Withanicandrin Isolated from Datura Ferox Promotes Antinociception by Modulating the Asics and TRPS Channels and Anti-Inflammation in Adult Zebrafish.

This is the first study to analyze the anti-inflammatory and antinociceptive effect of withanicandrin, isolated from Datura Ferox leaves, and the possible mechanism of action involved in adult zebrafish (ZFa). To this end, the animals were treated intraperitoneally (i. p.) with withanicandrin (4; 20 and 40 mg/kg; 20 µL) and subjected to locomotor activity and acute toxicity. Nociception tests were also carried out with chemical agents, in addition to tests to evaluate inflammatory processes induced by κ-Carrageenan 1.5 % and a Molecular Docking study. As a result, withanicandrin reduced nociceptive behavior by capsaicin at a dose of 40 mg/kg and by acid saline at doses of 4 and 40 mg/kg, through neuromodulation of TRPV1 channels and ASICs, identified through blocking the antinociceptive effect of withanicandrin by the antagonists capsazepine and naloxone. Furthermore, withanicandrin caused an anti-inflammatory effect through the reduction of abdominal edema, absence of leukocyte infiltrate in the liver tissue and reduction of ROS in thel liver tissue and presented better affinity energy compared to control morphine (TRPV1) and ibuprofen (COX-1 and COX-2).

Modified Procedure for Simple and Rapid Collection of Zebrafish Fluid Samples for Biochemical Analyses.

This study addresses the challenge of collecting blood samples from zebrafish for biochemical analysis. Traditional methods are cumbersome due to low blood flow and rapid coagulation. Based on a previously published technique, we simplified the process by applying an anticoagulant solution directly to the incision site. The modified protocol involves immersing the fish in an ice bath, making a cross-sectional incision, and immediately applying anticoagulant solution. Centrifugation of the specimens provides a streamlined and efficient approach to zebrafish fluid sample collection, compatible with classic biochemical marker analyses.

Neuromodulation of Acid-Sensitive Ion Channels (ASICs) and Anti-Inflammatory Potential by Lichenxanthone in Adult Zebrafish (Danio rerio): Experimental and Docking Studies.

The xanthone lichenxanthone did not show toxic effects (LC50>1.0 mg/mL). lichenxanthone prevented nociceptive behavior induced by acidic saline, and its analgesic effect was blocked by amiloride, highlighting the involvement of neuromodulation of acid-sensitive ion channels (ASICs). In the analysis of anti-inflammatory activity, concentrations of 0.1 and 0.5 mg/mL of lichenxanthone reduced the edema induced by k-carrageenan 3.5 %, observed from the fourth hour of analysis. This effect was similar to that observed with ibuprofen (positive control). No leukocyte infiltrates were observed in lichenxanthone, suggesting that the compound acts in the acute inflammatory response. The results of the molecular docking study revealed that lichenxanthone exhibited better affinity energy when compared to the ibuprofen control against the two targets evaluated.

Transcriptional Profiling of SARS-CoV-2-Infected Calu-3 Cells Reveals Immune-Related Signaling Pathways.

The COVID-19 disease, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), emerged in late 2019 and rapidly spread worldwide, becoming a pandemic that infected millions of people and caused significant deaths. COVID-19 continues to be a major threat, and there is a need to deepen our understanding of the virus and its mechanisms of infection. To study the cellular responses to SARS-CoV-2 infection, we performed an RNA sequencing of infected vs. uninfected Calu-3 cells. Total RNA was extracted from infected (0.5 MOI) and control Calu-3 cells and converted to cDNA. Sequencing was performed, and the obtained reads were quality-analyzed and pre-processed. Differential expression was assessed with the EdgeR package, and functional enrichment was performed in EnrichR for Gene Ontology, KEGG pathways, and WikiPathways. A total of 1040 differentially expressed genes were found in infected vs. uninfected Calu-3 cells, of which 695 were up-regulated and 345 were down-regulated. Functional enrichment analyses revealed the predominant up-regulation of genes related to innate immune response, response to virus, inflammation, cell proliferation, and apoptosis. These transcriptional changes following SARS-CoV-2 infection may reflect a cellular response to the infection and help to elucidate COVID-19 pathogenesis, in addition to revealing potential biomarkers and drug targets.

Subcutaneous, Oral, and Intranasal Immunization of BALB/c Mice with Leishmania infantum K39 Antigen Induces Non-Protective Humoral Immune Response.

Visceral leishmaniasis is a high-burden disease caused by parasites of the Leishmania genus. The K39 kinesin is a highly antigenic protein of Leishmania infantum, but little is known about the immune response elicited by this antigen. We evaluated the humoral immune response of female BALB/c mice (n = 6) immunized with the rK39-HFBI construct, formed by the fusion of the K39 antigen to a hydrophobin partner. The rK39-HFBI construct was administered through subcutaneous, oral, and intranasal routes using saponin as an adjuvant. We analyzed the kinetics of IgG, IgG1, and IgG2a production. The groups were then challenged by an intravenous infection with L. infantum promastigote cells. The rK39-HFBI antigen-induced high levels of total IgG (p < 0.05) in all groups, but only the subcutaneous route was associated with increased production of IgG1 and IgG2a 42 days after immunization (p < 0.05), suggesting a potential secondary immune response following the booster dose. There was no reduction in the splenic parasite load; thus, the rK39-HFBI failed to protect the mice against infection under the tested conditions. The results presented here demonstrate that the high antigenicity of the K39 antigen does not contribute to a protective immune response against visceral leishmaniasis.

Unveiling a differential metabolite modulation of sorghum varieties under increasing tunicamycin-induced endoplasmic reticulum stress.

Plants trigger endoplasmic reticulum (ER) pathways to survive stresses, but the assistance of ER in plant tolerance still needs to be explored. Thus, we selected sensitive and tolerant contrasting abiotic stress sorghum varieties to test if they present a degree of tolerance to ER stress. Accordingly, this work evaluated crescent concentrations of tunicamycin (TM µg mL-1): control (0), lower (0.5), mild (1.5), and higher (2.5) on the initial establishment of sorghum seedlings CSF18 and CSF20. ER stress promoted growth and metabolism reductions, mainly in CSF18, from mild to higher TM. The lowest TM increased SbBiP and SbPDI chaperones, as well as SbbZIP60, and SbbIRE1 gene expressions, but mild and higher TM decreased it. However, CSF20 exhibited higher levels of SbBiP and SbbIRE1 transcripts. It corroborated different metabolic profiles among all TM treatments in CSF18 shoots and similarities between profiles of mild and higher TM in CSF18 roots. Conversely, TM profiles of both shoots and roots of CSF20 overlapped, although it was not complete under low TM treatment. Furthermore, ER stress induced an increase of carbohydrates (dihydroxyacetone in shoots, and cellobiose, maltose, ribose, and sucrose in roots), and organic acids (pyruvic acid in shoots, and butyric and succinic acids in roots) in CSF20, which exhibited a higher degree of ER stress tolerance compared to CSF18 with the root being the most affected plant tissue. Thus, our study provides new insights that may help to understand sorghum tolerance and the ER disturbance as significant contributor for stress adaptation and tolerance engineering.

Quantum Biochemistry and MM-PBSA Description of the ZIKV NS2B-NS3 Protease: Insights into the Binding Interactions beyond the Catalytic Triad Pocket.

The Zika virus protease NS2B-NS3 has a binding site formed with the participation of a H51-D75-S135 triad presenting two forms, active and inactive. Studies suggest that the inactive conformation is a good target for the design of inhibitors. In this paper, we evaluated the co-crystallized structures of the protease with the inhibitors benzoic acid (5YOD) and benzimidazole-1-ylmethanol (5H4I). We applied a protocol consisting of two steps: first, classical molecular mechanics energy minimization followed by classical molecular dynamics were performed, obtaining stabilized molecular geometries; second, the optimized/relaxed geometries were used in quantum biochemistry and molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) calculations to estimate the ligand interactions with each amino acid residue of the binding pocket. We show that the quantum-level results identified essential residues for the stabilization of the 5YOD and 5H4I complexes after classical energy minimization, matching previously published experimental data. The same success, however, was not observed for the MM-PBSA simulations. The application of quantum biochemistry methods seems to be more promising for the design of novel inhibitors acting on NS2B-NS3.

A Redox-Probe-Free Immunosensor Based on Electrocatalytic Prussian Blue Nanostructured Film One-Step-Prepared for Zika Virus Diagnosis.

The Zika virus (ZIKV) is a great concern for global health due to its high transmission, including disseminating through blood, saliva, urine, semen and vertical transmission. In some cases, ZIKV has been associated with microcephaly, neurological disorders, and Guillain−Barré syndrome. There is no vaccine, and controlling the disease is a challenge, especially with the co-circulation of the Dengue virus, which causes a severe cross-reaction due to the similarity between the two arboviruses. Considering that electrochemical immunosensors are well-established, sensitive, and practical tools for diagnosis, in this study we developed a sensor platform with intrinsic redox activity that facilitates measurement readouts. Prussian blue (PB) has a great ability to form electrocatalytic surfaces, dispensing redox probe solutions in voltammetric measurements. Herein, PB was incorporated into a chitosan−carbon nanotube hybrid, forming a nanocomposite that was drop-casted on a screen-printed electrode (SPE). The immunosensor detected the envelope protein of ZIKV in a linear range of 0.25 to 1.75 µg/mL (n = 8, p < 0.01), with a 0.20 µg/mL limit of detection. The developed immunosensor represents a new method for electrochemical measurements without additional redox probe solutions, and it is feasible for application in point-of-care diagnosis.

ABO blood group and link to COVID-19: A comprehensive review of the reported associations and their possible underlying mechanisms.

ABO blood group is long known to be an influencing factor for the susceptibility to infectious diseases, and many studies have been describing associations between ABO blood types and COVID-19 infection and severity, with conflicting findings. This narrative review aims to summarize the literature regarding associations between the ABO blood group and COVID-19. Blood type O is mostly associated with lower rates of SARS-CoV-2 infection, while blood type A is frequently described as a risk factor. Although results regarding the risk of severe outcomes are more variable, blood type A is the most associated with COVID-19 severity and mortality, while many studies describe O blood type as a protective factor for the disease progression. Furthermore, genetic associations with both the risk of infection and disease severity have been reported for the ABO locus. Some underlying mechanisms have been hypothesized to explain the reported associations, with incipient experimental data. Three major hypotheses emerge: SARS-CoV-2 could carry ABO(H)-like structures in its envelope glycoproteins and would be asymmetrically transmitted due to a protective effect of the ABO antibodies, ABH antigens could facilitate SARS-CoV-2 interaction with the host' cells, and the association of non-O blood types with higher risks of thromboembolic events could confer COVID-19 patients with blood type O a lower risk of severe outcomes. The hypothesized mechanisms would affect distinct aspects of the COVID-19 natural history, with distinct potential implications to the disease transmission and its management.

Codium isthmocladum lectin 1 (CiL-1): Interaction with N-glycans explains antinociceptive and anti-inflammatory activities in adult zebrafish (Danio rerio).

Inflammation and oxidative stress are processes associated with different human diseases. They are treated using drugs that have several side effects. Seaweed are sources of potentially relevant natural compounds for use as treatment of these disorders. Lectins are able to reversibly interact with complex carbohydrates and modulate cell membrane glycosylated receptors through this interaction. This study aimed to determine the antinociceptive and anti-inflammatory potential of CiL-1 in adult zebrafish by modulation of TRPA1 through lectin-glycan binding. Possible neuromodulation by TRPA1 channel was also evaluated by camphor pretreatment. CiL-1 was efficacious at all tested doses, revealing anti-nociceptive and anti-inflammatory effects in adult zebrafish. This galactose-binding lectin was also able to reduce the content of ROS in brain and liver. In silico analyses showed CiL-1 interactions with both ligands tested. LacNac2 presents the most favorable binding energy with the protein. The interaction occurs at 4 subsites as an extended conformation at the site. LacNac2-Sia had a less favorable curved-shape interaction energy. Based on the predictions made for the oligosaccharides, a tetra-antenate putative glycan was schematically constructed, illustrating an interaction between TRPA1 N-glycan and CiL-1. This binding seems to be related to CiL-1 anti-inflammatory activity as result of receptor modulation.

New insights into the recombinant proteins and monoclonal antibodies employed to immunodiagnosis and control of Zika virus infection: A review.

An emergent positive-stranded RNA virus, transmitted by mosquitoes with its first case of vertical transmission confirmed in 2015 in Brazil. The Zika virus (ZIKV) fever has received particular attention, mainly related to neurological diseases such as microcephaly in newborns. However, the laboratory diagnosis for ZIKV still faces some challenges due to its cross-reactivity with other flaviviruses, requiring a correct and differential diagnosis, contributing to the good prognosis of patients, especially in pregnant women. Among these, for early diagnosis, the CDC considers the RT-PCR the gold standard, more sensitive and specific, but expensive. Serological tests for the diagnosis of ZIKV can also be found beyond the period when the viral components are detectable in the serum. Inputs to produce more sensitive and specific diagnostic kits and the possibility of viral detection in less invasive samples are among the objectives of recent research on ZIKV. This review outlines recent advances in developing recombinant antigen and antibody-based diagnostic tools for the main flaviviruses in Northeast Brazil, such as ZIKV and Dengue virus (DENV).

Combretum lanceolatum extract reverses anxiety and seizure behavior in adult zebrafish through GABAergic neurotransmission: an in vivo and in silico study.

Combretaceae are reported in the literature for presenting neuroprotective and anxiolytic effects in animal models. Combretum lanceolatum Pohl. has few scientific reports on its pharmacological effects. The aim of this study was to evaluate the anxiolytic and anticonvulsant effects of the ethanol extract from the leaves of C. lanceolatum Pohl. (EtFoCl) and its possible mechanism of GABAergic action in adult zebrafish. EtFoCl was subjected to determination of the total phenol concentration, identification of phytochemical flavonoids by HPLC and in vitro antioxidant activity test, open field test and 96-hour acute toxicity in zebrafish. Anxiolytic doses were tested for pentylenetetrazole-induced seizures in adult zebrafish. To study the mechanisms of action, molecular docking simulations were performed between the main phytochemicals and the GABAA receptor (anxiolytic activity) and carbonic anhydrase II (anticonvulsant). The non-toxic doses that caused motor impairment were assessed in acute and chronic anxiety using the light and dark test. EtFoCl had altered the animals' locomotion, presenting an effect similar to the anxiolytic and anticonvulsant. These effects were prevented with flumazenil (GABAA antagonist). The phytochemicals homoorientin and quercetin-3-O-galactoside coupling in a region close to that of the inhibitor diazepam (GABAA receptor). Regarding the anticonvulsant mechanism, Homoorientina and Isovitexina were identified as the most favorable for the complex form with the carbonic anhydrase enzyme. C. lanceolatum has pharmacological potential for the treatment of acute and chronic anxiety and seizures, which can be partially explained by an interaction with the GABAA receptor.Communicated by Ramaswamy H. Sarma.

Antinociceptive, anti-inflammatory and hypoglycemic activities of the ethanolic Turnera subulata Sm. flower extract in adult zebrafish (Danio rerio).

Turnera subulata Sm. belongs to the family Turneraceae and is found in Brazil. The present study evaluated the antinociceptive, anti-inflammatory, and hypoglycemic potential of T. subulata flower extract (EtFloTsu) in zebrafish (Danio rerio). The total phenol and flavonoid contents of EtFloTsu were determined and identified using the Folin Ciocalteu reagent and aluminum chloride (AlCl3), respectively. The constituents of the extract were identified by HPLC-DAD, and the in vitro antioxidant activity (DPPH) was determined, toxicity in brine shrimp, and acute toxicity of 96 h in adult zebrafish. In addition, adult zebrafish (n = 6/fish) were treated orally with EtFloTsu (4, 20, or 40 mg/kg; vo) and subjected to formalin-induced nociception tests (with its possible mechanism of action with camphor), carrageenan-induced inflammation, and D-glucose-induced hyperglycemia (111 mM). Oxidative stress in the liver and brain tissues was assessed. EtFloTsu showed high levels of phenolic and flavonoid compounds with antioxidant activity. The phytochemicals chlorogenic acid, luteolin-7-o-glucoside, vitexin, and apigenin-7-o-glucoside were also identified in EtFloTsu. The synergism between these constituents was possibly responsible for the antinociceptive (via TRPA1), anti-inflammatory, and hypoglycemic effects of EtFloTsu in adult zebrafish, without causing toxicity in animals. Therefore, T. subulata flowers have therapeutic agents that could treat pain, inflammation, diabetes, and related complications.Communicated by Ramaswamy H. Sarma.

Virucidal efficacy of chlorhexidine: a systematic review.

This study aimed to systematically review the literature about the virucidal efficacy of CHX in comparison to other substances used in the oral cavity. Electronic searches were performed in four databases (PubMed, Scopus, Embase, and Web of Science). Only studies that presented the following characteristics were included: (1) verified virucidal efficacy of CHX against Herpes Simplex Type-1 (HSV-1), any Influenza, or any human coronavirus (HcoV); and (2) compared the virucidal efficacy of CHX with essential oils (Listerine®), quaternary ammonium compounds, povidone-iodine, hydrogen peroxide, negative control substance, and absence of therapy. Two researchers independently selected the studies, extracted data and evaluated the risk of bias. A narrative data synthesis was used. Twenty-five studies were included, of which 21 were in vitro and four were randomized clinical trials (RCT). Studies assessed the virucidal efficacy of CHX against Herpes Simplex Type-1 (HSV-1) (10 studies), Influenza A (InfluA) (4 studies), human coronavirus (HCoV) (4 studies) and Severe Acute Respiratory Syndrome-Related Coronavirus (SARS-CoV-2) (11 studies). Most studies demonstrated that CHX has a positive virucidal efficacy against HSV-1 and InfluA strains. However, lower efficacy was shown to InfluA strain in comparison to povidone-iodine. Lower to none virucidal efficacy of CHX is expected for HCoV and SARS-CoV-2 strains for in vitro studies. Three RCT demonstrated that CHX was able to significantly reduce the viral load of SARS-CoV-2 for a short period. CHX may present an interesting virucidal efficacy against HSV-1 and InfluA viruses. CHX also presents transient efficacy against SARS-CoV-2 when used as a mouthwash.

A review of plant-based expression systems as a platform for single-domain recombinant antibody production.

Monoclonal antibodies have contributed to improving the treatment of several diseases. However, limitations related to pharmacokinetic parameters and production costs have instigated the search for alternative products. Camelids produce functional immunoglobulins G devoid of light chains and CH1 domains, in which the antigenic recognition site is formed by a single domain called VHH or nanobody. VHHs' small size and similarity to the human VH domain contribute to high tissue penetration and low immunogenicity. In addition, VHHs provide superior antigen recognition compared to human antibodies, better solubility and stability. Due to these characteristics and the possibility of obtaining gene-encoding VHHs, applications of this biological tool, whether as a monomer or in related recombinant constructs, have been reported. To ensure antibody efficacy and cost-effectiveness, strategies for their expression, either using prokaryotic or eukaryotic systems, have been utilized. Plant-based expression systems are useful for VHH related constructs that require post-translational modifications. This system has exhibited versatility, low-cost upstream production, and safety. This article presents the main advances associated to the heterologous expression of VHHs in plant systems. Besides, we show insights related to the use of VHHs as a strategy for plant pathogen control and a tool for genomic manipulation in plant systems.

SARS-CoV-2 Unrevealed: Ultrastructural and Nanomechanical Analysis.

The ongoing outbreak of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) started in late 2019 and spread across the world, infecting millions of people, with over 3.3 million deaths worldwide. To fight back the virus, it is necessary to understand how the main structures work, especially those responsible for the virus infectivity pathogenicity. Here, using the most advanced atomic force microscopy techniques, SARS-CoV-2 viral particles were analyzed, with a special focus on their ultrastructure, adsorption conformation, and nanomechanical behavior. The results uncovered the aspects of the organization and the spatial distribution of the proteins on the surface of the viral particles. It also showed the compliant behavior of the membrane and ability to recover from mechanical injuries. At least three layers composing the membrane and their thickness were measured, protecting the virus from external stress. This study provides new insight into the ultrastructure of SARS-CoV-2 particles at the nanoscale, offering new prospects that could be employed for mapping viral surfaces. The understanding of the viruses' capacity to survive mechanical disruptions at any level and their ability to recover from such injuries can shed a light on the structure-function relationship and help us to find targets for drug action, especially for this virus that, to this day, has no course of treatment approved.

Hypolipidemic and reduced nitrergic effects of p-hydroxycinnamic diesters extracted from Copernicia prunifera in mice challenged by a high-fat diet.

Dyslipidemia is a chronic non-transmissible condition that has increased due to an unhealthy lifestyle. Statins have been used as the standard treatment to control hyperlipidemia. However, side effects and high costs may be associated with its prolonged treatment, so plants derivatives have been an attractive therapy to overcome these problems. Among the compounds extracted from plants, the p-hydroxycinnamic diesters (HCE), present in carnauba wax (CW), have been found with good pharmacological properties. Therefore, this study aimed to evaluate the potential anti-hypercholesterolemic and possible toxicological effects of HCE in C57BL/6J mice under a high-fat (HF) diet. Male C57BL/6J mice were fed during 60 days under the HF diet and therefore were either treated with HCE (200 and 400 mg/kg) or simvastatin (20 mg/kg) or received saline (controls) by gavage for 30 days under the same diet. HCE treatment was able to reduce serum total cholesterol and LDL levels. Besides, this compound increased liver X receptor (LXR) and but not significantly affected IL-1ß and TNF-α liver mRNA transcription activity. In conclusion, HCE treatment was found safe and may attenuate the deleterious effects of dyslipidemia due to chronic feeding with western diets.

Non-structural protein 1 from Zika virus: Heterologous expression, purification, and potential for diagnosis of Zika infections.

Zika virus (ZIKV) infections were associated with neurological disorders only after the Brazilian outbreak in 2015. The lack of vaccines and precise diagnosis requires a precise method to detect ZIKV infection. This study aimed to evaluate three ZIKV recombinant proteins for the development of ZIKV infections. Here, it was purified stable recombinant ZIKV Capsid (r-ZIKV-c), non-structural proteins NS1 (r-ZIKV-NS1), and NS3 (r-ZIKV-NS3) for detection of the infection by ZIKV in blood sera of patients. A commercial polyclonal antibody recognized the r-ZIKV-NS1. Here, among three proteins, NS1 showed the best result for diagnostic purposes using serum samples, despite the high similarity with NS1 from DENV, and could differentiate the infections. The recombinant NS1 was used to produce a monoclonal antibody to differentiate between DENV and ZIKV NS1. As for recombinant proteins, the result for r-ZIKV-NS1 values showed 77% and 100% sensitivity and specificity, respectively, in the IgM assay. Our data showed the protein could successfully differentiate between sera of ZIKV infected patients from sera of those not infected with the virus and differentiate from sera of DENV infected patients. Thus, the generated recombinant proteins have great potential for serological diagnosis of ZIKV in Brazil, where it is indispensable.

Plant Expression of Hydrophobin Fused K39 Antigen for Visceral Leishmaniasis Immunodiagnosis.

Visceral leishmaniasis is a Neglected Tropical Disease of high mortality caused by the protozoan Leishmania infantum. Its transmission cycle is complex, and it has in the domestic dog its main reservoir. The diagnostic tests currently used rely on prokaryotic systems' proteins, but their low sensitivity increases the disease's burden. The plant transient expression of recombinant proteins allows the production of complex antigens. However, this system has limited competitiveness against the bacterial production of purified antigens. Thus, we have shown that the L. infantum K39 antigen's fusion to a hydrophobin allows its production for diagnostic tests without the need for intensive purification. The sera of naturally infected dogs specifically detect the semi-purified rK39-HFBI protein. The test validation against a panel of 158 clinical samples demonstrates the platform's viability, resulting in sensitivity and specificity of 90.7 and 97.5%, respectively. Thus, the use of semi-purified antigens fused to hydrophobins can become the standard platform for large-scale antigens production to expand diagnostic tests for other human and veterinary diseases worldwide.

Alpha Lipoic Acid Supplementation Improves Ovarian Tissue Vitrification Outcome: An Alternative to Preserve the Ovarian Function of Morada Nova Ewe.

This study evaluated the effect of adding alpha lipoic acid (ALA) to the vitrification solution of sheep ovarian tissue on 7 days of in vitro culture or 15 days of xenotransplantion. ALA was used at two different concentrations (100 µM: ALA100 and 150 µM: ALA150). Ovarian tissue was evaluated by classical histology (follicular morphology, development, and stromal cell density); immunohistochemistry for forkhead box O3a (FOXO3a); Ki67 (cell proliferation); cluster of differentiation 31 (CD31); and alpha smooth muscle actin (α-SMA). Reactive oxygen species (ROS) levels in ovarian tissue, as well as malondialdehyde (MDA) and nitrite levels in the culture medium, were assessed. Similar percentage of morphologically normal follicles was found in the vitrified ovarian tissue in the presence of ALA100 or ALA150 after in vitro culture or xenotransplantation. Follicular development from all treatments was higher (P < 0.05) than the control group. Moreover, an activation of primordial follicles was observed by FOXO3a. Stromal cell density and immunostaining for Ki67 and CD31 were significantly higher (P < 0.05) in ALA150 vitrified tissue. No difference (P > 0.05) was found in α-SMA between ALA concentrations after in vitro culture or xenograft. ROS levels in the ovarian tissue were similar (P > 0.05) in all treatments, as well as MDA and nitrite levels after 7 days of culture. We concluded that the addition of ALA 150 is able to better preserve the stromal cell density favoring granulosa cell proliferation and neovascularization.