{"paper_id":"2506d4b0-a068-4b3e-bcde-624b3df0d69f","body_text":"MMP-9 expression in rat pup incisor teeth is not altered by maternal hypertension or maternal atenolol treatment during pregnancy and lactation. | 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 MMP-9 expression in rat pup incisor teeth is not altered by maternal hypertension or maternal atenolol treatment during pregnancy and lactation. Gracieli Prado ELIAS, Haylla de Faria Horta, Alanna Ramalho MATEUS, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4966229/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Nov, 2024 Read the published version in Journal of Molecular Histology → Version 1 posted 10 You are reading this latest preprint version Abstract Hypertension alters tooth formation and Atenolol, a β-blocker, reduces the blood pressure of spontaneously hypertensive rats (SHR) during pregnancy and lactation, and as demonstrated before, increases the microhardness of the SHR offspring's teeth. We hypothesize hypertension alters the expression of MMP-9 in dental structures of SHR offspring and that treating female SHR with atenolol prevents this alteration. This study aimed to evaluate the expression of matrix metalloproteinase (MMP-9) in incisor teeth (IT) in male offspring of SHR (30 days old) treated or untreated with Atenolol (At, 100 mg/kg/day, orally) during pregnancy and lactation. MMP-9 expression was evaluated in ameloblasts (AM), enamel matrix (EM), odontoblasts (OD), and pre-dentin (PD) of IT through immunohistochemical reactions (immunoperoxidase). Data were analyzed by Shapiro-Wilk and Kruskal-Wallis (p < 0.05), with Dunn post-test. Histological differences were not observed between IT tissues of SHR and normotensive Wistar rats. For the first time, our data showed that MMP-9 expression in specific dental structures is not altered in SHR. Atenolol treatment increased MMP-9 immunostaining in EM of Wistar rat, however, Atenolol did not alter the MMP-9 in the IT tissues of SHR. Our results suggest that neither hypertension nor atenolol treatment alters MMP-9 expression in dental tissues of SHR. Atenolol Hypertension Dental Enamel Dentin Metalloproteinase Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Morphological abnormalities resulting from the underdevelopment of ameloblasts were observed in the teeth of spontaneously hypertensive stroke-prone rats (SHR-SP)(Ishibashi et al. 1990 ), which may be associated with reduced microhardness of enamel and dentin in incisors teeth of offspring of spontaneously hypertensive rats (SHR)(Elias et al. 2006 ). Treatment of female spontaneously hypertensive rats (SHR) during pregnancy and lactation with Atenolol (100 mg/kg/day, orally), a β1-adrenergic blocker used as an antihypertensive in human hypertension, increased the microhardness of enamel and dentin in incisors and molars teeth of male SHR offspring (Elias et al. 2020 ). Amelogenesis in rat teeth involves a cascade of events that begins with the secretion of the organic matrix, rich in amelogenin and ends with enamel mineralization when the organic matrix is strongly reduced (Robinson et al. 1995 ). Amelogenin is the most abundant protein during enamel formation. It is hydrolyzed within a few hours (Smith et al. 1989 ) after secretion into the matrix, along with other proteins, by the action of matrix metalloproteinases (MMPs), which regulate the mineralization process and influence the formation of dental tissues (Fanchon et al. 2004 ). MMPs degrade a series of components of the extracellular matrix. Collectively, they can degrade all matrix proteins (Souza and Line 2002 ). Previous studies on proteinases involved in enamel development suggest that MMPs are more important during the initial stages of enamel formation, while serine proteases would be involved in the later stages of maturation (Overall and Limeback 1988 ; Fukae et al. 1996 ; Tanabe et al. 1996 ; Robinson et al. 1998 ). MMPs are also active in the early stages of dentin mineralization, degrading dentin sialoproteins, the most abundant proteins in this tissue (Bourd-Boittin et al. 2005 ). When dentin and enamel are in the process of formation, two gelatinases (MMP-2 e MMP-9) and stromelysin-1 (MMP-3) are present, each with specific distributions, suggesting functional implications for each of them(Hall et al. 1999 ; Goldberg et al. 2003 ; Fanchon et al. 2004 ) in these processes. Although the expression of MMPs in dental tissues of humans (Palosaari et al. 2003 ) and animals(Hall et al. 1999 ; Goldberg et al. 2003 ; Bourd-Boittin et al. 2005 ) has been studied, the potential impact of systemic pathologies such as hypertension or antihypertensive therapies on the expression of these enzymes in teeth have not yet been evaluated. We hypothesize that hypertension alters the expression of MMP-9 in dental structures of SHR offspring and that treating female SHR with Atenolol prevents this alteration. This study aimed to evaluate the expression of MMP-9 in the enamel and dentin of male offspring from female SHR untreated or treated with atenolol during the pregnancy and lactation period, through immunohistochemical reactions. Methodology Animals Male offspring, 30 days old, of normotensive Wistar rats and Spontaneously Hypertensive Rats (SHR) treated or not with atenolol during the pregnancy and lactation periods were used in the experiments. The male and female Wistar rats were provided by the bioterium of the School of Dentistry – Araçatuba, and the females SHR were obtained from a strain from the Department of Basic Sciences. The animals were maintained under controlled temperature (22–24°C) and light cycle (12h/ light and 12h/darkness) in the bioterium, receiving standard food and water “ad libitum” . The experimental protocols for this study were previously approved by the Animal Research Ethics Committee of the School of Dentistry of Araçatuba, UNESP (process n. 37/03). Atenolol Treatment Atenolol is an antagonist of β1-adrenergic receptors, used in clinical practice as an antihypertensive, antianginal and antiarrhythmic medication. The chemical C 14 H 22 N 2 O 3 has a molecular weight of 266.34. In our study, A7655 from Sigma Aldrich, was used. The chemical structure of Atenolol is shown in Fig. 1 . Female normotensive Wistar rats and SHR were placed with their respective males for mating. Pregnancy was determined by the presence of spermatozoa in the rats’ vaginal smear. This day was labeled as day zero of pregnancy, and the treatment with Atenolol (diluted in drinking water) started. The volume of water offered during 24h to the rats was previously calculated. Treatment of rats with Atenolol (100 mg/kg/day, orally) was carried out throughout the pregnancy (21 days) and lactation (30 days) period. The weight of the rats was monitored daily to ensure adequate administration of the atenolol dose. The rats in the untreated groups received the same volume of water without Atenolol. The dose of Atenolol was calculated according to previous studies (Elias et al. 2006 , 2020 ). Systolic Blood Pressure The systolic blood pressure (SBP) in rats was recorded by tail plethysmography, using a plethysmograph adapted for rat measurements (Physiograph® MK-III-S, NBS – Narco Bio-Systems, Houston, Texas, USA). The recordings were made by a single operator in the morning in a specific laboratory with light and noise control to avoid animal stress. SHR with SBP greater than or equal to 150 mmHg were used, recorded before mating. The SBP values obtained at day zero of pregnancy and the end of pregnancy (20th day) and lactation (28th ) were expressed as mean ± SEM. Jaws removal Male Offspring (30 day-old) were killed with excess anesthesia (Halothane) to remove the hemi-jaws. The male offspring’s hemi-jaws were kept intact and used in the processing and analysis described below. Histological processing Immediately after removing the hemi-jaws (n = 5 per group), they were immersed in 10% buffered formalin solution for 48 hours for tissue fixation. After this period, the specimens were washed in running water for 8 hours and stored in 70% alcohol until completely dehydrated and embedded in paraffin. After embedding, the specimens were cut to a thickness of 5 µm. The specimens were stained with Hematoxylin and Eosin, and qualitative histological analysis was performed to evaluate the tissues and cells. Only slides with preserved tissues and cells were used in the processing and immunohistochemical analysis. Immunohistochemical technique The expression of MMP-9 protein was evaluated in dental tissues and cells of lower incisors teeth (IT) in SHR and Wistar male offspring of female rats treated or not treated with Atenolol. MMP-9 protein was determined by immunohistochemical reactions, using immunoperoxidase as the detection method. After embedding in paraffin, semi-serial sections of 5 µm from hemi-jaws were placed on silanized slides (Erviegas) and deparaffinized. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide (Perhidrol 30% H 2 O 2 – MERCK) for 40 minutes, and the sections underwent antigen retrieval using citrate buffer (pH 6.0–95°C). Subsequently, the primary antibody (1:50) was incubated in a humid chamber at 4°C for 18 hours. A polyclonal antibody produced in goats against MMP-9 was used (C-20: sc-6840, Santa Cruz Biotechnology, Inc.). As a second antibody, ImmunPure® Rabbit Anti-coat IgG (H + L, biotin-conjugated (PIERCE Biotechnology) was used. Afterward, incubation with (Avidin-Biotin Complex / HRP-Dako) was carried out for 45 minutes to amplify the markings’ signal. The chromogen used to reveal the reaction was 3-3-diaminobenzidine-HCl substrate (DAKO), and nuclear counterstaining was performed using Harris Hematoxylin. The MMP-9 expression was evaluated in distinct dental tissues: ameloblasts (AM), enamel matrix (EM), odontoblasts (OD), and pre-dentin (PD). Immunohistochemical analysis The slices were analyzed under an optical microscope, and scores of 0 (no staining), 1 (weak/diffuse staining), 2 (moderate staining), and 3 (intense staining) were assigned according to the pattern of immunostaining distribution to assess the presence of MMP-9 protein in the teeth of the studied groups. Three different observers determined the scores, and the median of the obtained values was expressed for each tissue (enamel, predentin, and dentin), cell type (ameloblasts, odontoblasts), or evaluated region (enamel matrix). Statistical analysis The results were evaluated using Bioestat Version 5.0. The normality of the data was checked using the Shapiro-Wilk test (Bioestat Version 5.0), and the comparison between groups was performed using the Kruskal-Wallis test, followed by Dunn’s post-test for multiple comparisons. The significance level was considered as p < 0,05. Results Untreated SHR females showed increased SBP values compared to normotensive rats on day zero of pregnancy (Fig. 2 ). Pregnancy reduced SBP in SHR, with the lowest SBP values observed on the 20th day of pregnancy. Treatment with atenolol reduced SBP values in SHR throughout pregnancy, with the lowest values observed in Atenolol-treated SHR at the end of pregnancy (20th day). SBP values remained low in treated SHR during the lactation period, an effect not observed in untreated SHR, as the increased SBP values at 28 days of lactation were restored and were similar to those found on day zero of pregnancy (Fig. 2 ). Analyzing the SBP of normotensive Wistar rats, no differences (p > 0,05) were observed in SBP values between the two groups, treated and untreated, during pregnancy or lactation. Histological analysis showed tissues and cells involved in dental formation (Fig. 3 ) of the IT of male Wistar and SHR offspring. As can be observed in the images of Fig. 3 , no morphological differences were observed between AM, EM, OD, and PD of the IT of Wistar and SHR rats. Capillary loop formation was observed in the odontoblastic layer, with a normal appearance of its cells (OD) and no apparent difference between the groups. It was noted that the AM exhibited similar morphology between groups, and they were in the amelogenesis secretory phase, as evidenced by the presence of Tomes’ processes. Tomes’ processes are conical projections formed from the distal cytoplasm, related to the release of metalloproteinases (Palosaari et al. 2003 ) enzymes into the enamel matrix. Immediately below the Tomes’ process, the enamel matrix (EM) is observed. Once the deposition of the enamel matrix is complete, the Tomes’ process will disappear. Analyzing the enamel and dentin layers, along with their respective cells (AM and OD), homogeneity and similarity to the pattern of normality (Fig. 3 ) between the groups can be observed, which were not altered by treatment with Atenolol. Immunostaining for MMP-9 was evident (brown staining) in AM (a), EM (b), OD (c), and PD (d) (Fig. 3 ). In Fig. 4 , the expression of MMP-9 was quantified (scores) in different tissues and compared between the groups. No alteration in immunostaining (p > 0,05) for MMP-9 was observed in the cells and tissues of IT in SHR offspring compared to Wistar offspring (Figs. 3 and 4 ). Treatment with Atenolol promoted increased MMP-9 expression only in the EM of IT in Wistar offspring but had no effect on other structures or cells (Fig. 4 ). Discussion The results presented in this study suggest that neither hypertension nor treatment with Atenolol, during pregnancy and lactation, alter the expression of MMP-9 in the dental tissues of SHR, leading to the rejection of the study hypothesis. The IT of hypertensive rats (SHR-SP) present changes in their structure, a more whitish color, and less resistance to wear (Elias et al. 2020 ). As observed by our group, 30-day-old SHR IT already show dental alterations (Elias et al. 2006 ). The suggestion that hypertension alters tooth formation is reinforced by the observation that the diameter of the crown of deciduous and permanent teeth in children of hypertensive mothers is reduced (Garn et al. 1979 ) compared to children of normotensive mothers. However, the mechanisms involved in the dental alterations associated with hypertension are not fully understood. In this study, when we analyzed the enamel and dentin and their respective cells, ameloblasts and odontoblasts, and the formation of capillaries in the odontoblastic layer of IT of SHR, we observed characteristics of homogeneity and similarity with the normal pattern observed in the tissues and cells of Wistar rats. Furthermore, the immunohistochemical reactions allowed us to observe that the presence of MMP-9 protein in the evaluated dental tissues and cells follows the pattern described in the literature (Goldberg et al. 2003 ). A weaker immunostaining was observed in odontoblasts and pre-dentin; however, strong staining was seen in the enamel matrix of the animals, a fact that differs from what has been described by some authors (Goldberg et al. 2003 ). It can be observed that the positive MMP-9 marking in ameloblasts, odontoblasts, and pre-dentin followed the data from the literature (Palosaari et al. 2003 ; Fanchon et al. 2004 ; Bourd-Boittin et al. 2005 ; Bei 2009 ), although variations in its expression can be detected in the representative figures of the groups (Fig. 3 ). MMP-9 has the function of degrading enamel proteins, favoring subsequent mineralization (Bartlett 2013 ). Previously, studies demonstrated that IT of SHR offspring exhibited lower enamel and dentin microhardness (Elias et al. 2006 ) compared to Wistar offspring’s IT. Thus, we expected changes in MMP-9 expression/immunostaining would favor altered removal of organic matrix and mineral deposition, reducing mineralization. However, when quantifying MMP-9 expression in cells and dental tissues, no significant difference was found between the IT of SHR and Wistar offspring. Although the results are suggestive, it is important to bear in mind that the presence of the protein in the tissue does not always imply its activity. A protein can be expressed but in its inactive form. For some authors, MMPs are mostly produced in their latent form, an inactive form, and their activation occurs subsequently (Cingolani and Houssay 2004 ). It is also necessary to consider that there are other MMPs involved in forming enamel and dentin in rat incisors, which may also be contributing, to a greater or lesser extent, to the observed changes in SHR. Specific studies to evaluate the expression of other MMPs, such as MMP-20 and MMP-2, should be conducted before conclusive statements about the role of MMPs in the studied alterations. If the removal of the organic matrix is not altered in the teeth of SHR offspring, mineral deposition may occur inadequately and at a speed different from matrix degradation. Furthermore, alterations in plasma concentrations of PTH and calcium found in SHR (Fanchon et al. 2004 ) could actively interfere with the mineralization process of SHR incisor teeth. As previously demonstrated (Elias et al. 2020 ), Atenolol is effective in reducing the SBP of SHR during pregnancy and lactation, and the treatment of female rats with Atenolol increased enamel and dentin hardness in the male offspring (p < 0.05) in both groups. We could not associate the effects of Atenolol with possible alterations in MMP-9 expression (Sorsa et al. 2004 ). As observed in this study, Atenolol treatment did not alter MMP-9 expression in the cells and tissues of SHR, and in Wistar rats, Atenolol increased MMP-9 expression only in the enamel matrix. These results suggest that the effect of Atenolol on the microhardness of enamel and dentin in SHR incisor teeth is most likely not due to mechanisms involving MMP-9. The mechanism by which Atenolol promotes increased dental mineralization has not yet been established; however, it is known that the parathyroid glands have adrenergic innervation, and the secretion of parathyroid hormone can be inhibited by beta-blockers (Elias et al. 2020 ). Specific studies should be conducted to determine with clarity the molecular mechanisms altered by Atenolol that would be associated with the dental mineralization process. At this point, we can suggest that MMP-9 does not participate in the mechanism by which Atenolol increases the microhardness of enamel and dentin in incisor teeth of SHR. Conclusion The results of this study suggest that neither hypertension nor Atenolol treatment alters MMP-9 expression in SHR incisor teeth cells and tissues. Declarations The authors declare no conflicts of interest. Funding This research was funded by the Coordination of Improvement of Higher Education Personnel (CAPES, finance code 001) and National Council for Scientific and Technological Development (CNPq). Author Contribution GPE: Conceptualization, Methodology, Formal Analysis, Investigation, Writing - Original Draft. HFH: Methodology, Formal Analysis, Writing - Original Draft. ARM: Formal Analysis, Writing - Original Draft. LVBP and AOL: Writing - Original Draft. FC: Formal Analysis, Statistics. ALCBD and KTS: Methodology and Writing - Original Draft. RO and CA: Conceptualization, Methodology, Validation, Data Curation, Writing - Original Draft, Supervision and Project Administration. Data Availability The data used to support the findings of this study can be made available upon request to the corresponding author. References Bartlett JD (2013) Dental Enamel Development: Proteinases and Their Enamel Matrix Substrates. 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Adv Dent Res 10:170–172. https://doi.org/10.1177/08959374960100020801 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 29 Nov, 2024 Read the published version in Journal of Molecular Histology → Version 1 posted Editorial decision: Revision requested 21 Sep, 2024 Reviews received at journal 20 Sep, 2024 Reviewers agreed at journal 11 Sep, 2024 Reviewers agreed at journal 07 Sep, 2024 Reviews received at journal 27 Aug, 2024 Reviewers agreed at journal 26 Aug, 2024 Reviewers invited by journal 25 Aug, 2024 Editor assigned by journal 25 Aug, 2024 Submission checks completed at journal 23 Aug, 2024 First submitted to journal 23 Aug, 2024 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. 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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-4966229\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":357307227,\"identity\":\"4eaa1320-7921-46ff-b1a2-1d3f6e2d67ba\",\"order_by\":0,\"name\":\"Gracieli Prado ELIAS\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Universidade Federal de Juiz de Fora- UFJF, Juiz de Fora Dental School, Juiz de 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(UNESP)\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Fernando\",\"middleName\":\"\",\"lastName\":\"CHIBA\",\"suffix\":\"\"},{\"id\":357307233,\"identity\":\"d80dafda-fa1b-41b4-a5eb-8d507468f76a\",\"order_by\":6,\"name\":\"Alberto Carlos Botazzo DELBEM\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"São Paulo State University (UNESP)\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Alberto\",\"middleName\":\"Carlos Botazzo\",\"lastName\":\"DELBEM\",\"suffix\":\"\"},{\"id\":357307234,\"identity\":\"8a135e5f-f4ca-4865-93e4-1d4119fb8606\",\"order_by\":7,\"name\":\"Kikue Takebayashi SASSAKI\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"São Paulo State University (UNESP)\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Kikue\",\"middleName\":\"Takebayashi\",\"lastName\":\"SASSAKI\",\"suffix\":\"\"},{\"id\":357307235,\"identity\":\"33aa687a-ec94-4118-8526-ee25db1df495\",\"order_by\":8,\"name\":\"Roberta OKAMOTO\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"São Paulo State University (UNESP)\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Roberta\",\"middleName\":\"\",\"lastName\":\"OKAMOTO\",\"suffix\":\"\"},{\"id\":357307236,\"identity\":\"d60f4338-71a9-4927-b89b-5c45a7693fd6\",\"order_by\":9,\"name\":\"Cristina ANTONIALI\",\"email\":\"data:image/png;base64,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\",\"orcid\":\"\",\"institution\":\"São Paulo State University (UNESP)\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Cristina\",\"middleName\":\"\",\"lastName\":\"ANTONIALI\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-08-23 20:21:12\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4966229/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4966229/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1007/s10735-024-10294-2\",\"type\":\"published\",\"date\":\"2024-11-29T15:56:52+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":65206575,\"identity\":\"cf6d4d5f-12f3-43ab-9ebd-d4ceacc14051\",\"added_by\":\"auto\",\"created_at\":\"2024-09-24 18:33:56\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":27910,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eChemical Structure of Atenolol (4-[2′-Hydroxy-3′-isopropylamino) propoxy] phenylacetamide) (https://www.sigmaaldrich.com).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4966229/v1/3ba9f798d7d675051a2b3304.png\"},{\"id\":65206574,\"identity\":\"d5e35bf4-b32c-4901-b2d2-a9907a895116\",\"added_by\":\"auto\",\"created_at\":\"2024-09-24 18:33:56\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":662442,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSystolic blood pressure (mmHg) of Wistar (n=10), SHR (n=10), Wistar (n=10) and SHR (n=10) treated with atenolol on different days of pregnancy and lactation. Bars represent the mean ± SEM of values. *p\\u0026lt;0.05 between groups (ANOVA).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4966229/v1/402d95d946690266b5ef846c.png\"},{\"id\":65206572,\"identity\":\"58d48a58-c7f6-430b-a7a9-c6b69fc62828\",\"added_by\":\"auto\",\"created_at\":\"2024-09-24 18:33:55\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2219319,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eImmunostaining of matrix metalloproteinase (MMP-9) in a) AM, ameloblasts, b) EM, enamel matrix, c) OD, odontoblasts, and d) PD, pre-dentin of incisors teeth of SHR (E) and Wistar (F) rats untreated and SHR (G) and Wistar (H) rats treated with atenolol (160x).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4966229/v1/d7f0e49f88b61f88fbee3bb6.png\"},{\"id\":65206573,\"identity\":\"af456d60-c95a-4c90-ad77-71b1b3fde706\",\"added_by\":\"auto\",\"created_at\":\"2024-09-24 18:33:56\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":958692,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eQuantitative immunostaining for MMP-9 in (A) AM, (B) OD, (C) PD, and (D) EM of incisor teeth of the different groups was expressed as median and demonstrated in the interleaved box \\u0026amp; whiskers graphics. *p\\u0026lt;0.05 (Kruskal-Wallis).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4966229/v1/a2f57f5426ac2bd297390928.png\"},{\"id\":70381174,\"identity\":\"e4fe614c-9ebb-42e2-bd1b-1d71e76c51db\",\"added_by\":\"auto\",\"created_at\":\"2024-12-02 16:02:29\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":4158373,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4966229/v1/73a52609-32ae-40e4-9150-444126fcdc11.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"MMP-9 expression in rat pup incisor teeth is not altered by maternal hypertension or maternal atenolol treatment during pregnancy and lactation.\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eMorphological abnormalities resulting from the underdevelopment of ameloblasts were observed in the teeth of spontaneously hypertensive stroke-prone rats (SHR-SP)(Ishibashi et al. \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e1990\\u003c/span\\u003e), which may be associated with reduced microhardness of enamel and dentin in incisors teeth of offspring of spontaneously hypertensive rats (SHR)(Elias et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e). Treatment of female spontaneously hypertensive rats (SHR) during pregnancy and lactation with Atenolol (100 mg/kg/day, orally), a β1-adrenergic blocker used as an antihypertensive in human hypertension, increased the microhardness of enamel and dentin in incisors and molars teeth of male SHR offspring (Elias et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eAmelogenesis in rat teeth involves a cascade of events that begins with the secretion of the organic matrix, rich in amelogenin and ends with enamel mineralization when the organic matrix is strongly reduced (Robinson et al. \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e1995\\u003c/span\\u003e). Amelogenin is the most abundant protein during enamel formation. It is hydrolyzed within a few hours (Smith et al. \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e1989\\u003c/span\\u003e) after secretion into the matrix, along with other proteins, by the action of matrix metalloproteinases (MMPs), which regulate the mineralization process and influence the formation of dental tissues (Fanchon et al. \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). MMPs degrade a series of components of the extracellular matrix. Collectively, they can degrade all matrix proteins (Souza and Line \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2002\\u003c/span\\u003e). Previous studies on proteinases involved in enamel development suggest that MMPs are more important during the initial stages of enamel formation, while serine proteases would be involved in the later stages of maturation (Overall and Limeback \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e1988\\u003c/span\\u003e; Fukae et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e1996\\u003c/span\\u003e; Tanabe et al. \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e1996\\u003c/span\\u003e; Robinson et al. \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e1998\\u003c/span\\u003e). MMPs are also active in the early stages of dentin mineralization, degrading dentin sialoproteins, the most abundant proteins in this tissue (Bourd-Boittin et al. \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e). When dentin and enamel are in the process of formation, two gelatinases (MMP-2 e MMP-9) and stromelysin-1 (MMP-3) are present, each with specific distributions, suggesting functional implications for each of them(Hall et al. \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e1999\\u003c/span\\u003e; Goldberg et al. \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Fanchon et al. \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e) in these processes.\\u003c/p\\u003e \\u003cp\\u003eAlthough the expression of MMPs in dental tissues of humans (Palosaari et al. \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e) and animals(Hall et al. \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e1999\\u003c/span\\u003e; Goldberg et al. \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Bourd-Boittin et al. \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e) has been studied, the potential impact of systemic pathologies such as hypertension or antihypertensive therapies on the expression of these enzymes in teeth have not yet been evaluated. We hypothesize that hypertension alters the expression of MMP-9 in dental structures of SHR offspring and that treating female SHR with Atenolol prevents this alteration. This study aimed to evaluate the expression of MMP-9 in the enamel and dentin of male offspring from female SHR untreated or treated with atenolol during the pregnancy and lactation period, through immunohistochemical reactions.\\u003c/p\\u003e\"},{\"header\":\"Methodology\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAnimals\\u003c/h2\\u003e \\u003cp\\u003eMale offspring, 30 days old, of normotensive Wistar rats and Spontaneously Hypertensive Rats (SHR) treated or not with atenolol during the pregnancy and lactation periods were used in the experiments.\\u003c/p\\u003e \\u003cp\\u003eThe male and female Wistar rats were provided by the bioterium of the School of Dentistry \\u0026ndash; Ara\\u0026ccedil;atuba, and the females SHR were obtained from a strain from the Department of Basic Sciences. The animals were maintained under controlled temperature (22\\u0026ndash;24\\u0026deg;C) and light cycle (12h/ light and 12h/darkness) in the bioterium, receiving standard food and water \\u003cem\\u003e\\u0026ldquo;ad libitum\\u0026rdquo;\\u003c/em\\u003e. The experimental protocols for this study were previously approved by the Animal Research Ethics Committee of the School of Dentistry of Ara\\u0026ccedil;atuba, UNESP (process n. 37/03).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAtenolol Treatment\\u003c/h2\\u003e \\u003cp\\u003eAtenolol is an antagonist of β1-adrenergic receptors, used in clinical practice as an antihypertensive, antianginal and antiarrhythmic medication. The chemical C\\u003csub\\u003e14\\u003c/sub\\u003eH\\u003csub\\u003e22\\u003c/sub\\u003eN\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e3\\u003c/sub\\u003e has a molecular weight of 266.34. In our study, A7655 from Sigma Aldrich, was used. The chemical structure of Atenolol is shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. Female normotensive Wistar rats and SHR were placed with their respective males for mating. Pregnancy was determined by the presence of spermatozoa in the rats\\u0026rsquo; vaginal smear. This day was labeled as day zero of pregnancy, and the treatment with Atenolol (diluted in drinking water) started. The volume of water offered during 24h to the rats was previously calculated. Treatment of rats with Atenolol (100 mg/kg/day, orally) was carried out throughout the pregnancy (21 days) and lactation (30 days) period. The weight of the rats was monitored daily to ensure adequate administration of the atenolol dose. The rats in the untreated groups received the same volume of water without Atenolol. The dose of Atenolol was calculated according to previous studies (Elias et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSystolic Blood Pressure\\u003c/h2\\u003e \\u003cp\\u003eThe systolic blood pressure (SBP) in rats was recorded by tail plethysmography, using a plethysmograph adapted for rat measurements (Physiograph\\u0026reg; MK-III-S, NBS \\u0026ndash; Narco Bio-Systems, Houston, Texas, USA). The recordings were made by a single operator in the morning in a specific laboratory with light and noise control to avoid animal stress. SHR with SBP greater than or equal to 150 mmHg were used, recorded before mating. The SBP values obtained at day zero of pregnancy and the end of pregnancy (20th day) and lactation (28th ) were expressed as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SEM.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eJaws removal\\u003c/h2\\u003e \\u003cp\\u003eMale Offspring (30 day-old) were killed with excess anesthesia (Halothane) to remove the hemi-jaws. The male offspring\\u0026rsquo;s hemi-jaws were kept intact and used in the processing and analysis described below.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eHistological processing\\u003c/h2\\u003e \\u003cp\\u003eImmediately after removing the hemi-jaws (n\\u0026thinsp;=\\u0026thinsp;5 per group), they were immersed in 10% buffered formalin solution for 48 hours for tissue fixation. After this period, the specimens were washed in running water for 8 hours and stored in 70% alcohol until completely dehydrated and embedded in paraffin. After embedding, the specimens were cut to a thickness of 5 \\u0026micro;m. The specimens were stained with Hematoxylin and Eosin, and qualitative histological analysis was performed to evaluate the tissues and cells. Only slides with preserved tissues and cells were used in the processing and immunohistochemical analysis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImmunohistochemical technique\\u003c/h2\\u003e \\u003cp\\u003eThe expression of MMP-9 protein was evaluated in dental tissues and cells of lower incisors teeth (IT) in SHR and Wistar male offspring of female rats treated or not treated with Atenolol. MMP-9 protein was determined by immunohistochemical reactions, using immunoperoxidase as the detection method. After embedding in paraffin, semi-serial sections of 5 \\u0026micro;m from hemi-jaws were placed on silanized slides (Erviegas) and deparaffinized. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide (Perhidrol 30% H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e \\u0026ndash; MERCK) for 40 minutes, and the sections underwent antigen retrieval using citrate buffer (pH 6.0\\u0026ndash;95\\u0026deg;C). Subsequently, the primary antibody (1:50) was incubated in a humid chamber at 4\\u0026deg;C for 18 hours. A polyclonal antibody produced in goats against MMP-9 was used (C-20: sc-6840, Santa Cruz Biotechnology, Inc.). As a second antibody, ImmunPure\\u0026reg; Rabbit Anti-coat IgG (H\\u0026thinsp;+\\u0026thinsp;L, biotin-conjugated (PIERCE Biotechnology) was used. Afterward, incubation with (Avidin-Biotin Complex / HRP-Dako) was carried out for 45 minutes to amplify the markings\\u0026rsquo; signal. The chromogen used to reveal the reaction was 3-3-diaminobenzidine-HCl substrate (DAKO), and nuclear counterstaining was performed using Harris Hematoxylin. The MMP-9 expression was evaluated in distinct dental tissues: ameloblasts (AM), enamel matrix (EM), odontoblasts (OD), and pre-dentin (PD).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImmunohistochemical analysis\\u003c/h2\\u003e \\u003cp\\u003e The slices were analyzed under an optical microscope, and scores of 0 (no staining), 1 (weak/diffuse staining), 2 (moderate staining), and 3 (intense staining) were assigned according to the pattern of immunostaining distribution to assess the presence of MMP-9 protein in the teeth of the studied groups. Three different observers determined the scores, and the median of the obtained values was expressed for each tissue (enamel, predentin, and dentin), cell type (ameloblasts, odontoblasts), or evaluated region (enamel matrix).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eThe results were evaluated using Bioestat Version 5.0. The normality of the data was checked using the Shapiro-Wilk test (Bioestat Version 5.0), and the comparison between groups was performed using the Kruskal-Wallis test, followed by Dunn\\u0026rsquo;s post-test for multiple comparisons. The significance level was considered as p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0,05.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003eUntreated SHR females showed increased SBP values compared to normotensive rats on day zero of pregnancy (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Pregnancy reduced SBP in SHR, with the lowest SBP values observed on the 20th day of pregnancy. Treatment with atenolol reduced SBP values in SHR throughout pregnancy, with the lowest values observed in Atenolol-treated SHR at the end of pregnancy (20th day). SBP values remained low in treated SHR during the lactation period, an effect not observed in untreated SHR, as the increased SBP values at 28 days of lactation were restored and were similar to those found on day zero of pregnancy (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Analyzing the SBP of normotensive Wistar rats, no differences (p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0,05) were observed in SBP values between the two groups, treated and untreated, during pregnancy or lactation.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eHistological analysis showed tissues and cells involved in dental formation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e) of the IT of male Wistar and SHR offspring. As can be observed in the images of Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e, no morphological differences were observed between AM, EM, OD, and PD of the IT of Wistar and SHR rats. Capillary loop formation was observed in the odontoblastic layer, with a normal appearance of its cells (OD) and no apparent difference between the groups. It was noted that the AM exhibited similar morphology between groups, and they were in the amelogenesis secretory phase, as evidenced by the presence of Tomes\\u0026rsquo; processes. Tomes\\u0026rsquo; processes are conical projections formed from the distal cytoplasm, related to the release of metalloproteinases (Palosaari et al. \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e) enzymes into the enamel matrix. Immediately below the Tomes\\u0026rsquo; process, the enamel matrix (EM) is observed. Once the deposition of the enamel matrix is complete, the Tomes\\u0026rsquo; process will disappear. Analyzing the enamel and dentin layers, along with their respective cells (AM and OD), homogeneity and similarity to the pattern of normality (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e) between the groups can be observed, which were not altered by treatment with Atenolol.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eImmunostaining for MMP-9 was evident (brown staining) in AM (a), EM (b), OD (c), and PD (d) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). In Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, the expression of MMP-9 was quantified (scores) in different tissues and compared between the groups. No alteration in immunostaining (p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0,05) for MMP-9 was observed in the cells and tissues of IT in SHR offspring compared to Wistar offspring (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e and \\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Treatment with Atenolol promoted increased MMP-9 expression only in the EM of IT in Wistar offspring but had no effect on other structures or cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThe results presented in this study suggest that neither hypertension nor treatment with Atenolol, during pregnancy and lactation, alter the expression of MMP-9 in the dental tissues of SHR, leading to the rejection of the study hypothesis.\\u003c/p\\u003e \\u003cp\\u003eThe IT of hypertensive rats (SHR-SP) present changes in their structure, a more whitish color, and less resistance to wear (Elias et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). As observed by our group, 30-day-old SHR IT already show dental alterations (Elias et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e). The suggestion that hypertension alters tooth formation is reinforced by the observation that the diameter of the crown of deciduous and permanent teeth in children of hypertensive mothers is reduced (Garn et al. \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e1979\\u003c/span\\u003e) compared to children of normotensive mothers. However, the mechanisms involved in the dental alterations associated with hypertension are not fully understood.\\u003c/p\\u003e \\u003cp\\u003eIn this study, when we analyzed the enamel and dentin and their respective cells, ameloblasts and odontoblasts, and the formation of capillaries in the odontoblastic layer of IT of SHR, we observed characteristics of homogeneity and similarity with the normal pattern observed in the tissues and cells of Wistar rats. Furthermore, the immunohistochemical reactions allowed us to observe that the presence of MMP-9 protein in the evaluated dental tissues and cells follows the pattern described in the literature (Goldberg et al. \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e). A weaker immunostaining was observed in odontoblasts and pre-dentin; however, strong staining was seen in the enamel matrix of the animals, a fact that differs from what has been described by some authors (Goldberg et al. \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e). It can be observed that the positive MMP-9 marking in ameloblasts, odontoblasts, and pre-dentin followed the data from the literature (Palosaari et al. \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Fanchon et al. \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e; Bourd-Boittin et al. \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e; Bei \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e), although variations in its expression can be detected in the representative figures of the groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eMMP-9 has the function of degrading enamel proteins, favoring subsequent mineralization (Bartlett \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e). Previously, studies demonstrated that IT of SHR offspring exhibited lower enamel and dentin microhardness (Elias et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e) compared to Wistar offspring\\u0026rsquo;s IT. Thus, we expected changes in MMP-9 expression/immunostaining would favor altered removal of organic matrix and mineral deposition, reducing mineralization. However, when quantifying MMP-9 expression in cells and dental tissues, no significant difference was found between the IT of SHR and Wistar offspring. Although the results are suggestive, it is important to bear in mind that the presence of the protein in the tissue does not always imply its activity. A protein can be expressed but in its inactive form. For some authors, MMPs are mostly produced in their latent form, an inactive form, and their activation occurs subsequently (Cingolani and Houssay \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). It is also necessary to consider that there are other MMPs involved in forming enamel and dentin in rat incisors, which may also be contributing, to a greater or lesser extent, to the observed changes in SHR. Specific studies to evaluate the expression of other MMPs, such as MMP-20 and MMP-2, should be conducted before conclusive statements about the role of MMPs in the studied alterations. If the removal of the organic matrix is not altered in the teeth of SHR offspring, mineral deposition may occur inadequately and at a speed different from matrix degradation. Furthermore, alterations in plasma concentrations of PTH and calcium found in SHR (Fanchon et al. \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e) could actively interfere with the mineralization process of SHR incisor teeth.\\u003c/p\\u003e \\u003cp\\u003eAs previously demonstrated (Elias et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e), Atenolol is effective in reducing the SBP of SHR during pregnancy and lactation, and the treatment of female rats with Atenolol increased enamel and dentin hardness in the male offspring (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) in both groups. We could not associate the effects of Atenolol with possible alterations in MMP-9 expression (Sorsa et al. \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). As observed in this study, Atenolol treatment did not alter MMP-9 expression in the cells and tissues of SHR, and in Wistar rats, Atenolol increased MMP-9 expression only in the enamel matrix. These results suggest that the effect of Atenolol on the microhardness of enamel and dentin in SHR incisor teeth is most likely not due to mechanisms involving MMP-9.\\u003c/p\\u003e \\u003cp\\u003eThe mechanism by which Atenolol promotes increased dental mineralization has not yet been established; however, it is known that the parathyroid glands have adrenergic innervation, and the secretion of parathyroid hormone can be inhibited by beta-blockers (Elias et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). Specific studies should be conducted to determine with clarity the molecular mechanisms altered by Atenolol that would be associated with the dental mineralization process. At this point, we can suggest that MMP-9 does not participate in the mechanism by which Atenolol increases the microhardness of enamel and dentin in incisor teeth of SHR.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eThe results of this study suggest that neither hypertension nor Atenolol treatment alters MMP-9 expression in SHR incisor teeth cells and tissues.\\u003c/p\\u003e \"},{\"header\":\"Declarations\",\"content\":\" \\u003cp\\u003eThe authors declare no conflicts of interest.\\u003c/p\\u003e\\u003ch2\\u003eFunding\\u003c/h2\\u003e \\u003cp\\u003eThis research was funded by the Coordination of Improvement of Higher Education Personnel (CAPES, finance code 001) and National Council for Scientific and Technological Development (CNPq).\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eGPE: Conceptualization, Methodology, Formal Analysis, Investigation, Writing - Original Draft. HFH: Methodology, Formal Analysis, Writing - Original Draft. ARM: Formal Analysis, Writing - Original Draft. LVBP and AOL: Writing - Original Draft. FC: Formal Analysis, Statistics. ALCBD and KTS: Methodology and Writing - Original Draft. RO and CA: Conceptualization, Methodology, Validation, Data Curation, Writing - Original Draft, Supervision and Project Administration.\\u003c/p\\u003e\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\u003cp\\u003eThe data used to support the findings of this study can be made available upon request to the corresponding author.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eBartlett JD (2013) Dental Enamel Development: Proteinases and Their Enamel Matrix Substrates. ISRN Dent 2013:1\\u0026ndash;24. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1155/2013/684607\\u003c/span\\u003e\\u003cspan address=\\\"10.1155/2013/684607\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBei M (2009) Molecular genetics of ameloblast cell lineage. 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Anat Rec 224:292\\u0026ndash;316. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1002/ar.1092240219\\u003c/span\\u003e\\u003cspan address=\\\"10.1002/ar.1092240219\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSorsa T, Tj\\u0026auml;derhane L, Salo T (2004) Matrix metalloproteinases (MMPs) in oral diseases. Oral Dis 10:311\\u0026ndash;318. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1111/j.1601-0825.2004.01038.x\\u003c/span\\u003e\\u003cspan address=\\\"10.1111/j.1601-0825.2004.01038.x\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSouza A, Line S (2002) The biology of matrix metalloproteinases. Rev FOB 10:1\\u0026ndash;6\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTanabe T, Fukae M, Shimizu M (1996) Possible Actions of Metalloproteinases Found in Porcine Enamel in an Early Secretory Stage. Adv Dent Res 10:170\\u0026ndash;172. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1177/08959374960100020801\\u003c/span\\u003e\\u003cspan address=\\\"10.1177/08959374960100020801\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\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\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-molecular-histology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"hijo\",\"sideBox\":\"Learn more about [Journal of Molecular Histology](https://www.springer.com/journal/10735)\",\"snPcode\":\"10735\",\"submissionUrl\":\"https://submission.springernature.com/new-submission/10735/3\",\"title\":\"Journal of Molecular Histology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Atenolol, Hypertension, Dental Enamel, Dentin, Metalloproteinase\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4966229/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4966229/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eHypertension alters tooth formation and Atenolol, a β-blocker, reduces the blood pressure of spontaneously hypertensive rats (SHR) during pregnancy and lactation, and as demonstrated before, increases the microhardness of the SHR offspring's teeth. We hypothesize hypertension alters the expression of MMP-9 in dental structures of SHR offspring and that treating female SHR with atenolol prevents this alteration. This study aimed to evaluate the expression of matrix metalloproteinase (MMP-9) in incisor teeth (IT) in male offspring of SHR (30 days old) treated or untreated with Atenolol (At, 100 mg/kg/day, orally) during pregnancy and lactation. MMP-9 expression was evaluated in ameloblasts (AM), enamel matrix (EM), odontoblasts (OD), and pre-dentin (PD) of IT through immunohistochemical reactions (immunoperoxidase). Data were analyzed by Shapiro-Wilk and Kruskal-Wallis (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), with Dunn post-test. Histological differences were not observed between IT tissues of SHR and normotensive Wistar rats. For the first time, our data showed that MMP-9 expression in specific dental structures is not altered in SHR. Atenolol treatment increased MMP-9 immunostaining in EM of Wistar rat, however, Atenolol did not alter the MMP-9 in the IT tissues of SHR. Our results suggest that neither hypertension nor atenolol treatment alters MMP-9 expression in dental tissues of SHR.\\u003c/p\\u003e\",\"manuscriptTitle\":\"MMP-9 expression in rat pup incisor teeth is not altered by maternal hypertension or maternal atenolol treatment during pregnancy and lactation.\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-09-24 18:33:51\",\"doi\":\"10.21203/rs.3.rs-4966229/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2024-09-21T21:03:51+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-09-20T16:35:19+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"169417123097798385342793828288457221858\",\"date\":\"2024-09-11T14:39:20+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"288264416191789508506978134584140843776\",\"date\":\"2024-09-08T00:29:50+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-08-27T07:37:29+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"295666607369988769140873760191469344408\",\"date\":\"2024-08-27T00:19:59+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-08-25T13:36:30+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-08-25T13:30:14+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-08-24T03:32:32+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Journal of Molecular Histology\",\"date\":\"2024-08-23T20:18:38+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-molecular-histology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"hijo\",\"sideBox\":\"Learn more about [Journal of Molecular Histology](https://www.springer.com/journal/10735)\",\"snPcode\":\"10735\",\"submissionUrl\":\"https://submission.springernature.com/new-submission/10735/3\",\"title\":\"Journal of Molecular Histology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"b4996e9d-1c4d-4a3a-acb9-8e49d5948857\",\"owner\":[],\"postedDate\":\"September 24th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-12-02T15:58:49+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-4966229\",\"link\":\"https://doi.org/10.1007/s10735-024-10294-2\",\"journal\":{\"identity\":\"journal-of-molecular-histology\",\"isVorOnly\":false,\"title\":\"Journal of Molecular Histology\"},\"publishedOn\":\"2024-11-29 15:56:52\",\"publishedOnDateReadable\":\"November 29th, 2024\"},\"versionCreatedAt\":\"2024-09-24 18:33:51\",\"video\":\"\",\"vorDoi\":\"10.1007/s10735-024-10294-2\",\"vorDoiUrl\":\"https://doi.org/10.1007/s10735-024-10294-2\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4966229\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4966229\",\"identity\":\"rs-4966229\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}