition. Representative images of neurons from CA1 and CA3 hippocampal regions (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) reveal marked differences in neuronal density among the different experimental conditions. Data from morphometric analysis showed significant differences among groups in both CA1 and CA3 regions (CA1: F\u003csub\u003e(6,14)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;89.47, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001; CA3: F\u003csub\u003e(6,14)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;252.1, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001). \u003cem\u003ePost hoc\u003c/em\u003e Tukey tests revealed a significant loss of neurons after kainic treatment in both CA1 and CA3 regions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 in both cases), which was prevented by the independent injection of E2 or PRL. Surprisingly, and only observed in lesioned animals, the combination of these hormones resulted in an increase of neuronal density over the control (VEH) group in both regions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe main findings derived from this work are: 1) E2 or PRL administration prevented the KA-induced cognitive deficit in the NOR; 2) the combination of E2 plus PRL in KA-lesioned animals resulted in an improvement of long-term memory over those groups treated only with E2 or PRL, which was accompanied by 3) an increase of neurons density at CA1 and CA3 regions.\u003c/p\u003e \u003cp\u003eAs mentioned previously, the pro-cognitive and neuroprotective actions of E2 have been deeply explored in a variety of animal models. For instance, it has been established that it promotes synaptic plasticity of hippocampal neurons [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], attenuates apoptosis and KA-induced excitotoxicity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], increases spinogenesis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and improves several types of memory, specifically long-term memory [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Similarly, although research on the role of PRL in cognition and neuroplasticity is rather scarce, it has been recently reported that PRL, like E2, facilitates axogenesis, synaptic plasticity, dendrite growth and neurotrophic factors synthesis in the hippocampus [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough the present findings appear to point to a pro-cognitive effects of PRL, its role in this regard remains controversial. Some authors have found that high levels of PRL are associated to poor performance in several memory tasks. For instance, in male rats, hyperprolactinemia was shown to impair object recognition and declarative memory but not spatial learning [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. By contrast, the low levels of this hormone observed in PRL null mice, were related to spatial learning deficit, which could be rescued by chronic (28 days) infusion of PRL into the hippocampus [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. On a different level, lactation seems to be associated to neuroprotection and cognitive improvement in rats [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In this sense, recent research from our group has shown that the number of pregnancies in these animals was related to an increase in dendritic spines of neurons from prefrontal cortex and CA1 hippocampal subfield [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], along with improved performance in the Morris Water-Maze task [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Additionally, other authors have found a close correlation between poor recognition memory and low spine density in both PFC and hippocampus from ovariectomized rats [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These studies suggest that the lack of PRL may be correlated with impaired learning and memory and supports the role for this hormone as both a neuroprotective and pro-cognitive agent. Our results showing that PRL prevents excitotoxic damage and improves object-recognition memory are in line with the beneficial effects previously reported for this hormone (for a review see Duc Nguyen et al., [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]).\u003c/p\u003e \u003cp\u003eThe neuroprotective and proliferative actions of E2 have been explained considering its antioxidant activity, its stimulation of intracellular and membranal E2 receptors and its effects on different growth factors and neuronal signaling pathways, among others [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, E2 has been shown to reduce inflammation, apopotosis, gliosis, and induces neurogenesis. In this regard, E2-induced cell proliferation in the hippocampus seems to be enhanced by ischemic processes, epilepsy, stress, kainic acid, etc. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, it is known that neuronal damage itself triggers cell proliferation processes. For instance, Chamma et al., [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] showed that low doses of KA into the anterior thalamic nucleus boost proliferation in the dentate gyrus, an effect mediated by thalamo-hippocampal connections.\u003c/p\u003e \u003cp\u003eFurthermore, it has been reported that KA-induced epileptic seizures are followed by enhanced neurogenesis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], while KA lesions in neonatal rats induce cell proliferation in CA1 and CA3 hippocampal subfields [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne of the most intriguing findings was the fact that a low dose of E2 administered daily for three days, attenuated KA-induced lesion at the hippocampus. Previously we reported a similar effect injecting a high dose of this hormone (100 ug/rat) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The data reported herein suggest that low doses of E2 are sufficient to promote neuroprotection and proliferative effects when combined with PRL. In line with this, there are reports where even physiological doses of E2 protect neurons against several harmful agents [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and increase neurogenesis in the dentate gyrus [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the other hand, the pro-cognitive and proliferative actions of PRL could be linked to the fact that it facilitates axogenesis, dendrite growth and neurotrophic factors synthesis, which seem to be associated with hippocampal dependent memory [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Previously we reported that in OVX rats, this hormone prevents the PRL receptors reduction induced by KA and increases the vesicular glutamate transporter 1 (VGLUT1) as a long-term bounce response to the lesion by KA in CA1 subfield [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Accordingly, subchronic (7 days) administration of PRL increases the number of hippocampal neurosphere cells in the adult mouse, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eE2 and PRL activate different biochemical pathways to mitigate neural damage and induce cell proliferation. Thus, it is possible that the long-term memory recovery observed in lesioned rats after the combined treatment, together with the cell density increase observed in CA1 and CA3 hippocampal subfields, are due to synergistic effects of both hormones. The fact that an effect was observed only in lesioned animals is in line with Turner et al., [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] who reported that a high dose of PRL rescues adult hippocampal neurogenesis during chronic stress. These findings open the possibility of implementing new substitution therapies based not only on the combination of E2 and progesterone but also including PRL.\u003c/p\u003e \u003cp\u003eTo our knowledge, this is the first report about the improvement at long-term of DI together with cellular increase, over the control group, produced by the combined E2-PRL treatment. We propose that this effect is due to different mechanisms of action for E2 and PRL. However, further experiments are needed to test this hypothesis.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe present study shows, for the first time, the improvement of long-term memory along with the increase of hippocampal cell density produced by the combined treatment of E2-PRL. We propose that this effect is due to a synergistic action of these hormones through different mechanisms. However, further experiments are needed to test this hypothesis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis study was partially supported by CONACYT (286446), COFAA, SIP-IPN and DGAPA PAPIIT UNAM grant numbers IN224019 to GRR and IN227123 to JPM.\u003c/p\u003e\n\u003cp\u003eDisclosure of potential conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003eResearch involving Human Participants and/or Animals.\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were performed in accordance with the guidelines and standards established by the Ethics Committee of the Faculty of Medicine, UNAM, Mexico City, project registration number FM/DI/046/2018 and by the Internal Committee for Use and Care of Laboratory Animals No. 006/CIC/2018.\u003c/p\u003e\n\u003cp\u003eInformed consent.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSince this research only included animals, this process does not apply.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePan Q, Guo K, Xue M, Tu Q (2020) Estrogen protects neuroblastoma cell from amyloid-\u0026beta; 42 (A\u0026beta;42)-induced apoptosis via TXNIP/TRX axis and AMPK signaling. 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The Journal of Neuroscience 40:7355\u0026ndash;7374. https://doi.org/10.1523/JNEUROSCI.0115-20.2020\u003c/li\u003e\n\u003cli\u003eDuarte-Guterman P, Yagi S, Chow C, Galea LAM (2015) Hippocampal learning, memory, and neurogenesis: Effects of sex and estrogens across the lifespan in adults. Horm Behav 74:37\u0026ndash;52. https://doi.org/10.1016/j.yhbeh.2015.05.024\u003c/li\u003e\n\u003cli\u003eChen Y, Guo W, Xu L, et al (2016) 17 \u003cem\u003e\u0026beta;\u003c/em\u003e -Estradiol Promotes Schwann Cell Proliferation and Differentiation, Accelerating Early Remyelination in a Mouse Peripheral Nerve Injury Model. 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The Journal of Neuroscience 23:1742\u0026ndash;1749. https://doi.org/10.1523/JNEUROSCI.23-05-01742.2003\u003c/li\u003e\n\u003cli\u003eTorner L, Karg S, Blume A, et al (2009) Prolactin Prevents Chronic Stress-Induced Decrease of Adult Hippocampal Neurogenesis and Promotes Neuronal Fate. The Journal of Neuroscience 29:1826\u0026ndash;1833. https://doi.org/10.1523/JNEUROSCI.3178-08.2009\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"hormones","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"HORM","sideBox":"Learn more about [Hormones](https://www.springer.com/journal/42000)","snPcode":"42000","submissionUrl":"https://www.editorialmanager.com/horm/default2.aspx","title":"Hormones","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"estradiol, prolactin, kainic acid, recognition memory, hippocampus","lastPublishedDoi":"10.21203/rs.3.rs-3409192/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3409192/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThe neuroprotective actions of the ovarian hormone 17β-estradiol (E2) against different brain lesions have been continuously confirmed in a variety of models including kainic acid (KA) lesions. In the same line, the pituitary hormone prolactin (PRL), traditionally associated to lactogenesis, has recently been linked to a great diversity of functions, including neurogenesis, neuroprotection, and cognitive processes. While the mechanisms of actions of E2 regarding its neuroprotective and behavioral effects have been extensively explored, the molecular mechanisms of PRL related to these roles remain under investigation. The aim of the current study was to explore if the simultaneous administration of PRL and a low dose of E2 prevents the KA-induced cognitive deficit and if this action is associated to changes in hippocampal neuronal density.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eOvariectomized (OVX) rats were treated with saline, PRL and/or E2 in the presence or absence of KA. Neuroprotection was assessed by Nissl staining and neuron counting. Evaluation of memory was carried out by means of the novel object recognition test (NOR).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThese findings indicate that both PRL and E2 prevent short- and long-term memory deficits in lesioned animals. In addition, both hormones exert neuroprotection against KA-induced excitotoxicity in the hippocampus. Interestingly, the combined hormonal treatment was superior at improving the behavioral performance of rats in the NOR and neuronal survival than either treatment administered separately.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eTaken together, these results suggest that these hormones act in different ways at the hippocampus to produce their behavioral, proliferative, and neuroprotective effects.\u003c/p\u003e","manuscriptTitle":"Synergistic neuroprotective action of prolactin and 17β-estradiol on kainic acid-induced hippocampal injury and long-term memory deficit in ovariectomized rats.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-19 17:51:16","doi":"10.21203/rs.3.rs-3409192/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2024-01-17T05:26:47+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-11-22T13:24:36+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-10-17T08:31:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-10-13T07:41:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Hormones","date":"2023-10-12T16:01:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"hormones","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"HORM","sideBox":"Learn more about [Hormones](https://www.springer.com/journal/42000)","snPcode":"42000","submissionUrl":"https://www.editorialmanager.com/horm/default2.aspx","title":"Hormones","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9eacc8f3-3079-4bb8-9682-84058c47d04a","owner":[],"postedDate":"October 19th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-03-19T23:16:25+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-19 17:51:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3409192","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3409192","identity":"rs-3409192","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}