Animal Research Research Articles

Exploring distinct and joint contributions of the locus coeruleus and the substantia nigra/ventral tegmental area complex to reward and valence processing using high-resolution fMRI

Abstract Dopaminergic neurons in the substantia nigra and the ventral tegmental area (SN/VTA) are classically viewed as key mediators in reward processing, while noradrenergic cells in the locus coeruleus (LC) are thought to modulate (negative) saliency processing. However, this conventional distinction is being revised by more recent research in animals. To explore the respective contributions of both the LC and SN/VTA in reward and valence processing in humans, we assessed fMRI data during stimulus encoding and response phase of a rewarded emotion-discrimination task (n = 38). Participants responded significantly faster to reward predicting and negative valence stimuli compared with their non-salient counterparts. LC activity was overall higher during trials involving reward prospect, and in particular for reward trials featuring positive valence, demonstrating an additive effect of reward and positive valence in LC. Moreover, LC activity was differentially increased for negative compared with positive valence in the response phase, indexing its role in invigorating responses to negative events. The SN/VTA showed increased activity in the response phase of reward trials (neutral valence) and negative valence trials (no reward), which aligns with coding relative saliency of these events in their respective contexts. LC modulations were accompanied by covariations in occipital cortex, suggesting noradrenergic contributions to visual prioritization of salient events. The findings underscore the sensitivity of both LC and SN/VTA to reward prospects and negative valence, challenging the dominant view of SN/VTA’s involvement in merely positive events and emphasizing their essential role in action invigoration above and beyond mere stimulus encoding. The intricate roles of the DA and NA system in reward and emotional valence processing in humans warrant further exploration and validation, given the limitations inherent to neuroimaging of deep brain structures.

Effects of SGLT2 inhibitors on the cardiovascular outcomes in patients with cancer: a systematic review and meta-analysis

Abstract Introduction The incidence of cardiovascular diseases in cancer survivors has gradually increased due to well-documented drug-associated cardiotoxicity and the expanding population resulting from advances in cancer treatment. Sodium-glucose cotransporter type 2 inhibitors (SGLT2i) have demonstrated efficacy in reducing cardiovascular risk in patients with type 2 diabetes mellitus, independent of glycemic control. Previous bench and animal research, along with several cohort studies, suggest potential benefits of SGLT2 inhibitors on cardiovascular outcomes in patients with cancer, with or without anthracycline therapy. This systematic review and meta-analysis aim to explore the potential of SGLT2 inhibitors as a therapeutic intervention to reduce cardiovascular risks in cancer patients, especially those undergoing chemotherapy. Methods We systematically searched Cochrane, PubMed, and Embase from inception until February 29, 2024, without language restrictions. Observational cohort studies with a follow-up period of at least 1 year were included to analyze the effects of SGLT2 inhibitors on cardiovascular outcomes in type 2 diabetes patients with cancer. Inclusion and exclusion criteria were predefined. Using random-effects models, we calculated odds ratios (OR) and 95% confidence intervals (CI). Subgroup and sensitivity analyses were conducted to identify potential sources of heterogeneity and explore the impact of SGLT2 inhibitors on mitigating cardiotoxicity. Primary outcomes of interest were mortality, heart failure hospitalization, sepsis, and diabetic ketoacidosis. Results Six studies involving 2959 patients in the SGLT2i group and 29169 patients in the non-SGLT2i group were included. Type 2 diabetes patients receiving SGLT2i exhibited significantly lower odds of mortality than those without SGLT2i (OR=0.34, 95% CI=[0.26-0.46], I²=77%). Subgroup analysis revealed a similar reduction in mortality among patients on SGLT2i treated with anthracyclines (OR=0.44, 95% CI=[0.23-0.82], I²=69%). Odds ratios for heart failure hospitalization and sepsis were also significantly lower in the SGLT2i group than the non-SGLT2i group (OR=0.46, 95% CI=[0.35-0.60], I²=0%, and OR=0.28, 95% CI=[0.20-0.40], I²=0%, respectively). The risk for diabetic ketoacidosis was not increased in the SGLT2i group (OR=0.66, 95% CI=[0.20-2.17], I²=0%). Conclusion SGLT2 inhibitors demonstrate a reduction in mortality, heart failure hospitalization, and sepsis in type 2 diabetes cancer patients treated with or without anthracyclines in cohort studies. Our findings support the need for further investigation through randomized controlled trials.Forest plots of mortality with subgroupForest plots of other primary outcomes

Experimental models of antibiotic exposure and atopic disease

In addition to numerous clinical studies, research using experimental models have contributed extensive evidence to the link between antibiotic exposure and atopic disease. A number of mouse models of allergy have been developed and used to uncover the specific effects of various microbiota members and perturbations on allergy development. Studies in mice that lack microbes entirely have also demonstrated the various components of the immune system that require microbial exposure. The importance of the early-life period and the mechanisms by which atopy “protective” species identified in human cohorts promote immune development have been elucidated in mice. Finally, non-animal models involving human-derived cells shed light on specific effects of bacteria on human epithelial and immune responses. When considered alongside clinical cohort studies, experimental model systems have provided crucial evidence for the link between the neonatal gut microbiota and allergic disease, immensely supporting the stewardship of antibiotic administration in infants. The following review aims to describe the range of experimental models used for studying factors that affect the relationship between the gut microbiota and allergic disease and summarize key findings that have come from research in animal and in vitro models.

Animal researchers' views on the publication of negative results and subsequent policy adoptions.

For over a decade, the non-publication of negative results from preclinical studies has been identified as a significant concern in biomedical research. Such underreporting is considered a contributor to the reproducibility crisis in the field and has been recognized by significant journals such as Science and Nature. In response to the consistently high non-publication rates of preclinical animal research in Europe, a survey was conducted among the biomedical research community to gather their views on publishing negative results. Using the EUSurvey platform, over 200 researchers directly working with animals were surveyed. The study aimed to understand the frequency of negative results, the reasons behind their non-publication, and the perceived pros and cons of making such results public. Insights from the survey could guide steps toward promoting transparency in science, refining research methodologies, reducing animal usage in experiments and minimizing research waste.

Tryptophan Metabolites Improve Intestinal Mucosal Barrier via the Aryl Hydrocarbon Receptor-Interleukin-22 Pathway in Murine Dextran Sulfate Sodium-Induced Pouchitis.

Pouchitis is the commonest complication after ileal pouch-anal anastomosis for ulcerative colitis. The protective effect of tryptophan metabolites on the mucosal barrier may be an effective method for treating pouchitis. The role of tryptophan metabolites on pouchitis remained unclear. We aimed to establish a murine model of dextran sulfate sodium-induced pouchitis to examine the roles of tryptophan metabolites in its pathogenesis. This is a study combined clinical patient data and animal research. A total of 22 patients were enrolled: 5 with familial adenomatous polyposis after ileal pouch-anal anastomosis, eight ulcerative colitis patients after ileal pouch-anal anastomosis patients with pouchitis, and 9 ulcerative colitis patients after ileal pouch-anal anastomosis with normal pouch. The demographic data and fecal samples of patients were collected. Male C57BL/6 mice were purchased from a licensed breeder and underwent ileal pouch-anal anastomosis to establish murine model of pouch. The bloods, feces, tissues of mice were collected. This study was performed in an academic medical center in China. The demographic data of patients were observational collected. The mice underwent ileal pouch-anal anastomosis were divided into six groups: control group with chow diet, dextran sulfate sodium, 6-formylindolo[3,2-b] carbazole + dextran sulfate sodium, high tryptophan diet + dextran sulfate sodium, CH-223191 + dextran sulfate sodium, indole-3-carboxaldehyde + dextran sulfate sodium. Animals were sacrificed after dextran sulfate sodium for 7 days. Fecal tryptophan metabolite level and microbiome composition, the severity of pouchitis, intestinal mucosal barrier function, and activation of the aryl hydrocarbon receptor-interleukin 22 pathway were assessed. Patients with pouchitis had lower fecal microbial diversity and indole-3-acetic acid levels. In murine pouchitis model, high-tryptophan diet increased fecal levels of 3-indoleglyoxylic acid, indole-3-aldehyde, and indole. A high-tryptophan diet and intraperitoneal aryl hydrocarbon receptor ligand 6-formylindolo[3,2-b] carbazole injection alleviated pouchitis. Tryptophan metabolites improved pouch mucosal barriers. aryl hydrocarbon receptor inhibitors exacerbated experimental pouchitis and disrupted the mucosal barrier; however, the aryl hydrocarbon receptor ligand indole-3-carboxaldehyde reversed this effect. This study was limited by small human sample size and lacking an Aryl hydrocarbon receptor knockout mouse model. A high-tryptophan diet and aryl hydrocarbon receptor ligand alleviated dextran sulfate sodium-induced pouchitis in murine ileal pouch-anal anastomosis model, which might be through regulating epithelial tight junctions and promoting goblet-cell differentiation, as well as maintaining the integrity and function of the mucosal barrier. This study provides a rationale for the clinical application of Aryl hydrocarbon receptor ligands in the treatment of pouchitis. See Video Abstract.

Spontaneous hepatocellular and gastric tumours in African clawed frogs (Xenopus laevis).

Three seven-year-old African clawed frogs (Xenopus laevis) from an animal research facility showed weight loss, coelomic distention and an abnormal swimming gait were euthanised on welfare grounds. A complete necropsy of each animal showed a focal, firm, dark brown to green mass with multifocal haemorrhages in the left liver lobe in two animals and a transmural, firm, beige, multilobulated gastric mass in another animal. Additionally, one of the frogs with the hepatic mass had haemocoeloma. Histologically, the hepatic masses were diagnosed as hepatocellular adenomas and the gastric mass as a gastric carcinoma with trans-coelomic metastases. The three tumours were immunophenotyped using the following antibodies: cytokeratin AE1/AE3, vimentin, E-cadherin, P53 and Ki67 and, additionally, for the hepatic tumours only, synaptophysin, Prox-1, S100 and Sox-9. Masson's trichrome, Periodic acid Schiff and Gram stains was also performed in selected cases. One of the hepatocellular adenomas was weakly positively labelled with E-cadherin whereas the other showed variable positivity for Sox-9 only. Vimentin labelled the stroma and sinusoidal endothelia. Interestingly in the liver, the cytokeratin AE1/AE3 labelling was restricted to the biliary epithelium and sinusoidal endothelia. The gastric carcinoma labelled positively with cytokeratin AE1/AE3 only. This report aims to guide laboratory animal veterinarians to accurately diagnose multi-organ masses in amphibians. To the authors' knowledge, this is the first comprehensive morphological study on a case series of hepatocellular and gastric neoplasia in X. laevis.

Defining social reward: A systematic review of human and animal studies.

Social rewards are strong drivers of behavior and fundamental to well-being, yet there is a lack of consensus regarding what actually defines a reward as "social." Because a systematic overview of existing social reward operationalizations is currently absent, a review of the literature seems necessary to advance toward a unified framework and to better guide research and theory. To bridge this gap, we preregistered and conducted the first comprehensive systematic review of human and animal experimental studies that used the term "social reward" and charted existing operationalizations, revealing the implicit and explicit definitions used in the field. Stimulus characteristics and measures of social reward were extracted from a total of 384 studies encompassing 42,118 participants and subjects. We provide detailed summaries of these elements, stratified by species (human/animal) and study type (behavioral, brain imaging, pharmacological, and physiological). Two main aspects were found to account for most of the difference in operationalizations: the sensory richness of a stimulus (intimacy) and engagement in social interaction (i.e., the synchronous observation and action between at least two individuals, viz., immediacy). Drawing insights from second-person neuroscience approaches and theoretical models in the field of human-computer interaction, we propose that human and animal research can greatly benefit from considering these properties, as they have important theoretical and practical consequences for human and translational research, with far-reaching implications for neighboring research fields such as those pertaining to social media and the development of artificial intelligence. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Hypercholesterolemia and Alzheimer's Disease: Unraveling the Connection and Assessing the Efficacy of Lipid-Lowering Therapies.

This article examines the relationship between cholesterol levels and Alzheimer's disease (AD), beginning with the early observation that individuals who died from heart attacks often had brain amyloid deposition. Subsequent animal model research proved that high cholesterol could hasten amyloid accumulation. In contrast, cholesterol-lowering treatments appeared to counteract this effect. Human autopsy studies reinforced the cholesterol-AD connection, revealing that higher cholesterol levels during midlife significantly correlated with higher brain amyloid pathology. This effect was especially pronounced in individuals aged 40 to 55. Epidemiological data supported animal research and human tissue observations and suggested that managing cholesterol levels in midlife could reduce the risk of developing AD. We analyze the main observational studies and clinical trials on the efficacy of statins. While observational data often suggest a potential protective effect against AD, clinical trials have not consistently shown benefit. The failure of these trials to demonstrate a clear advantage is partially attributed to multiple factors, including the timing of statin therapy, the type of statin and the appropriate selection of patients for treatment. Many studies failed to target individuals who might benefit most from early intervention, such as high-risk patients like APOE4 carriers. The review addresses how cholesterol is implicated in AD through various biological pathways, the potential preventive role of cholesterol management as suggested by observational studies, and the difficulties encountered in clinical trials, particularly related to statin use. The paper highlights the need to explore alternate therapeutic targets and mechanisms that escape statin intervention.

Representation of Anticipated Rewards and Punishments in the Human Brain.

Subjective value is a core concept in neuroeconomics, serving as the basis for decision making. Despite the extensive literature on the neural encoding of subjective reward value in humans, the neural representation of punishment value remains relatively understudied. This review synthesizes current knowledge on the neural representation of reward value, including methodologies, involved brain regions, and the concept of common currency representation of diverse reward types in decision-making and learning processes. We then critically examine existing research on the neural representation of punishment value, highlighting conceptual and methodological challenges in human studies and insights gained from animal research. Finally, we explore how individual differences in reward and punishment processing may be linked to various mental illnesses, with a focus on stress-related psychopathologies. This review advocates for the integration of both rewards and punishments within value-based decision-making and learning frameworks, leveraging insights from cross-species studies and utilizing ecological gamified paradigms to reflect real-life scenarios.

Comments to the article submitted by А.А. Kovalev et al. “The intraluminal administration of Indocyanine green as a method of intraoperative diagnostics of machine suture incompetence in experimental cases of longitudinal gastric resection”

The editorial policy of the “Clinical Practice” journal is supposed to be oriented to Applied Clinical Researches, the results of which practicing physicians could use in their routine work. The main objective of creating the journal was informing the physicians on the novel approaches in the diagnostics and treatment of various diseases, as well as rehabilitation and restoration of the quality of life. For this reason, the editorial board had often refused to publish the articles from the authors of well-planned and performed animal research works, even despite the fact that the clinical specialty certificate 3.1.9 “Surgery” supposes the experimental development of new methods.

Methionine supply during mid-gestation modulates the bovine placental mTOR pathway, nutrient transporters, and offspring birth weight in a sex-specific manner.

The mechanistic target of rapamycin (mTOR) predominantly regulates the expression and activity of placental nutrient transporters. The mTOR pathway can be activated by several nutrients, including the essential amino acid methionine. Additionally, previous research in nonruminant animals suggests that mTOR is influenced in a sexually dimorphic manner. In bovine, there is limited understanding of how maternal nutrition and offspring sexual dimorphism affect the placental transfer of nutrients. Thus, we investigated the effects of increasing the supply of dietary methionine to beef cattle heifers during mid-gestation on mTOR signaling, placental nutrient transporters, and fetal growth in male and female offspring. Forty purebred Angus heifers were used in a randomized complete block design experiment. From day 90 to 180 of gestation, heifers received a basal diet with no added methionine (CON, n = 20), or the basal diet plus 8.3g of rumen-protected methionine (MET, n = 20) per animal daily. All animals received a basal diet in the first and third trimesters of gestation. Cotyledonary tissue samples were collected at parturition and utilized to examine the mTOR pathway and nutrient transporters through protein and gene expression analysis. The offspring's body weight was measured at birth. Data were analyzed using a mixed model that included the fixed effect of treatment, offspring sex, their interactions, and the random effect of block. At day 170 of gestation, MET-supplemented heifers showed higher plasma concentrations of methionine and glutamate (P < 0.01) and lower glycine and proline levels (P ≤ 0.01) compared to the CON group. A treatment × sex interaction was observed for calf birth weight (P = 0.03). In heifers that delivered male calves, MET supplementation increased the birth weight of the calves (P < 0.01). However, the dietary treatments had no effect on the birth weight of female calves (P = 0.32). The increase in birth weight of male calves from MET-fed heifers resembles with the upregulation of placental mTOR and phosphorylated mTOR (P ≤ 0.03), as well as the amino acid transporters SLC1A5, SLC7A5, SLC38A6, and SLC38A11, and the glucose transporters SLC2A1 and SLC2A8 (P ≤ 0.05). Our findings suggest that increasing the supply of methionine to beef heifers during mid-gestation can modulate placental nutrient transport and fetal growth in a sex-dependent manner and that these effects are mediated, at least in part, by the mTOR pathway.

Development of a center for automated equipment processing to transform the research enterprise configuration.

The strategy of centralizing equipment sanitation and processing was developed by a top-tier public university to address the growing physical infrastructure and human resource challenges of its expanding in-vivo research enterprise. Subsequently, a center for automated equipment processing physically separate from all animal research facilities was developed and has operated consistently since 2016. The facility incorporates systems such as process automation to sanitize and sterilize equipment as efficiently as possible. Analysis of the differences between the new centralized and old distributed research enterprise configurations shows a total estimated fiscal benefit of almost US$54 m to date projected out to US$124 m through 2028. Utility consumption of operations over nine years in the new configuration was estimated to be decreased by over 125 million gallons of water, 14 million gallons of chilled water, 121 million pounds of steam, and almost 3.6 million kilowatts of electricity, with consumption savings significantly increased projected out to 2028. Additional operational and organizational benefits as well as direct research benefits were identified. The new configuration has functioned for nine years without detectable cross contamination within the research enterprise thus providing evidence that the location of equipment processing can be less of a "microbial" risk factor than previously attributed.

Genetic contribution to sleep homeostasis in early adolescence.

The sleep homeostatic process in adults is moderately stable over time and unique to an individual. Work in transgenic mice has suggested a role of genes in sleep homeostasis. The current study quantified the genetic contribution to sleep homeostasis in adolescence. We use slow wave energy (SWE) as a metric for sleep pressure dissipation during sleep. This measure reflects both sleep intensity and duration. High-density (58 derivations) sleep electroencephalogram (EEG) was recorded in 14 monozygotic and 12 dizygotic adolescent twin pairs (mean age = 13.2 years; standard deviation [SD] = 1.1; 20 females). SWE at the end of sleep was quantified as the cumulative delta power (1-4.6Hz) over the night. We also examined the time constant of the decay and the level of slow wave activity (SWA) at the beginning of the sleep episode. Structural equation modelling was used to quantify the amount of variance in SWE and the dissipation of sleep pressure due to genes. We found that most (mean = 76% across EEG derivations) of the variance in SWE was due to genes. In contrast, genes had a small (mean = 33%) influence on the rate of dissipation of sleep pressure, and this measure was largely (mean = 67%) driven by environmental factors unique to each twin. Our results show that the amount of dissipated sleep pressure is largely under genetic control; however, the rate of sleep pressure dissipation is largely due to unique environmental factors. Our findings are in line with research in animals and suggest that the heritability of the rate of sleep pressure dissipation is limited.

The Safety of Alcohol Pharmacotherapies in Pregnancy: A Scoping Review of Human and Animal Research.

Alcohol pharmacotherapies pose unknown teratogenic risks in pregnancy and are therefore recommended to be avoided. This limits treatment options for pregnant individuals with alcohol use disorders (AUD). The information on the safety of these medications during pregnancy is uncertain, prompting a scoping review. The objective of this review was to investigate available information on the safety of alcohol pharmacotherapies in pregnancy. Studies published between January 1990 and July 2023 were identified through searches in BIOSIS, Embase, PsycINFO and MEDLINE databases, using terms related to pregnancy and alcohol pharmacotherapies. The alcohol pharmacotherapies investigated were naltrexone, acamprosate, disulfiram, nalmefene, baclofen, gabapentin and topiramate. Studies were screened by two independent reviewers. Covidence software facilitated the management, screening and extraction of studies. A total of 105 studies were included in the review (naltrexone: 21, acamprosate: 4, disulfiram: 3, baclofen: 3, nalmefene: 0, topiramate: 55, gabapentin: 32) with some studies investigating multiple medications. Studies investigating naltrexone's safety in pregnancy focussed on opioid use disorders, with limited evidence regarding its safety in the context of AUD. Despite concerns about higher rates of some pregnancy complications, studies generally indicate naltrexone as a safer option compared with opioid agonists or alcohol during pregnancy. Acamprosate was not clearly associated with adverse effects of exposure in pregnancy, with two pre-clinical studies suggesting potential neuroprotective properties. Disulfiram has a high risk of congenital anomalies when used in pregnancy, believed to be due to its mechanism of action. Prenatal topiramate has also been associated with an increased risk of congenital anomalies, particularly oral clefts. There were mixed results concerning the safety of prenatal gabapentin and little to no literature investigating the safety of baclofen or nalmefene during pregnancy. There is insufficient research on the safety of alcohol pharmacotherapies in pregnancy. Despite this, given alcohol's teratogenic effects, naltrexone could be considered to help maintain abstinence in pregnant individuals with AUD, particularly when psychosocial treatments have failed.

Interdisciplinary Animal Research Ethics-Challenges, Opportunities, and Perspectives.

Can nonhuman animals be used for the benefit of humans in a scientifically and morally justified manner and, if yes, how? Based on our own experiences as scholars from various academic backgrounds, we argue that this question can only be answered as an interdisciplinary and international endeavor, considering insights from research ethics and animal ethics as well as scientific and legal aspects. The aim of this article is to contribute to the foundation of the emerging field of animal research ethics. In doing so, we describe the following seven phases of animal research experiments: ethical, legal and social presumptions (phase 0), planning (phase I), review (phase II), conduct of experiments (phase III), publication/dissemination (phase IV), further exploitation of results (phase V), and evaluation (phase VI). In total, 20 key ethical, legal, and practical challenges that an ethical framework for the use of animals in research needs to address are identified and analyzed. Finally, we characterize the following four meta-challenges and opportunities associated with animal research ethics as a field: (1) moral pluralism, (2) the integration of views and positions outside the laboratory, (3) international plurality of conduct, standards, and legal norms, and (4) interdisciplinary education.

Decision trees for determining the fate of laboratory animals.

Facilities involved in laboratory animal research often face ethical challenges such as: what should I do with the animals that are no longer suitable for experimental purposes? One of the common answers to this question is to kill them. And while numerous scientifically justifiable reasons exist for killing laboratory animals, we must not overlook our ethical responsibility towards these sentient beings. Animal facility managers and scientists frequently find themselves in a moral dilemma, torn between furthering their research and addressing the well-being of experimental animals required for their studies. We elaborated a concept consisting of six decision trees and recommendations for making informed decisions about the need to kill laboratory animals in research facilities, considering legal and ethical considerations. The concept is based on the German regulatory perspective. However, the measures and decisions for animal welfare can be implemented in all laboratory animal facilities. These recommendations suggest several courses of action, including implementing consistent breeding plans, exploring alternative uses, reassigning surplus animals and their organs, and establishing appropriate housing capacity limits that ensure species-appropriate care. We encourage scientists and animal facility managers to develop and implement decision-making frameworks and procedures tailored to their specific facilities, in the hope that this work will promote a thoughtful and responsible approach to the complex challenges associated with the killing of laboratory animals, advancing scientific progress and the humane treatment of these animals.

A guide to improving the use of activity data in animal research

A guide to improving the use of activity data in animal research

An Easily Customizable Approach for Automated Species-Specific Detection of Anuran Calls Using the European Green Toad as an Example

Machine learning approaches for pattern recognition are increasingly popular. However, the underlying algorithms are often not open source, may require substantial data for model training, and are not geared toward specific tasks. We used open-source software to build a green toad breeding call detection algorithm that will aid in field data analysis. We provide instructions on how to reproduce our approach for other animal sounds and research questions. Our approach using 34 green toad call sequences and 166 audio files without green toad sounds had an accuracy of 0.99 when split into training (70%) and testing (30%) datasets. The final algorithm was applied to amphibian sounds newly collected by citizen scientists. Our function used three categories: “Green toad(s) detected”, “No green toad(s) detected”, and “Double check”. Ninety percent of files containing green toad calls were classified as “Green toad(s) detected”, and the remaining 10% as “Double check”. Eighty-nine percent of files not containing green toad calls were classified as “No green toad(s) detected”, and the remaining 11% as “Double check”. Hence, none of the files were classified in the wrong category. We conclude that it is feasible for researchers to build their own efficient pattern recognition algorithm.

8415 Rapid Generation Of An In Vivo Loss-Of-Function Genetic Model Of sdhb Zebrafish: Functional And Metabolic Characterization

Abstract Disclosure: S. Parisien-La Salle: None. F. Nobilleau: None. J. Lamontagne: None. S. Éric: None. I. Bourdeau: None. Introduction: Animal research has been limited in pheochromocytomas and paragangliomas (PPGLs). Recently, Dona et al., described a sdhb mutated zebrafish model by introducing a 13 bp frameshift mutation at the sdhb exon1—intron1 boundary into the zebrafish sdhb gene that recapitulated features of PPGLs. Objective: To generate a new model of mutated sdhb zebrafish to further investigate the underlying metabolic and biochemical perturbations in vivo. Methods: We took advantage of the genetic accessibility of zebrafish embryo to generate loss-of-function model for sdhb using CRISPR/Cas9 technology. Three guide RNAs were designed and microinjected in one-cell stage zebrafish embryos to target the coding sequence of zebrafish sdhb gene. In order to control for the stress brought on by injections, we injected a group of zebrafish with Cas9 endonuclease as a control. At five days post fertilization, mutant and control zebrafish were flash frozen and sent for liquid chromatography mass spectrometry measurements of Krebs cycle metabolites and normetanephrines/metanephrines. All results were normalized to the control groups consisting of wild-type or Cas9 injected larvae. Sdhb expression was analyzed by RT-qPCR in sdhb mutants and wild-type zebrafish larvae. We also monitored survival and heart rate in sdhb mutant zebrafish, compared to wild-type and Cas9. Results: Firstly, we confirmed the significant reduction of sdhb expression in CRISPR-injected larvae compared to wild-type larvae at 5 dpf (0.0613 vs 1.000 p&amp;lt;0.0001). Our metabolic panel showed that succinate was up to 26 times more elevated in sdhb mutants than in wild-type fish extracts (sdhb: 26.51 p&amp;lt;0.0001). Lactate (sdhb: 5.27 p&amp;lt;0.0001), leucine (sdhb: 2.88 p&amp;lt;0.0001), arginine (sdhb: 1.50 p=0.0034) and HMG-CoA (sdhb: 1.36 p=0.0033) were also higher in sdhb mutants. Whereas aspartate (sdhb: 0.244 p&amp;lt;0.0001) and oxidized gluthiatone (sdhb: 0.64 p=0.0011) were significantly lower in sdhb mutants than in wild-types. In regards to functionality, normetanephrine and metanephrine levels were at least four folds higher in sdhb mutants than in Cas9 injected larvaes (4.06 p&amp;lt;0.0001 and 3.42 p&amp;lt;0.0001 respectively). Interestingly, this difference was more noticeable in the fish bathing medium than in larvae tissue extracts, suggesting an increased hormonal excretion (normetanephrines: 4.81 p&amp;lt;0.0001 and metanephrines: 9.40 p&amp;lt;0.0001). Finally, sdhb mutated zebrafish presented with a higher heart rate (148 vs 133 p&amp;lt;0.0001) and reduced lifespan (p&amp;lt;0.0001) when compared to the wild-type/Cas9 group. Conclusion: These findings confirm that the zebrafish is an ideal model for studying PPGLs and their associated genetic defects. Our approach allows the in vivo assessment of a large metabolic panel, opening avenues on new potential clinical metabolic biomarkers in a loss-of-function of the SDHB gene. Ref: Dona M, et al. Endocr Relat Cancer. 2021 Presentation: 6/1/2024

9233 Sperm-carried IGF2: a Spark Contributing To Embryo Growth And Development

Abstract Disclosure: R. Cannarella: None. O.J. Rando: None. R.A. Condorelli: None. C. Sandrine: None. S. Romano: None. A. Guglielmino: None. Q. Yin: None. H. Gustafsson: None. F. Mancuso: None. C. Bellucci: None. G. Luca: None. S. La Vignera: None. A.E. Calogero: None. Background: Sperm RNAs can regulate early embryo development. A potential key regulator of growth is insulin-like growth factor 2 (IGF2), a highly conserved paternally-imprinted gene involved in cell growth and proliferation that is expressed in human spermatozoa. Animal research supports the positive impact of IGF2-supplemented cultures on embryo quality. However, the role of sperm-carried IGF2 in the early development of the human embryo is unknown. Objective: To study whether sperm-carried IGF2 mRNA influences the morphokinetics of the human embryo. Design: Prospective, uncontrolled, observational pilot study. Setting: University-affiliated fertility center. Subjects: We enrolled 16 patients undergoing assisted reproductive techniques (ART) for idiopathic or male factor infertility and 10 healthy fertile controls with proven paternity. Couples with female factor infertility due to reduced oocyte reserve were excluded, while those with tubal factor or anovulation were included. Main Outcome Measure(s): After clinical use, an aliquot of the sperm samples employed for ART was used to evaluate IGF2 mRNA levels. Embryo kinetics were recorded and correlated with the amount of IGF2 mRNA, correcting for female age, body mass index (BMI), anti-Müllerian hormone (AMH) levels, and oocyte quality. To provide mechanistic insights into the human data, a transcriptome analysis was performed of parthenotes obtained from twelve-week-old superovulated mice injected with Igf2 mRNA plus Gfp mRNA (experimental group, n=20) or Gfp mRNAs (control group, n=20). Pathway enrichment analysis was then performed using g:Profiler g:GOSt and EnrichR. Results: Sperm IGF2 mRNA negatively correlated with time to 2-cell stage (t2), t3, t4, t5, and time to expanded blastocyst (tEB), independently of maternal age, BMI, AMH, and oocyte quality. IGF2 mRNA index &amp;gt;4.9 predicted the ability of the embryo to reach the blastocyst stage at day 5, with a sensitivity of 100% and specificity of 71.6% (AUC 0.845; p&amp;lt;0.001). The distribution of sperm IGF2 levels differs significantly between morphologically good or poor embryos, with the lowest levels more frequently associated with morphologically poor embryos (61.9%) than with good ones (38.1%) (p=0.0091). Furthermore, sperm-derived IGF2 levels were significantly lower in subfertile men compared to fertile controls with proven paternity. Transcriptome analysis of Igf2-injected embryos demonstrated differential expression of 101 genes activating pathways regulating early embryo development. Conclusion: Sperm-carried IGF2 mRNA can support early embryo development, independently of female factors. Larger studies are needed to confirm this finding and to investigate its role as a clinical marker to predict the chances of obtaining blastocysts that can be implanted in infertile couples undergoing ART. Presentation: 6/1/2024