The multifunctional role of the human skin is well known. It acts as a sensory and immune organ that protects the human body from harmful environmental impacts such as chemical, mechanical, and physical threats, reduces UV radiation effects, prevents moisture loss, and helps thermoregulation. In this regard, skin disorders related to skin integrity require adequate treatment. Lipid nanoparticles (LN) are recognized as promising drug delivery systems (DDS) in treating skin disorders. Solid lipid nanoparticles (SLN) together with nanostructured lipid carriers (NLC) exhibit excellent tolerability as these are produced from physiological and biodegradable lipids. Moreover, LN applied to the skin can improve stability, drug targeting, occlusion, penetration enhancement, and increased skin hydration compared with other drug nanocarriers. Furthermore, the features of LN can be enhanced by inclusion in suitable bases such as creams, ointments, gels , lotions, etc. This review focuses on recent developments in lipid nanoparticle systems and their application to treating skin diseases. We point out and consider the reasons for their creation, pay attention to their advantages and disadvantages, list the main production techniques for obtaining them, and examine the place assigned to them in solving the problems caused by skin disorders. Skin diseases cause significant discomfort to millions of people around the world daily. Various studies show that between 30 and 70% of the world’s population suffers from skin diseases . In mostThis review focuses on the recent advances of lipid-based nanosystems in the treatment of skin disorders. We discuss the most common types of lipid-based nanocarriers researched for dermal drug delivery and used for the treatment of skin disorders by incorporation into appropriate dosage forms, namely: Nanovesicular carriers, lipid nanoparticulate carriers, microemulsions, and nanoemulsions. The considered compositions are intended principally for local treatment and are an attempt to reflect the current picture of development in this field.The skin is the largest metabolically active organ of the human body. Its vital functions include protection from external environmental threats, vitamin D synthesis, and the maintenance of the body’s dynamic balance ,8,9. MorIn short, the skin consists of three main layers—epidermis, dermis, and hypodermis (subcutaneous fat tissue) .The superficial part of the skin is called the epidermis. In essence, it is a laminar, squamous corneal epithelium composed mostly of two types of cells: Keratinocytes and dendritic cells (antigen-presenting cells) .The epidermis consists of five layers—the stratum corneum, stratum lucidum, stratum granulosum (granular ply), stratum spinosum (spinous ply), and stratum germinativum  . No bloostratum corneum (SC) (10–20 µm), plays a fundamental role as the body’s first and main physical skin barrier from external menaces [The outermost sublayer of the epidermis, named  menaces ,20,21. I menaces . The SC  menaces . The strThe permeation of matter through the SC is possible primarily through passive diffusion in three ways: Transcellular (the bulk of flux), intercellular, and appendageal ,25,26. FKeratinocytes, melanocytes (melanin producers), Merkel cells (sensory receptors), and Langerhans cells (immunocompetent cells) are epidermal formations—essential for the skin’s vitality . In addiThe skin can be affected by various pathological changes, i.e., inflammatory, neoplastic, traumatic, hormonal, degenerative, and even hereditarily determined . InfectiIn practice, most of the skin disorders are complicated, polygenic, and multifactorial . This inThe human skin creates a vast opportunity for drug delivery application. In general, dermal  and transdermal skin drug delivery can be differentiated. Dermal delivery is the application of the drug directly at the place of action—on the skin’s surface. Transdermal drug delivery is an alternative, painless, and non-invasive approach used to deliver drugs for therapeutic use .Many conventional topical preparations are intended for topical delivery of the drug and not for systemic action. This skin preparation delivers a concentrated amount of the active ingredient for absorption via the application layer . In addiSome of the commonly used therapeutic solutions are listed in For chronic inflammatory skin diseases, a topical treatment is often not too efficient. Therefore, more efficacious medicinal products (MP) are given systemically, where they can be immunosuppressive, and their long-term usage is not recommended as they suppress the affected area .A large part of therapeutic indications is treated by MP, which are intended for topical administration . These MThe barrier function of the targeted biologic membrane provides a significant challenge for optimal therapy. The efficacy of drug delivery and the therapeutic effect depend mainly on the diffusion affinity of the drug substance and the interaction between the excipients of the formulation and the membrane components. Therefore, the conducive balance between potency and deliverability has to be ensured through the design and development of a delivery system to reach optimum therapeutic levels at the site of infection .Biologics have become increasingly popular as a targeted treatment. Biologics are products composed of sugars, proteins, nucleic acids or complex combinations of these substances. Several biologics that target specific subgroups of cells in the skin have been tested .Empirical experience shows that conventional topical preparations suffer from certain limitations and are compromised in patient compliance, safety, and efficacy of therapy . AgainstAn inventive strategy for improving the penetration of molecules through the epidermal barrier is the application of nanocarriers due to the advantage of their lipophilicity, which mediates the passage through the intact lipid layer. The use of lipid nanoparticles in dermal formulations provides several benefits: Chemical protection of the incorporated drug molecules, application to the skin of labile drug substances, improved bioavailability of drugs, and the ability for better release by provision penetration and retention in the skin .Lipid-based nanosystems have proven to be suitable for dermal carriers due to their biocompatibility, efficient delivery of active ingredients, and stability. In addition, their enhanced surface leads to the improved penetration of active ingredients .Lipid-based drug delivery systems (LBDDS) are formulations containing a dissolved or suspended drug substance in lipidic excipients . LBDDS aLiposomes  are considered to be the first generation of nanovesicular carriers. They are small artificial vesicles of the spherical shape created from cholesterol and natural, non-toxic phospholipids with an enclosed inner aqueous core . There aThe second generation of nanovesicular carriers—transfersomes were developed in 1992 by Cevc et al. They are modified liposomes with an average diameter below 300 nm and contain an edge activator that makes transfersomes nearly eight times more flexible than the conventional liposomes ,86,87. EThe usefulness of different lipid vesicles prompted researchers to experiment with modifications to give them specific structural or application properties . HoweverAnother nanovesicular carrier widely used for dermal drug delivery is called niosomes, with an average particle size between 50 and 200 nm . These aPsoriasis is a skin disorder characterized by impaired epidermal differentiation, commonly treated by systemic methotrexate, an effective cytotoxic drug. Abdelbary and Abou Ghaly generated topical methotrexate-loaded niosomes for the influence on psoriasis . A thin-Perez et al. prepared ultra-deformable liposomes containing amphotericin B to treat cutaneous fungal infections and leishmaniasis . LiposomGarg et al. developed ethosome-based nanohydrogel formulations of methoxsalen to effectively treat vitiligo with enhanced topical delivery . The foracne vulgaris include:propionibacterium acnes bacteria in pilosebaceous units of the skin;The proliferation of Local inflammation .The two main processes that are typical for propionibacterium acne [®.Traditional topical anti-acne compositions mainly cause burning, erythema, photosensitivity, scaling, and bacterial resistance . In 2008ium acne . The eth® [Acyclovir has been investigated for the topical treatment of viral infections for more than four decades . In 1999® . Recentl® . The surIn the experimental work, Babaie et al. prepared lidocaine-loaded nanoethosomes for penetration into the deep strata of the skin with a particle size around 100 nm . IncreasIn 2005, Godin et al. developed an ethosomal system for the dermal delivery of antibiotics to improve their penetration through the SC and the bacterial membrane/cell wall ,143. In A report by Cosco et al. represented the formation of transfersomes for the combined delivery of resveratrol and 5-fluorouracil. The co-encapsulation of the drugs synergistically improved their anti-cancer activity on skin cancer cells .Solid lipid nanoparticles (SLN), as well as nanostructured lipid carriers (NLC), are extensively employed in cutaneous delivery systems. Since their creation in the nineties, lipid nanoparticles (SLN and NLC) are well known by the research and pharma technology community. Easily available raw materials, relatively simple production methods, biocompatibility, and non-toxicity as their advantages over other colloidal carriers can be mentioned as the main reasons for this .Therefore, these two types of lipid nanoparticles—SLN and NLC, are classified according to their structure. First, SLN are developed with a composition of solid lipids only. Then, to upgrade to SLN, NLC were created by representing a mixture of solid and liquid lipids, with a predominant solid lipid .® from Dr. Rimpler GmbH  and Nanobase® from Yamanouchi  [The dermal use of SLN and NLC is proving to be one of the most convenient for therapeutic and cosmetic purposes, despite various applications to date. Lipid nanoparticles are aqueous dispersions with low viscosity for successful direct application to the skin, which implies their incorporation in semisolid systems based on SLN or NLC. One of the first successful administered and marketed products based on NLC is Cutanova, Japan) .Generally speaking, SLN are nanometric colloidal carriers composed of a solid lipid core with an incorporated active pharmaceutical ingredient(s) (API) and a surfactant-stabilized shell ,178,179.The second generation of lipid nanoparticles—NLC, are composed of a mixture of solid lipids and liquid lipids in the nanocore, usually in a ratio of 7:3 to 9:1 . This leHot homogenization—the lipids are heated above their melting point;Cold homogenization—takes place at low temperatures and is suitable for hydrophilic and temperature-sensitive API ,187.The literature describes a significant number of production methods and many different combinations of lipids to obtain SLN and NLC. Nevertheless, the most common technique used today is high-pressure homogenization (HPH). The procedure is divided into two stages:Other commonly used techniques are: Sonication/ultra-sonication ,189, memIn dermal applications, SLN and NLC create a thin hydrophobic monolayer during skin contact, which has a pointed occlusive effect that settles the API penetration and prevents water loss from the skin .When applied topically, the lipid nanoparticles interact with the sebum and specific skin lipids, provoking a change in the natural arrangement of corneocytes. As a result of this interaction, the encapsulated molecules are released, and their penetration into the lower layers of the epidermis and dermis is potentiated, depending on their lipophilicity, of course .Okonogi and Riangjanapatee formulated NLC loaded with lycopene through a hot HPH. It has been found that the NLC with the highest concentration of lycopene had the slowest release rate and better antioxidant activity .In another study, Shrotriya et al. reported the development of SLN loaded with resveratrol (entrapment efficiency of 86–89%) to treat irritant contact dermatitis (chronic skin disorder with eczematous injuries). The composition was realized by incorporation into a Carbopol gel and showed increased antioxidant activity compared with a conventional resveratrol gel .Furthermore, Montenegro et al. designed a novel Idebenone (IDE)-loaded NLC containing tocopheryl acetate (VitE) as a liquid component to obtain a synergic effect between IDE and VitE .Pivetta et al. formulated NLC with thymol for the local treatment of inflammatory skin diseases (entrapment efficiency of 89%). The NLC were incorporated into a gel and showed anti-inflammatory activity and healing of induced psoriasis in mice .Gad et al. reported the encapsulation of chamomile oil in SLN for the local treatment of wounds. The composition contained stearic acid and chamomile oil and was prepared by the method of hot homogenization. Wound reduction was shown in the topical application in rats .Butani et al. developed a stable SLN system, containing amphotericin B with an enhancing antifungal activity (entrapment efficiency of 94%). The formulation indicated higher drug permeation and drug accumulation in the skin than the conventional gel in rats. A solvent diffusion technique was used for the preparation of the SLN .NLC, for the treatment of candidiasis with Mediterranean essential oils and clotrimazole, were designed by Carbone et al. As a result, they are obtained as a stable NLC, without an initial burst effect and with prolonged release of clotrimazole, as well as an enhanced antifungal activity .Tretinoin-loaded NLC with anti-aging and anti-acne activities were reported by Ghate et al. The hot melt probe sonication and hot melt microemulsion methods were used to prepare the NLC. The tretinoin-loaded NLC in Carbopol gels showed no irritation or erythema after the application in rats .Malik and Kaur developed the azelaic acid-loaded NLC, prepared by the melt emulsification and ultra-sonication method (entrapment efficiencies greater than 80%). NLC were incorporated into aloe-vera-based Carbopol hydrogels and demonstrated a deeper skin penetration than the commercial product (Aziderm 10%). Furthermore, the in vivo experiment in mice showed a higher effect of NLC incorporated into a gel than the plain drug suspended in the gel .In general, microemulsions and nanoemulsions are dispersion systems composed of two immiscible liquid phases that can penetrate deeper levels of the skin . EvidencIt has been found that microemulsions are spontaneously formed, transparent, and isotropic thermodynamically stable dispersion systems. The composition of the droplets is carried out by the precise mixing of volumes of immiscible liquids  and the interfacial film of stabilizing surfactants at specific pressures and temperatures . Short aOil-in-water (O/W) microemulsion;Water-in-oil (W/O) microemulsion;Bicontinuous microemulsion .Three various structural types of microemulsions can be formed:Nanoemulsions typically contain 20–500 nm large droplets and have a different appearance depending on their size. Traditionally, they are stabilized by surfactants and do not change in the long term . HoweverThe clobetasol propionate- and calcipotriol-loaded nanoemulsion gel for the topical treatment of psoriasis is reported, developed, and optimized by Kaur et al. The spontaneous emulsification method was used for the preparation. Compared with the other commercial MP , the nanRecently, Rajitha et al. reported the preparation of loaded nanoemulsion based on chaulmoogra oil, which is based on the self-emulsification method. Compared with the conventional methotrexate solution, the nanoemulsion showed enhanced skin permeation and retention of methotrexate in the deep skin layers .® cream, the optimized microemulsion-loaded hydrogels showed higher in vitro flux values, higher release rate, and higher in vitro antifungal activity against Candida albicans [In another study, Coneac et al. reported the development of microemulsion-loaded hydrogels for the topical delivery of fluconazole. Nonionic surfactants have been used to stabilize the microemulsions, which then were incorporated in Carbopol gels. Compared with the conventional hydrogel and Nizoralalbicans .Two nanoemulsion systems for the dermal application of natural or synthetic mixtures of pentacyclic triterpenes, with an anti-inflammatory effect, were reported by Alvarado et al. . Slightl®) [In another study, Goindi et al. reported an ionic liquid-in-water microemulsion formulation that can solubilize etodolac, a poorly water-soluble anti-inflammatory drug. An effective permeation profile, as well as anti-arthritic and anti-inflammatory activities are evaluated in vivo in different models compared with a marketed formulation of etodolac (Proxym gel®) .Lv et al. reported the preparation of essential oil-based microemulsions for topical application in order to improve the solubility, photostability, and skin permeation of quercetin. First, self-micro emulsifying DDS were prepared and then formed microemulsions. The microemulsions protected quercetin from degradation in an alkaline environment and under UV radiation. In these formulations, the in vitro skin permeation study on rats showed 2.5–3 times enhanced permeation capacity of quercetin compared with the conventional aqueous solution .To optimize the percutaneous absorption of lidocaine and prilocaine, Negi et al. formed nanoemulsions using the high-shear mixing method followed by the HPH. The optimized nanoemulsion systems showed higher permeation rates and permeability coefficient values in parallel with the marketed cream and were further incorporated into a Carbopol hydrogel. In addition, the nanoemulsions and the nanoemulsion gel had a stronger anesthetic effect in vivo than the commercial product .® for the accompanying therapy of skin carcinomas. Different preparation methods were used. The technique, combining a single-phase in-version temperature homogenization method with ultrasonication, produced a stable Tocomin®-loaded nanoemulsion, which demonstrated an exceeding cytotoxic profile against two human cutaneous carcinoma cell models [In the last few years, Pham et al. developed a nano-emulsification approach to optimize the incorporation of Tocominl models .The size of Global Topical Drug Delivery Market was estimated at USD 95.08 billion in 2020 and expected to reach USD 101.10 billion in 2021 and USD 140.01 billion by 2026. The current market situation  for the topical skin products, shows domination of generic products—about 74% of all the approved topical products are generic equivalents of reference medicines (RLD). According to the collected data, gels, creams, ointments, lotions, and solutions dominate the market for both topical reference and topical generic products. The available semi-solid, solid, and liquid topical products contain different combinations of surfactants, oils, water, colloidal, and solid ingredients in solutions or dispersions .Specific needs of skin, affected by inflammation, acne, or infections require adequate drug therapy with appropriate topical dosage forms. For example, the adhesive patch can provide a sustained and controlled release, while the gel can provide a faster and more intense action. On the other hand, some topical dosage forms may not be most suitable for application to certain areas of the skin, around the eyes, for example .One of the most important considerations in the development of the topical dosage forms is the patient need. The second consideration is the drug’s physicochemical properties. In general, regulatory procedures for the registration of topical products are slow, as clinical equivalence studies  involve a high number of participants, require time and significant costs to ensure a sufficiently objective assessment of the final therapeutic effect. Technological or cost problems are among the reasons for additional difficulties in the implementation of promising dermatological products .Precise delivery across the skin and to certain skin strata, depending on the final target;Successful elimination of lipid nanomaterial toxicity threats in topical medical formulations and cosmetics;Ensuring improved permeation and low skin irritability as a result of the use of lipid nanocarriers;Improved cutaneous release of incorporated API with a broad spectrum of physiological and physicochemical properties.It can be generalized that the main challenges in the development of cutaneous lipid nanometric delivery systems include:Lipid nanoparticulate DDS can be employed intensively for the delivery of phytomedicines intended for topical administration. The approach can be promising in this regard, considering the difficulties in their delivery which is caused by their physicochemical properties.The formulation of phytopreparations with lipid nanoparticles would find a useful application in nanomedicine at the desired targeted delivery, for example, in cancer treatments.Skin disorders represent a progressively emerging clinical public health problem. Treatment strategies based on conventional formulations are non-specific and can lead to considerable systemic toxicity. The progressive approach of the use of lipid nano-formulations as skin drug delivery systems can provide an incomparable prospect for the application of highly competent and safe treatments with the improved benefit-risk ratio.The use of lipid nanoparticlulate DDS is favored recently due to the GRAS status of the excipients. Lipid nanocarriers can effectively protect the API from degradation on the skin’s surface, increase their concentration gradient in the upper skin layers, and enable gradual release. Lipid nanoparticles for topical application could be formulated with the high content of lipid matrix or dispersed in different foundations.Lipid nanosystems provide a promising, flexible platform for the safe, effective, and biocompatible topical delivery of the API, as they do not cause cytotoxicity or morphological changes in the skin layers. The interest shown by pharmaceutical scientists, in the development of lipid nanoparticle delivery systems, may offer a future that provides sufficiently efficient lipid nanoparticle products for needy users.


Adrenomedullin (AM) has high expression in the spinal cord. In this study, we investigated the expression of AM and its receptor components, including calcitonin receptor-like receptor (CLR) and receptor activity modifying proteins (RAMPs) in dorsal root ganglion (DRG) and spinal motor (SM) neurons. Furthermore, the effects of AM on cAMP/cAMP response element-binding protein (CREB), AKT/glycogen synthase kinase-3 beta (GSK-3β) signaling pathways, and expressions of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were evaluated. Rat embryonic DRG and SM neurons were isolated, purified, and cultured. Real-time PCR was used to assess expressions of AM, CLR, and RAMPs. cAMP levels, p-CREB, BDNF, and NT-3 were determined using an enzyme-linked immunosorbent assay. p-AKT and p-GSK-3β levels were determined by western blotting. Real-time PCR showed expressions of AM, CLR, RAMP2, and RAMP3 in both DRG and SM neurons. AM increased cAMP accumulation and p-CREB levels in DRG and SM neurons. AM increased p-AKT and p-GSK-3β in DRG, but not SM neurons. AM significantly increased BDNF expression in both DRG and SM neurons. There was also an increase in NT-3 level in both DRG and SM neurons, which is statistically significant in SM neurons. These results showed both DRG and SM neurons are targets of AM actions in the spinal cord. An increase in BDNF expression by AM in both DRG and SM neurons suggests the possible beneficial role of AM in protecting, survival, and regeneration of sensory and motor neurons.  However22–52 -8. Anima22–52 , formali22–52 , capsaic22–52 , suggest22–52 . cAMP/pr22–52 , stimularalgesia . Brain-derived neurotrophic factor (BDNF) and Neurotrophin 3 (NT-3) are members of the neurotrophins family, essential for the growth, differentiation, and survival of neurons via activating tropomyosin receptor kinase receptors. BDNF is normally expressed in DRG neurons as well as sensory and motor neurons of the spinal cord. BDNF expression increases in the DRG and sensory neurons in response to various nociceptive stimuli, suggesting its role in pain processing . Motor nTo the best of our knowledge, no study has addressed the expression of AM and its receptor components, including CLR and RAMPs in the motor neurons of the spinal cord, yet. Herein, we evaluated and compared the expression of AM, CLR, and RAMP2-3 in dissociated rat DRG and motor neurons. We also investigated the effects of AM on the activation of cAMP/CREB and AKT/GSK-3β pathways in both DRG and motor neurons and the expression of BDNF and NT-3. ChemicalsRat AM and AM 22-52 were obtained from Bachem Americas, Inc. . All chemicals and western blot materials used in this study were obtained from Sigma-Aldrich, Santa Cruz, and Abcam. ELISA kit was obtained from Promega.Animalsad libitum under a 12 hr light/dark cycle. On day 14 rats were decapitated after CO2 inhalation. All experimental protocols were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Medical and Research Ethics Committee of the Shiraz University of Medical Sciences, Shiraz, Iran. Female Sprague-Dawley rats, weighing 200–250 g (n=120), were provided by the Laboratory Animal Center of Shiraz University of Medical Sciences, Shiraz, Iran. Rats were mated, and detection of the vaginal plaque was considered the first day of pregnancy. Rats were housed 4 to a cage in transparent cages (59×38×20 cm) and received water and food Isolation and culture of embryonic rat DRG and SM neurons et al.  and uridine at a final concentration of 20 mM for 72 hr. Anti-β III-tubulin  monoclonal fluorescent antibody and DAPI staining were used to determine the purity of the DRG neurons . Three distinct cellular layers  were separated  and uridine at a final concentration of 20 mM for 72 hr  t values of two genes were calculated as the percentage or fold difference compared with control.The expression of AM, CLR, RAMP2, and RAMP3 was measured using quantitative real-time PCR. Briefly, total RNA from DRG and SM neurons were extracted, using BIOZOL-RNA; RNA extraction reagent , and the cDNA was synthesized by a cDNA Synthesis Kit , using the manufacturers’ instructions. SYBR Green Real-time PCR  was conducted to determine gene expressions of AM, CLR, RAMP2, RAMP3, and β-Actin, using specific primers . Each gecAMP assay −11 to 10− 6 M) in the presence or absence of AM receptor antagonist (AM 22-52) for 10 min before terminating the reaction by adding ice-cold ethanol (100%). The cells treated with adenylyl cyclase activator, forskolin (1 μM), served as the positive controls. The cAMP level was measured by a specific ELISA kit  and in accordance with the manufacturer’s instructions.To evaluate AM’s effect on cAMP accumulation , DRG andQuantification of phosphorylated-CREB (p-CREB) in DRG and SM neuronsThe aforementioned protocol  was used to determine AM’s effect on the level of p-CREB/CREB. Briefly, DRG and spinal cells were incubated with AM (25 nM) for 20 min. The p-CREB (Ser133) level was assayed in the cell lysate by DuoSet IC Phospho-CREB (S133) ELISA kit (R&D Systems). Western blot °C for 5 min, the proteins (20 µg) were separated by 12.5% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Non-fat dry milk (5%) and TBST  were used as blocking buffer (1 hr at room temperature). Incubation with primary antibodies was performed overnight at 4 ºC. HRP-conjugated secondary antibody  was applied for 2 hr at room temperature. Washing with 1×TBST was performed before adding primary and secondary antibodies. Enhanced chemiluminescence (ECL) reagent (ab133406) was finally added to the membranes for 5 min. The ChemiDoc TM MP System  was used to visualize the bands. Protein expression was estimated by the dosimetry software Image Lab (Bio-Rad).Western blotting was used to detect AKT , p-AKT (Ser 473) , GSK-3β , and p-GSK-3β (Ser 9) . β-Tubulin  was used for normalization. The protocol was conducted as previously reported . NP40 lyEnzyme-linked immunosorbent assayBDNF and NT3 concentrations were measured using ELISA kits . Total protein was measured by the Bradford method. Results were normalized to the total protein concentration.Statistical analysispost hoc analysis. Differences were considered significant at P<0.05. pro-BDNF protein expression’s densitometric data were analyzed using the Mann Whitney U test, and P<0.05 was considered a significant difference between the groups. EC50 antagonist/EC50 agonist was calculated by plotting sigmoidal concentration-response curves. The pA2 values were calculated by log[antagonist] - log(EC50 antagonist/EC50 agonist - 1).Graph pad Prism program  and SPSS software (version 21) were used for statistical analysis. Data are presented as mean ± SEM. Differences between groups were assessed by ANOVA followed by the Student-Newman-Keuls Purification and culture of DRG and spinal motor neurons . To prepare DRG neurons’ culture, DRGs were isolated from rat embryos  in DRG and SM neurons. The data revealed expressions of AM, CLR, and RAMP-2 and -3 in both neurons . No signCharacterization of AM receptors using the cAMP assay50=85.76 nM) and SM neurons (EC50=103.3 nM). The peak of cAMP levels was observed at 1 μM AM, which were 16.6 and 9.5 times higher than the basal levels in DRG and SM neurons, respectively , an adenylyl cyclase activator, significantly elevated cAMP accumulation in both DRG (54.04±0.7) and SM neurons (53.62±45). AM increased cAMP accumulation dose-dependently in both DRG   and p-GSThe effects of AM on BDNF and NT3 expression in DRG and spinal motor neurons To investigate the effects of AM on expression of BDNF and NT3, DRG and SM neurons were treated with AM (25 nM), and the levels of these proteins were measured using specific ELISA methods. The result of ELISA showed a significant increase in the BDNF protein level in both DRG and SM neurons after treatment with AM (25 nM) . There w50=85.76 nM) and SM neurons (EC50=103.3 nM) in a manner that was sensitive to antagonistic effects of AM22-52, suggesting receptor-mediated AM effects. Furthermore, the p-CREB level increased in response to AM treatment, suggesting the cAMP/CREB signaling pathway’s possible role in AM-mediated effects in both DRG and SM neurons. An increase in phosphorylated AKT/GSK-3β following AM treatment suggests that this pathway might also be involved in AM effects in DRG neurons but not in SM neurons. The expression of BDNF increased in both DRG and SM neurons following treatment with AM. As far as we know, this is the first report on AM expression and function in the SM neurons. In the present study, the data revealed expression of AM and its receptor components, CLR and RAMP-2 and -3, in both DRG and spinal motor neurons. Dose dependently, AM increased cAMP accumulation in both DRG  and p-CREB level in the DRG neurons. Similarly, AM increased cAMP accumulation (EC50=103.3 nM) and p-CREB level in the SM neurons. These effects were antagonized by AM22-52, a selective antagonist for AM receptors  and SM neurons (EC50=103.3 nM), suggesting the presence of various kinds of receptors with different affinities for AM in DRG and SM neurons. Finally, although the expression of AM receptor components was not significantly different at the mRNA level in the DRG and SM neurons, however, the expression of these components may differently be regulated at the protein and post-translational levels in the DRG and SM neurons, leading to formation of AM receptors with various affinities that activate various signaling pathways. It was reported that activation of the AKT/GSK-3β signaling pathway contributes to the protective effects of AM against apoptosis induced by several conditions, such as hypoxia-induced apoptosis in mesenchymal stem cells , lipopolBNDF and NT3 are normally expressed in both DRG and SM neurons , 48. OurOur findings revealed expression of AM and its receptor components in both DRG and SM neurons. Moreover, AM increased cAMP and p-CREB accumulation in both DRG and SM neurons. AM also induced phosphorylation and inactivation of GSK-3β in DRG, but not SM neurons. Finally, AM induced BDNF expression in both DRG and spinal motor neurons. Considering the known activities of BDNF, AM can serve as a protective and surviving factor for both DRG and SM neurons.


Further, we show that MRGPRX2-containing macropinosomes undergo resolution by a mechanism that involves dynamin and LC3, giving rise to the incorporation of both LC3 and MRGPRX2 into the SGs. SP then promotes the acidification of the LC3-associated SGs, presumably by stimulating their fusion with lysosomes. Taken together, our results reveal a unique mode of MRGPRX2 trafficking that complements endocytosis and involves macropinocytosis, autophagic machinery-assisted macropinosome resolution and receptor delivery to the SGs.MRGPRX2, the human member of the MAS-related G protein coupled receptors (Mrgprs), serves as the cellular target of human mast cells (MCs) for innate ligands, including neuropeptides and antimicrobial peptides. In addition, MRGPRX2 also functions as the receptor for multiple FDA-approved drugs. As such, MRGPRX2 is a mediator of MC responses in neurogenic inflammation, host defense and pseudoallergy. We analyzed the spatiotemporal patterns of MRGPRX2 following its binding of the neuropeptide substance P (SP). Herein, we show that MRGPRX2 internalizes  Taken tIn particular intriguing are the responses of a subset of MCs to a variety of ligands, including neuropeptides, antimicrobial peptides and toxins, that share commonly only their positive charge , 10. MC The cellular functions of G protein coupled receptors (GPCRs) are tightly linked with their cellular positioning. Internalization and post-endocytic trafficking may terminate GPCR signaling or may prolong or diverse it –18. Ther® 488 Goat Anti-Rabbit IgG H&L  and secondary Alexa Fluor® 647 Goat Anti-Mouse IgG H&L (cat no. ab150115) were from Abcam . Substance P (cat no. S6883), pitstop2 (cat no. SML1169) and saponin (cat no. S4521-25G) were purchased from Sigma-Aldrich . Cytochalasin D (cat no. 1233), dynasore (cat no. 2897) and EIPA (cat no. 3378) were from Tocris Bioscience . Tetramethylrhodamine (TRITC)- labeled 70 kDa dextran was purchased from Thermo Fischer Scientific .Anti-human MRGPRX2 antibody (cat no. 359002) and anti-HA.11 Epitope Tag antibody (cat no. 901513) were from Biolegend . Anti-syntaxin 3 antibody (cat no. ab133750), secondary Alexa Fluorhttp://n2t.net/addgene:21073; RRID : Addgene_21073) and LC3-EGFP-mRFP (ptfLC3) was a gift from Dr. R. Pinkas Kramarski , originally a gift from Dr. Tamotsu Yoshimori . Lifeact-GFP was a gift from Dr. B.-Z. Shilo  and pcDNA3-EGFP-Rac1-T17N was a gift from Dr. Gary Bokoch . pcDNA3-EGFP was a gift from Dr. J. Silvio Gutkind .The following expression plasmids were used in this study: Neuropeptide Y (NPY)–mRFP and NPY Venus were a gift from Dr. U. Ashery , NPY-CFP was a gift from Dr. P. Blinder, . pEGFP-LC3 was a gift from Dr. A. Ashkenazi , originally a gift from Tamotsu Yoshimori  supplemented with 10% FBS , 2 mM L-Glutamine , 100 μg/mL streptomycin and 100 U/mL penicillin, 12.5 U/mL nystatin  and 1 mg/mL of G418 . LAD-2 cells  were cultured in StemPro-34  supplemented with 1x StemPro-34 Nutrient, 2 mM L-Glutamine , 100 U/mL penicillin and 100 μg/mL streptomycin , and 100 ng/mL hSCF .RBL cells stably expressing N-terminally tagged hemagglutinin (HA) human MRGPRX2 (RBL-MRGPRX2) were generated as previously described . Cells w7) were transfected with 30-50 µg cDNA by electroporation at 300 V for 20 ms, using an ECM 830 electroporator . The cells were immediately replated in either 24-well (1 x 105 cells/well) tissue culture dishes for confocal imaging or in 6-well (0.2 x 106 cells/well) tissue culture dishes for flow cytometry experiments containing growth medium and used within 20-24 h after transfection.Transient transfection of RBL-MRGPRX2 cells was performed as previously described . Briefly5 cells/well) were incubated with 0.1 mg/mL of TRITC- labeled 70 kDa dextran  in Tyrode’s buffer  for the desired time periods, without or with 10 µM SP, in 24-well dishes at 37°C and 5% CO2. Where indicated, cells were preincubated for 30 minutes (min), prior to SP trigger, with vehicle or with the indicated inhibitor(s). Cells were washed three times with ice-cold PBS and fixed for 20 min at room temperature (RT) with 4% paraformaldehyde  followed by permeabilization for 15 min with 0.1% Triton X-100, 5% FBS, and 2% BSA diluted in PBS. Subsequently, cells were labeled with mouse anti-HA (1:100 dilution) antibody for 1 hour (h) at RT, followed by three washes and a 1-h incubation with Alexa Fluor® 647 Goat Anti-Mouse IgG H&L secondary antibody (1:500 dilution).RBL-MRGPRX2 cells (1 x 105) or LAD-2 (6 x 105) cells were stimulated at 37°C and 5% CO2, in Tyrode’s buffer for the desired time periods. Cells were then washed three times with ice-cold PBS containing 0.5% BSA and 2 mM sodium azide (FACS buffer) followed by fixation in 4% paraformaldehyde for 15 min at RT. For analyses of cell surface MRGPRX2, cells were stained for 30 min at RT with anti-human MRGPRX2 antibody (1:300 dilution). Cells were subsequently washed three times and stained with Alexa Fluor® 647-conjugated Goat Anti-Mouse IgG H&L secondary antibody (1:2000 dilution) for 30 min at RT, in the dark. For total receptor expression, cells were washed and fixed for 15 min at RT with 4% paraformaldehyde. Cells were subsequently washed and permeabilized with 0.1% saponin and 10% FBS containing PBS for 15 min at RT, after which the cells were washed and stored in the permeabilization buffer overnight at 4°C. The permeabilized cells were then stained for 30 min at RT with anti-human MRGPRX2 antibody (1:300 dilution), washed three times with the permeabilization buffer and stained with the secondary antibody as above. Cells were washed three times with FACS buffer and analyzed by flow cytometry using a CytoFLEX LX flow cytometer . A minimum of 10,000 cells was acquired per sample. Cells were gated on single cells and were additionally gated on GFP-positive cells in experiments involving transient transfection with either dominant negative (DN) Rac1-GFP or control GFP. Data was analyzed using the FlowJo™ Software Version 10 .RBL-MRGPRX2 cells were washed three times in Tyrode’s buffer to remove dead, non-adherent cells. RBL-MRGPRX2 (5 x 105) and LAD-2 (0.6 x 105) cells grown on untreated (RBL-MRGPRX2) or fibronectin  coated (LAD-2) 12-mm round glass coverslips  were washed three times in Tyrode’s buffer, the cells were stimulated as indicated in the same buffer at 37°C and 5% CO2. The cells were subsequently washed three times with ice-cold PBS and fixed for 20 min at RT with 4% paraformaldehyde  in PBS. Fixed cells were permeabilized for 15 min at RT with 0.1% Triton X-100, 5% FBS, and 2% BSA diluted in PBS. Cells were then incubated for 1 h at RT with the desired primary antibodies followed by three washes and a 1-h incubation with the appropriate secondary antibody (1:500 dilution). After washing, cells were mounted in mounting medium  and analyzed with a Leica SP5 or SP8 TCS laser scanning confocal microscope  equipped with a HyD detector and a ×63 oil/1.4 NA objective or a Zeiss LSM 710 confocal microscope equipped with a 63 x1.4 oil Plan-Apochromat objective. Colocalization analysis of NPY-CFP with immunostained MRGPRX2 was quantified as the Manders’ correlation coefficient with Costes’ automatic threshold using the JaCoP plugin of the extended ImageJ version Fiji . One-way analysis of variance (ANOVA) with repeated measures followed by Dunnett’s post-test or Student’s or Welch’s t-test was used for comparing means, respectively according to the statistic requirements. Results were considered significant when P values were smaller than 0.05.Given the profound differences between the human MRGPRX2 and its rodent orthologs, we used the RBL-MRGPRX2 cells , that stFurther analysis of the first phase of MRGPRX2 internalization in RBL-MRGPRX2 cells revealed that MRGPRX2 endocytosis was sensitive to pitstop2, an inhibitor of both clathrin-dependent endocytosis and clathrin-independent and Arf6-dependent endocytosis , 27 Fig, signifiCell surface expression of MRGPRX2 was also reduced by dynasore, an inhibitor of dynamin, that executes membrane fission events  , a commonly used probe for measuring macropinocytosis , and comocytosis , 33  mutant of Rac1 [i.e. Rac1(T17N)], a key player of actin rearrangements during macropinocytosis , 35, on We have previously shown that early endosomes fuse with the SGs in MCs , thus prTo further decipher the connection between macropinocytosis and MRGPRX2 trafficking to the SGs, we tested the impact of dynasore, that inhibits endocytosis, on the cellular distribution of MRGPRX2 in the LC3-GFP-expressing cells. Consistent with its profound impact on the macropinosomes’ size (-/NPY+) may still contain LC3-GFP, the fluorescence of which is quenched  and the inhibition of its targeting to macropinosomes by either EIPA or expression of DN Rac1, both known inhibitors of macropinocytosis , and grant 2017182 by the United States– Israel Binational Science Foundation (to RS-E). PL-H was supported through The American Association of Immunologists Careers in Immunology Fellowship Program. This work was also supported by National Institutes of Health grant R01-AI124182 (to HA).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Vaccination is the most effective intervention to prevent influenza. Adults at risk of complications are among the targets of the vaccination campaigns and can be vaccinated with different types of quadrivalent influenza vaccines (QIVs). In the light of assessing the relative immunogenicity and efficacy of different QIVs, a systematic review was performed. Randomized controlled trials conducted in adults aged 18–64 years until 30 March 2021 were searched through three databases . Twenty-four RCTs were eventually included. After data extraction, a network meta-analysis was not applicable due to the lack of common comparators. However, in the presence of at least two studies, single meta-analyses were performed to evaluate immunogenicity and efficacy; on the contrary, data from single studies were considered. Seroconversion rate for H1N1 was higher for standard QIVs, while for the remaining strains it was higher for low-dose adjuvanted QIVs. For seroprotection rate, the recombinant vaccine recorded the highest values for H3N2, while for the other strains, the cell-based QIVs achieved better results. In general, standard and cell-based QIVs showed an overall good immunogenicity profile. Nevertheless, as a relative comparative analysis was not possible, further research would be deserved. Influenza is a major cause of disease burden globally . WorldwiTwo influenza A subtypes (A/H1N1 and A/H3N2), and two antigenically and genetically distinct B lineages (B/Victoria and B/Yamagata) are the current seasonal influenza strains circulating since 1985. Both influenza A subtypes and both B lineages co-circulate, and their frequency distribution varies widely by season and geographic region .Influenza vaccination is the most effective intervention available to prevent influenza infection and its complications and represents a major public health initiative .The Advisory Committee on Immunization Practices (ACIP) recommends influenza vaccination to all people aged 6 months and older, without any contraindications .As influenza viruses rapidly mutate, and the prophylactic effects of vaccination wane over time, vaccine formulations are updated each year in compliance with the World Health Organization (WHO) and Committee for Medicinal Products for Human Use (CHMP) recommendations (in the EU) ,5.Up to 2012, influenza vaccines have been produced to protect against three different seasonal influenza viruses, namely A/H3N2, pandemic A/H1N1, and one out of the two influenza B lineages .The first quadrivalent influenza vaccines (QIVs) were licensed in the US in 2012 ,8. CurreEven if it is challenging to compare the immunogenicity and efficacy of different types of influenza vaccines , there aTogether with children aged 6 months–5 years, pregnant women, and the elderly, the adult population also represents a target group of influenza vaccination [In adulthood, inactivated QIVs have already been compared with inactivated trivalent influenza vaccines (TIVs) with respect to immunogenicity and safety . NeverthThe systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analyses 2020 (PRISMA 2020) guidelines  and regiA comprehensive literature search was carried out from databases’ inception up to 30th March 2021. Medline, Scopus and Cochrane Central Register of Controlled Trials databases were used to identify all the original articles investigating the immunogenicity or the efficacy of any QIV in people aged from 18 to 64 years. The medical subject headings (MeSH) and key words used for the search are reported in the Evaluated any QIV compared to placebo or TIV or another QIV in adults aged 18–64 years;Adopted a randomized controlled trial (RCT) study design that compared two or more groups, one of which receiving any QIV as intervention;Issued results on immunogenicity, in terms of seroconversion (SCR) or seroprotection rate (SPR), and efficacy, in terms of laboratory-confirmed influenza.Two authors  screened the articles on the basis of titles and abstracts first, using Rayyan. Any double publication of a study was recorded and thereafter removed. Full texts of potential eligible papers were then obtained and checked for final inclusion according to the inclusion and exclusion criteria. Articles were included if they met the following criteria:The SCR was defined as the percentage of participants with either a pre-vaccination HI titer <10 and a post-vaccination HI titer ≥40, or a pre-vaccination HI titer ≥10 and a 4-fold increase in hemagglutination inhibition (HI) antibodies titer after vaccination. The SPR was defined as the percentage of participants with a HI titer ≥40. If the same study was reported in several publications, we selected the most recent publication. Reviews and meta-analyses were excluded. Disagreements were resolved by discussion or in consultation with a third author (CdW or MC).For each included study, two authors  independently carried out data extraction and quality assessment. From each included study, the following information was extracted: first author’s last name, year of publication, trial status, country, study population, population characteristics , participants in the experimental and control arms (number and characteristics), type of intervention, type of control, study endpoints with definition, and results for each endpoint stratified by influenza strains . Data on immunogenicity and efficacy, referring only to adult populations, were extracted from per protocol populations. For studies with missing or incomplete information, authors were contacted. If attempts to contact authors were not successful, such studies were excluded from the quantitative analyses. If raw data were not available, they were calculated from the percentages reported in the study.The study quality was assessed through the Cochrane Risk of Bias Tool (RoB-Tool 2)  by two aThe NMA was carried out using the Markov Chain Monte Carlo engine WinBUGS free software if the assumption of transitivity was satisfied . In the Results were reported as percentage with 95% confidence intervals (95% CI); the width and potential overlaps of 95%CI were considered to elaborate on the comparisons among QIVs.2) method [2 was <25%, and the fixed-effect model was then applied. In case of moderate (25–50%) and high heterogeneity (>50%), the random-effect model was used.Meta-analyses were performed using StatsDirect statistics software version 3, and a fixed- or random-effect model was used to pool data based on the assessment of heterogeneity. Heterogeneity was measured using Cochran-Q test and inconsistency index (I) method ,20. An i) method . Low hetFrom the literature search through Medline (n = 518), Scopus (n = 1090) and Cochrane (n = 757) databases, and after removing duplicates n = 930), we identified 1435 records for title and abstract screening . Among t, we idenAmongst the included studies, all RCTs have been completed. Eleven studies 45.8%) were published between 2011 and 2016 ,30,31,32.8% were As far as the intervention was concerned, 14 studies (58.3%) assessed the standard-dose egg-based QIVs ,41,43,44The control arm received the analogous TIV except for two studies on recombinant QIVs that compared it with the standard-dose egg-based QIVs ,35, and All the studies evaluated the SCR. The serological outcome was determined by HI assay at 21–28 days after vaccination. The SPR was evaluated in most studies, excluding Dunkle et al.   and reported by only two studies ,45. The Nineteen studies (79.2%) had a low risk of bias ,41,43,45The assumption of transitivity was not satisfied and the NMA was not applicable.As far as the meta-analysis of immunogenicity and efficacy endpoint was concerned, 14 studies on the standard-dose egg-based QIV, and 2 on the cell-based QIV were considered for both SCR and SPR. For the latter, two studies on the live attenuated QIV were also considered for meta-analysis. For other QIVs, data extracted from single studies were reported . The comThe SCR for strains A/H1N1 ranged from 5% for live attenuated QIVs to 65% for standard-dose egg-based QIVs, whereas values for A/H3N2 ranged from 5% for live attenuated QIVs to 66% for low-dose adjuvanted QIVs; for B/Yamagata strain, SCR ranged from 9% for live attenuated QIVs to 79% for low-dose adjuvanted QIVs; eventually, for B/Victoria strain, SCR ranged from 10% for live attenuated QIVs to 65% for low-dose adjuvanted QIVs. Looking at the 95%CI, it can be observed that standard-dose egg-based, low-dose adjuvanted, cell-based, recombinant and intradermal QIVs showed similar SCRs with respect to both A strains, apart from cell-based QIVs with respect to H3N2 that showed a lower immunogenicity. With respect to B lineages, results are less conclusive, but it appears that overall low dose adjuvanted QIVs showed a better profile with respect to B Yamagata.Regarding SPR, values ranged from 25% to 98% for A/H1N1, respectively, for live attenuated and cell-based QIVs, and from 26% to 100% for A/H3N2, respectively, for live attenuated and recombinant QIVs. As for B lineage, the lowest values of SPR were observed for recombinant QIV and plant-derived QIV, namely 68% for B/Yamagata and 41% for B/Victoria. The highest values were attained by the cell-based QIVs (99% for B/Yamagata and 98% for B/Victoria). Looking at the 95%CIs, cell-based and recombinant QIVs showed similar SPR with respect to A/H1N1. Low-dose adjuvanted QIVs added to them regarding A/H3N2. With respect to B lineages, low-dose adjuvanted and cell-based QIVs showed similar results. Additionally, the standard-dose egg-based QIVs showed similar results as compared to them with respect to B/Yamagata.With respect to laboratory-confirmed influenza, for plant-derived QIVs, the attack rate in the vaccine group was 4.5% (215/4812), and in the placebo group it was 7.3% (251/4812), with an absolute vaccine efficacy of 38.8% (95%CI 17.9–48.7) to prevent influenza caused by any strain and of 35.1% (95% CI 17.9–48.7) to prevent influenza caused by vaccine-matched strain. Regarding the recombinant QIV, in the 50–64-year-old subgroup, the attack rate was 1.7% for the recombinant QIV group, and 2.9% for the standard QIV control group, leading to a relative vaccine efficacy of 42% (95% CI 15–61).Influenza continues to be a major public health problem worldwide, and current influenza vaccines remain a valuable public health tool to counter it.In this regard, the CDC estimated that influenza vaccination averted 60,500 deaths in the United States between the 2010–2011 and 2019–2020 seasons .In Europe, it is estimated that seasonal influenza vaccination prevents an annual average of 1.6–2.1 million cases of influenza, 45,300 to 65,600 hospitalizations, and 25,200 to 37,200 deaths .QIVs have currently replaced TIVs in several countries, but national immunization advisory technical groups do not generally make specific recommendations for the preferential use of one over other. Nevertheless, the assessment of their relative immunogenicity and efficacy could be useful to inform decisions.Our systematic review and meta-analysis collated together immunogenicity and efficacy data of different QIVs. The analysis of data issued by included RCTs basically showed that QIVs have different immunogenicity. In particular, differences can be observed among QIVs in terms of SCR and SPR against the same vaccine strain, but also within the same QIV, with respect to different endpoints and vaccine strains.In general, live attenuated QIVs did not show good immunogenicity, whereas standard-dose egg-based QIVs showed better SCR, and cell-based QIVs showed better SPR for all four strains. In addition, the recombinant QIV induced high SPR for A/H3N2. Actually, the development of vaccines based on recombinant protein and viruses grown on cell lines has been pursued to overcome the problem of mismatch between vaccine and circulating strains, which mostly regards H3N2 strains  and is dIt is known that SCR and SPR are considered by regulatory agencies to decide upon market authorization of influenza vaccines. In fact, the relationship between HI antibody titer and clinical protection has been well-established. In healthy adults, a serum HI titer ≥40 is associated with a >50% reduction in influenza infection or disease and is considered as a surrogate correlate of protection ,50. ThisAccording to the results of our systematic review, and considering that an HI titer ≥40 is considered the best available parameter for predicting protection from influenza infection, it can be said that the cell-based QIVs showed the best immunogenicity profile even though other QIVs, with the exception of the live attenuated, also achieved high SPR with some differences across strains. However, considerations should be given to the criticalities of the evaluation of immunogenicity, such as the low sensitivity to B strains and the high degree of interlaboratory variability . FurtherAs a matter of fact, since 2017, new CHMP guidelines recommended the measurement of neutralizing antibodies in addition to the HI titer and encouraged the assessment of broader immune responses through anti-neuraminidase antibodies, antibody kinetics, and cell-mediated immunity . This isBeyond the assessment of immunogenicity, efficacy and effectiveness data on laboratory-confirmed influenza and influenza-related clinical endpoints could further provide evidence for the relative comparison of QIVs. In this regard, a recent retrospective study assessed the relative effectiveness of cell-based QIVs, compared to the standard egg-based QIVs, among people aged 4–64 years in the United States during the 2019–2020 influenza season. Results showed that cell-based QIVs were significantly more effective than standard-dose egg-based QIVs in preventing influenza-related and respiratory-related hospitalizations/emergency room visits .In discussing the results of our systematic review, several limitations should be taken in consideration. The most important is represented by the potential heterogeneity among studies due to different influenza seasons and different participants’ history before vaccination. Another relevant aspect is that all the included studies were conducted in the Northern Hemisphere, and this might limit the generalizability of results. Furthermore, even though HI antibody responses were commonly assessed at 21 days post-vaccination, in a few studies, they were assessed at 28–30 days post-vaccination.Among the strengths of our systematic review, the following could be included: the search was performed on the most relevant databases, and the selection of studies and data extraction was performed by taking appropriate measures to prevent potential errors. Therefore, selection bias can be ruled out. Furthermore, included studies were mostly at a low risk of bias, and this enables us to consider their results robust.To the best of our knowledge, this is the first study that collates immunogenicity data of the different QIVs that are either in use or under study for the immunization of the adult population. Although a relative comparison among QIVs was not possible, the assessment of SCR and SPR and their 95%CI provides an overview of their respective potentiality in adulthood.


Tympanometry plays a fundamental role in the identification of middle ear alterations, which are frequent in the population with cleft lip and palate.do a retrospective analysis of the otoscopy and tympanometric exams of infants with cleft lip and palate who were not operated. Retrospective study.we analyzed 273 charts from infants with cleft lip and palate whom, from March 1996 to April of 2002 underwent pneumatic otoscopy and tympanometry with a 226 Hz probe.We did not find statistical significance in the otoscopic and tympanometric findings considering ears and genders. We observed 84% of alterations in otoscopy  and 65% in tympanometric curves (B/38%), A/36.5%, As/21%, C/4% and Ad/0.5%).female and male infants with cleft lip and palate did not differ as far as otoscopic and tympanometry findings are concerned. All types of tympanometric curves were present, and types A and B were the most frequent ones. Ear drum opacification was the most frequent otoscopic finding. Pneumatic otoscopy identified a larger number of alterations when compared to conventional tympanometry. Studies have reported that children with craniofacial anomalies, especially those with cleft lip and palate, have a high incidence of middle ear alterations.5During decades, tympanometry has been a broadly accepted method to assess middle ear functionConsidering the fundamental role played by tympanometry in the identification of middle ear alterations, which have a high incidence in the population with cleft lip and palate, we thought it was necessary to carry out a retrospective study of tympanometric findings in infants with this congenital malformation in order to help characterize the person's audiologic profile.The goal of this study is to carry out a retrospective analysis of the results of otoscopic and tympanometric exams in infants with cleft lip and palate that were not operated.After proper approval by the Ethics in Research Committee (protocol # 140/2005UEPCEP), we carried out a retrospective study of 300 medical charts from infants with a unilateral trans-foramen cleft in the left incisive toothFrom the medical charts we studied data associated with gender, surgical condition, results from tympanometric and otoscopic exams and patient's age at the time of the exams.For the tympanometry, we used the Grason Stadler Middle Ear Analyzer version 2 impedanciometer. The immittance tone frequency was of 226Hz . The tympanometric measures were automatically carried out by the equipment at a speed of 50 deca-Pascals per second (daPa/s). The type of tympanometric curve followed the classification proposed.For the pneumatic otoscopic exams, we used the Heine otoscope (Diagnostik-Otoskope K 100).The exams (tympanometric and otoscopic) were carried out one day before the lip surgery.The otoscopic findings were classified in: without alteration, when through the otoscopy we saw an intact tympanic membrane ; and with alteration, when there was fluid in the middle ear, opacification, retraction and immobility of tympanic membrane during inflation.Tympanometric curves were classified in normal and altered. It was normal when the A-curve was obtained, and altered for the other types found .The data obtained was organized in Tables to facilitate analysis and presentation. We used the chi-squared statistical treatment for the data. The significance level adopted was 1% (p < 0.01).The random choice of medical charts showed that 27 infants had already been submitted to some surgical procedure and were taken off the study, thus making up a study group of 273 infants (546 ears), being that 161 (59%) were males and 112 (41%) were females.The age of the infants at the time of the exams varied between 3 and 5 months.The statistical study did not show significant statistical difference between the genders (p=0.13763) and ears (p=0.58783) for otoscopic findings, as well as for tympanometric curves (p=0.45534) and (p=0.52375), respectively.,,,The results from this study show that tympanic membrane opacification, with or without other alterations, had the higher incidence (83.4%) in the population with cleft lip and palate that we studied. Such otoscopic finding was also noticed in other papers published in the literature.Melker and Harris et al. studied and support the usefulness of pneumatic otoscopy as a low cost and adequate diagnostic tool able to predict the presence of fluid (effusion) in the middle ear, and ear drum positionIn eighty-seven ears (16%) of this present study, no alteration was determined by otoscopy, which can reflect lack of middle ear effusion.In regards of tympanometric exams, 38% (209 ears) of type-B tympanometric curves were identified by tympanometry with a low frequency probe in the present study with infants from three to five months of age with cleft lip and palate and who were not operated. This type of tympanogram is a strong evidence of otitis media with effusionAndrews et al. also identified a type-B curve in 83% of 40 infants with 3 months of age who had cleft palate, however using tympanograms obtained from a high frequency probe.,However, type-A tympanograms, indicating normal middle ear function were also found in 36.5% of the population sampled in the present investigation. An incidence of 64.1% of type-A tympanometric curve was reported by Namyslowski and Kubik in a study carried out with 85 children with cleft palate, using tympanometry with the 226HZ probe.Type As is a curve that presents a reduced maximum compliance peak, seen in middle ears with some fluid or with ossicle fixation, which partially reduces mobilitySeen in only 4% of the infants in our sample, the type-C tympanometric curve, not usually found in patients with cleft lip and palateHarris et al. proved that the types As and C tympanogram curves may be associated with normal middle ear function, as well as the presence of fluid. They also reported that some children with normal low frequency tympanograms had abnormal results found by the multifrequency probe, resulting from monomeric tympanic membranes or mechanical disorders in the middle ear.A low incidence (0.5%) of the type Ad tympanometric curve was seen in our study. In the literature surveyed, this type of alteration was not reported in infants with cleft palate and lip.,,Looking at It is important to highlight that for the tympanometric findings obtained in this study, we used conventional tympanometry, in other words, a low frequency probe (226HZ). Thus, it is highly important to continue this study by making a comparative analysis using the multifrequency probe for tympanometry, to help characterize the audiologic profile of the population sampled, thus contributing to identify the most effective method to be used in the auditory evaluation of infants with cleft lip and palate in their first years of life.Male and female infants with cleft lip and palate were not different in their tympanometric curves, or in their otoscopic findings. All types of tympanometric curves were present, and type A was the most frequently found. Tympanic membrane opacification was the most frequent otoscopic finding. Pneumatic otoscopy identified a greater number of alterations when compared to conventional tympanometry.


Gestational diabetes mellitus (GDM) plus rectus abdominis muscle (RAM) myopathy predicts long-term urinary incontinence (UI). Atrophic and stiff RAM are characteristics of diabetes-induced myopathy (DiM) in pregnant rats. This study aimed to determine whether swimming exercise (SE) has a therapeutic effect in mild hyperglycemic pregnant rats model. We hypothesized that SE training might help to reverse RAM DiM. Mild hyperglycemic pregnant rats model was obtained by a unique subcutaneous injection of 100 mg/kg streptozotocin (diabetic group) or citrate buffer (non-diabetic group) on the first day of life in Wistar female newborns. At 90 days of life, the rats are mated and randomly allocated to remain sedentary or subjected to a SE protocol. The SE protocol started at gestational day 0 and consisted of 60 min/day for 6 days/week in a period of 20 days in a swim tunnel. On day 21, rats were sacrificed, and RAM was collected and studied by picrosirius red, immunohistochemistry, and transmission electron microscopy. The SE protocol increased the fiber area and diameter, and the slow-twitch and fast-twitch fiber area and diameter in the diabetic exercised group, a finding was also seen in control sedentary animals. There was a decreased type I collagen but not type III collagen area and showed a similar type I/type III ratio compared with the control sedentary group. In conclusion, SE during pregnancy reversed the RAM DiM in pregnant rats. These findings may be a potential protocol to consider in patients with RAM damage caused by GDM.  GDM can cause damage to the skeletal muscle and extracellular matrix (ECM) health and function, i.e. maternal hyperglycemic myopathy4. Maternal GDM leads to long-term pelvic floor muscle (PFM) dysfunction and urinary incontinence (UI)7, having pregnancy specific-urinary incontinence as a risk factor5. It is unknown how this link occurs4 and how swimming exercise (SE) may alleviate this damage in mild hyperglycemic pregnancy (MHP).Gestational diabetes mellitus (GDM) is known as a serious global health problem11. The latter characteristics mimic those found in human hyperglycemic-associated PFM and RAM myopathy and are considered a profile of skeletal muscle injury caused by GDM during pregnancy in humans 13. Despite these observations, the link between GDM and maternal hyperglycemic myopathy remains largely unexplored and without an effective treatment 14.Rat models of MHP also revealed myopathy of the PFM and rectus abdominis muscle (RAM) induced by diabetes (DiM). DiM characterized by muscle atrophy, a shift in the maternal fiber type composition, increased collagen deposition, and higher collagen type I/III ratio 18. However, different exercise patterns improve maternal glucose control in some but not all pregnant women with GDM 21. Thus, lifestyle interventions during pregnancy alone may not be sufficient to decrease the risk of developing GDM-associated alterations in maternal health 27.Exercise has beneficial impacts on maternal glycaemia control and several lifestyle interventions have clarified mechanisms underlying GDM for preventing or minimizing this disease’s associated complications 27. In addition, SE and resistance training influence the gastrocnemius and soleus muscles myopathy in rat models of type 2 diabetes mellitus (T2DM)29. However, there is limited evidence describing the effect of SE on diabetic myopathy in GDM 30. There are no studies revealing whether the exercise intervention during pregnancy recovers the muscle damage associated with GDM, even considering the regenerative skeletal muscle fibers potential 31.Considering that pregnant women should avoid high-impact exercises with the risk of falling and with the risk of abdominal trauma, SE is considered ideal for pregnant women. Also, SE in a low-moderate intensity is effective in promoting glycemic control and preventing GDM The aim of this study was to determine whether SE has a therapeutic effect in MHP rats model resulting in attenuation of RAM DiM. We hypothesized that SE training would mitigate RAM DiM, thus reversing the RAM injury. A potential reversal of RAM DiM by SE may improve the handling of maternal hyperglycemic myopathy in women that developed GDM.The experimental design included three control groups, i.e., non-diabetic sedentary (NDsed), non-diabetic exercised (NDex), and diabetic sedentary allowing the analysis of the SE intervention in DiM in the authentic study group: the diabetic exercised (Dex). The OGTT showed that Dsed and Dex groups had two or more blood glucose measurements > 140 mg/dl. These data confirm that the generation of MHP rats model was efficient, including the animals in the experimental groups. The aquatic exercise practice during pregnancy did not promote changes in blood glucose levels  displays similar fiber area and diameter, slow-twitch, fast-twitch fiber area and diameter, total collagen area and type I/III collagen ratio compared to NDsed group Figs.  and 3.We investigated whether swimming exercise (SE) would alter the RAM DiM in rats. This preclinical study of SE started on the first day in MHP rats detailed the reversal of RAM skeletal atrophic and stiff muscle through an integrative morphological, ultrastructural and extracellular matrix (ECM) assessment. The main findings of the present study are as follows: first, the RAM DiM was confirmed, demonstrated by lower total muscle area and fiber diameter and increased type I collagen and type III collagen deposition in Dsed compared with NDsed group. These findings were already reported by our group. Second, SE during the whole pregnancy in MHP rats model reversed the RAM skeletal atrophy through an increase in fiber area and diameter, slow-twitch fiber area and diameter, fast-twitch fiber area and diameter in Dex group compared with NDsed group; and reduction in type I collagen area and type I/III collagen ratio compared with Dsed. Also, the similarity in total collagen area, type I collagen area and type I/III collagen ratio with NDsed rats were demonstrated. The close resemblance between RAM morphological, and ECM profile of MHP rats model submitted to SE (Dex group) and NDsed, suggest that SE since the beginning of pregnancy reversed the DiM. These findings support the hypothesis that DiM is inhibited by 21 days of SE training program through the entire pregnancy. Indeed, SE training plays a critical role in the reversion of DiM. It may be a sufficient duration and intensity to induce recovery of morphological changes in skeletal muscle.32. The underlying mechanism linking GDM and long-term UI seems to be diabetic myopathy, characterized by loss of muscle mass and strength 34. Many clinical and experimental studies have associated both type 1 and type 2 diabetes to muscle structural changes, including a reduction in myofiber and myofibrilar diameter, reduced muscle mass 33, reduced muscle fiber size 35, and decreased capacity to repair from damage 33.Women who develop GDM are more likely to develop type 2 diabetes and UI later in life10 and in urethral striated muscle 9, either in women or in rats 11. These structural harms may be related to functional changes as well as a decline in muscle strength and increased fatigability, which contributes to decreased physical capacity 35. Therefore, this damage to muscles involved in urinary continence may contribute to increased incidence of PS-UI, a predictor of long-term UI in previous GDM women 5. These findings allow us to adopt MHP’ rats model for studying the effects of SE on the RAM fiber-ECM structure, as it mimics GDM in women 11. Therefore, further study is necessary to elucidate the influence of exercise types and duration on the muscle fiber cross-sectional area of MHP rats' skeletal muscle.MHP rats is an experimental model that induces structural changes including reduced muscle area and increased slow-twitch fiber in pregnant rats in RAM 37.SE started at the first day of pregnancy in MHP rats may be considered a preclinical study being developed as a prevention strategy of hyperglycemia in pregnancy for long-term UI. Our intention was for longer-lasting interventions leading to significant downstream impact in promoting long-term women’s health. The goals of this early intervention program in pregnancy are related to improving future maternal health in GDM women, one of the United Nation’s Eight Millennium Development Goals (MDGs) 37. However, there are few studies evaluating the impact of hyperglycemia in pregnancy on structural muscles damage involved in urinary continence. This study tested the hypothesis that SE daily, since the beginning of pregnancy in MHP rats could act as a protective non-pharmacological intervention, so we studied its effects on RAM muscle looking at the reversal of muscle atrophy and stiffness.Pregnancy is the center of a program established by the International Federation of Gynecology and Obstetrics (FIGO) in the Non-Communicable Disease (NCD)—as early as possible but focusing on the first-trimester and follow-up post-pregnancy for mother and offspring, leading to prevent NCD. Pregnancy, in particular the first trimester, receive not only substantial attention but also many points of intervention, from pre-conception to postpartum 38. The results of this study revealed that SE daily, since the beginning of pregnancy in MHP rats, reverses the muscle atrophy caused by the maternal hyperglycemia environment. In short, these findings indicated that SE could reverse muscle atrophy in MHP rats, back into the NDsed group profile. In contrast, sarcomeres disruption areas were observed in both diabetic groups, which means that exercise had no impact on this parameter. Previous results demonstrate that RAM exposed to a hyperglycemic environment is characterized by a decrease in the number and area of the fast fiber and an increase in the number of slow fibers 10. In our study, daily SE during all pregnancy had an effect not only on increasing fast-twitch fiber area and diameter but also on slow-twitch fiber area and diameter of RAM muscle in both exercised groups.Increased muscle fiber cross-sectional area, fiber area and diameter in RAM of diabetic pregnant rats after 21 days of SE is similar to the reported after 8 weeks of aerobic training 39. Different SE modalities increased diameter of both slow-twitch and fast-twitch fibers on gastrocnemius and soleus muscles in the type 2 diabetes animal model 28. Thus, the high slow-twitch fiber area and diameter in the RA muscle of both exercised groups regardless of diabetes and high fast-twitch fiber area and diameter in diabetic exercised rats could be attributed to physiological adaptations from aerobic exercise during the whole pregnancy. Consistent with previous studies, we demonstrated that SE training during the whole pregnancy induces an increase not only in the cross-sectional area and slow-twitch fiber, but also the fast-twitch fiber 41. Indeed, SE applied in this study showed a significant reversal of muscle atrophy in pregnant hyperglycemic rats.The present study shows that both exercised groups regardless of diabetes had increased fast-twitch fiber area and diameter. These findings support the possibility that DiM model is inhibited by exercise training 29. Conversely, the fast and slow-twitch fiber area and diameter increase may be related to improved muscle endurance, possibly caused by mitochondrial biogenesis and increased mitochondrial enzymes activities. These findings enhance aerobic capacity and provide muscle contraction with less energy expenditure and greater oxygen uptake 42.Exercise training plays an important role in the mitigation of muscular atrophy in DiM. Coherent with previous studies, a resistance training protocol enhanced muscle strength on extensor digitorum longus muscle, gastrocnemius and soleus muscles of male diabetic rats, improving muscle physical and motor function, minimizing the deleterious effect of diabetes on muscles43. In prior translational studies in rats, it was shown that diabetes during pregnancy results in damaged extracellular matrix (ECM) and urethral striated muscle suggestive of the high long-term UI prevalence in women 9. Other studies examined the relationship between ECM components (particularly collagens) and diabetes, PFDs and pregnancy, although few comparative data are available 18. The results in the present study show that SE in MHP rats did not reduce the total collagen area compared with the three other groups. However, SE in MHP rats decreases type I collagen area to similar levels of non-diabetic sedentary group and maintain high levels of type III collagen similar to SE MHP and SE non-diabetic group. The decrease in collagen type I/III ratio in SE-MHP suggests the reversal of a stiff into a soft healthy skeletal muscle. Our findings complement the results of four previously experimental studies, either in RAM or PFM 43 indicating a complete muscle remodeling. It confirms the importance of this biological approach in DiM. This changeover in collagen type I/III ratio in SE in MHP favors normal soft RAM muscle, with likely normalization of biomechanical properties in muscles involved in urinary continence. All these changes allow suggesting SE for assessment as a potential treatment for maternal UI injured by the maternal hyperglycemic environment.The urethral extracellular matrix also plays a role in the mechanism of urinary continence 44. Further studies should address this fundamental mechanism. This study as a preclinical model in the obstetrical area, the maternal-placental and fetal (MPF) unit needs to be carefully considered. Although we conducted this experiment by using the same swimming protocol established previously 47, the whole MPF unit analysis was not performed. Thus, our successful results with SE in MHP rats environment may be analysed with caution to be considered in GDM women. Also, the present study has limitations regarding the experimental diabetes induction, which represents GDM blood glucose levels, but occurred before pregnancy 49. In addition, the guidelines recommend that pregnant women should exercise at least 150 min/week or 30 min/day most days of the week 25. Also, specific analysis of aerobic capacity and muscle function should be explored.Although this set of results related to SE in mitigating the negative effects of the maternal hyperglycemic environment on DiM in the transformation into a soft health muscle, the molecular regulations of this gain muscle mass is unclear. It was suggested that distinct mechanisms regulate skeletal muscle mass recovery and hypertrophy In conclusion, hyperglycemia exerts an impact on DiM generating an atrophic and stiff muscle. SE intervention training during whole pregnancy in rats imposes a positive influence on DiM being capable of reversing this muscle atrophy and transforming a stiff into a soft healthy skeletal muscle. Therefore, these findings of reversal myopathy suggest that SE training during the whole pregnancy may play a therapeutic role in regulating the damages caused by the maternal hyperglycemic environment and may be considered as a potential treatment for RAM damage caused by GDM.4. According to the requirement to develop innovative treatments for DiM, this translational project was developed.This current project corresponds to the translational part of the Diamater Study, a cohort Thematic Project with the financial support of São Paulo Research Foundation (FAPESP 2016/01743-5), a state research foundation in Brazil. The Diamater cohort project has been developed to investigate biomolecular muscle profiles as predictors for long-term urinary incontinence in women with Gestational Diabetes Mellitus All animal experiments were approved by the Institutional Animal Care and Use Committee, Faculdade de Filosofia e Ciências, São Paulo State University (UNESP), in accordance with the Brazilian Council for Control of Animal Experimentation (CONCEA) (protocol number 007/2016). The study is reported in accordance with the ARRIVE guidelines.Female and male Wistar rats obtained from ANILAB were housed in a facility with constant temperature (22 ± 2 ˚C) and humidity 55 ± 5%) on a controlled 12 h light–12 h dark cycle with food and water ad libitum, in individual plastic cages during all experimental protocol. The experimental sequence is shown in Fig. 5 ± 5% on47. To induce MHP rats’ model, newborn female Wistar received in the first day of life subcutaneously injection of STZ (Sigma®) diluted in citrate buffer (0.1 mol/l pH 4.5) in a dose of 100 mg/kg. Non-diabetic rats received subcutaneous injection of citrate buffer (0.1 mol/l pH 4.5) 50. All female newborn rats were maintained with their mothers until the end of the lactation period (21 days). After this period, the mother rats were euthanized by sodium thiopental injection(Thiopentax®—80 mg/kg). The female newborns were maintained until adulthood. Fasting blood glucose level was determined in adult life, and used for inclusion or exclusion criteria in the study. Diabetic animals should present blood glucose level between 120 mg/dl and 300 mg/dl, and non-diabetic animals blood glucose level < 120 mg/dl 51.The MHP model is the same as previous reports At approximately day 90 of age, four diabetic and non-diabetic female rats were housed overnight with one adult male rat. The presence of spermatozoa in the vaginal smear was considered gestational day 0 [52]. After, rats were housed in individual cages until 21º day of pregnancy.On gestational day 0, female rats were randomly allocated into four experimental groups according to sedentary lifestyle or swimming exercise: Non-Diabetic Sedentary (NDsed) (n = 13), Non-Diabetic Exercised (NDex) (n = 13), Diabetic Sedentary (Dsed) (n = 13) and Diabetic Exercised (Dex) (n = 13). Dex is the study group and the other three  are control groups.47 and considered to be a moderate intensity exercise protocol 45. The exercised animals were exposed to water daily temperature 31 ± 1°C for 6 days/week, from gestational day 0 until gestational day 20 on a pool at a depth of 40 cm at the water. The first training session started with 20 min, progressively increasing 10 min/day until 60 min. The sedentary rats were exposed to water daily for 15 min, at a depth of 10 cm at water temperature 31 ± 1 °C for 6 days/week, from gestational day 0 until gestational day 20, aiming not to promote physiological adaptations from exercise practice.The swimming exercise protocol was based on previous studies 50. Fasting glycemia and at 10, 20, 30, 60 and 120 min after administration of an intragastric glucose solution (0.2 g/mL) in a dose of 2.0 g/kg were measured. Diabetes diagnosis was confirmed with two or more blood glucose measurements > 140 mg/dL 51.On gestational day 17, an OGTT was performed to confirm the glucose intolerance in diabetics 2. Then, a C-section was performed and fetuses and placentas were separately analyzed in different projects.At the end of pregnancy  the dams were euthanized by sodium thiopental injection . An abdominal incision was performed for RAM sample collection. The lower third on the right side of RAM was exposed, dissected, and removed for integrative morphological analysis. The fragments had approximately 0.25 cmThe samples obtained were selected into separate parts according to methodological procedures. The integrative morphological analysis is composed by morphological, morphometric, immunohistochemistry and ultrastructural RAM analysis.®/BX41TF coupled with DP25-4 digital câmera). The photographs were obtained with cell Sens Ver 1.18 Olympus Corporation® software.For morphological and morphometric analysis, RAM samples were immersed for 24 h in neutral 10% buffered formaldehyde, transferred to 70% alcohol and maintained at room temperature and then embedded in paraffin. The 4-µm-thick sections were cut in microtome (Reichert-Jung model 820) and fixed on microscope glass slides stained with Hematoxylin & Eosin (H&E) and Picrosirius Red. H&E-stained slides were used to observe the general morphology of the RAM. Picrosirius Red-stained slides were analyzed with the color-segmentation method to determine the red (collagen) and yellow (muscle fiber) -stained tissue in the same section and used to determine muscle, fiber and collagen area and fiber diameter. The slides were analyzed in a light microscope (Olympus Corporation®) image analysis software (20 × magnification).For morphometric analysis of muscle and collagen area, 40 sections/group  were selected. For morphometric analysis of fiber area and diameter, 100 muscle fibers/group were selected. All analyzes were performed using CellSens Dimension Version 1.16 . After washing, the sections were incubated with bovine serum albumin solution (BSA 3%) for 1 h. The sections were incubated overnight at 4 °C with primary antibodies against myosin heavy chain, slow  and fast  muscle fibers, type I Collagen  and type III Collagen . After incubation, the sections were washed three times for five minutes with PBS and incubated with secondary antibodies for 1 h 30 min . For staining, the sections were incubated with diaminobenzidine for 1 min and hematoxylin for 10 min. The slow-twitch and fast-twitch muscle fibers, and type I and III Collagen were analyzed using a light microscope  and 40 sections/group  were selected to quantify the fiber type area and collagen type area. Also, each muscle fiber was manually selected  and fiber type area and diameter were quantified using CellSens Dimension (Olympus Corporation®) Version 1.16 image analysis software (20 × magnification). The fiber type area was quantitatively determined, as described previously10.Immunohistochemical analysis was used to stain slow-twitch and fast-twitch muscle fibers and type I and III collagen. For immunohistochemistry (N = 5 samples/group), the samples were immersed for 24 h in neutral 10% buffered formaldehyde, transferred to 70% alcohol and maintained at room temperature and then embedded in paraffin. The 4-µm-thick sections were cut in the microtome. Sections were deparaffinized and incubated with antigenic recovery for 35 min in a pressure cooker. Endogenous peroxidase was blocked using H, the RAM tissues obtained to ultrastructural analysis (3 samples/group) were cut into small strips and immediately immersed in Karnovsky fixative for 3 h at room temperature and transferred to the refrigerator to post-fixation in 1% osmium tetroxide for 24 h and afterward, the samples were embedded in epoxy resin. Ultra-thin sections were obtained at a longitudinal orientation and stained sections were examined using transmission electron microscopy using .As previously describedGraphPad Prism® v.8.0 software was used to analyze the data. Data are expressed as mean ± standard deviation (SD). Comparisons of measurements among groups were performed by two-way ANOVA followed by Tukey’s multiple comparison tests. For all statistical comparisons, p < 0.05 was considered statistically significant.