Medulla Tissue Research Articles

7698 A case of Pheochromocytoma in a patient with severe aortic stenosis

Abstract Disclosure: C. Lane: None. L.E. Aguirre: None. J. Azocar-Villalobos: None. R.I. Dorin: None. Background: Pheochromocytomas are rare neuroendocrine tumors derived from chromaffin tissue of the adrenal medulla and can secrete catecholamines. Pre-operative management of pheochromocytoma involves initiation of a- followed by b-adrenergic receptor blockade. In critical aortic stenosis (AS), adrenergic blockade may be complicated by decreased cardiac output and hypotension owing to dependence of venous return on maintenance of high afterload (1). We report a case of critical AS and incidentally discovered pheochromocytoma and describe our management strategy, which involved prioritization of aortic valve repair prior to adrenal surgery and multidisciplinary approach to initiation and titration of a- and b-adrenergic blockade prior to cardiac catheterization and transcutaneous aortic valve replacement (TAVR) procedures. Clinical Case: A 75-year-old Caucasian male with past medical history of obstructive sleep apnea, tobacco use, and cocaine use disorder was admitted with progressive dyspnea, decreased exercise tolerance, and involuntary weight loss of 18 kg over 3 months, and community acquired pneumonia. On admission, he was found to have critical aortic stenosis (valve area of 0.59 cm2, mean gradient 83 mmHg) and new HFrEF (EF 40-45%). CT chest demonstrated incidental right adrenal mass (5.6 x 5.0 x 4.7 cm) with central necrosis, HU units >10, and evidence of internal hemorrhage. Subsequent laboratory and functional imaging studies confirmed diagnosis of pheochromocytoma. Pre-procedural a-adrenergic blockade with doxazosin followed by beta-blockade was managed by a multidisciplinary team with ICU admission for monitoring and uncomplicated completion of cardiac catheterization and TAVR. Conclusions: Pre-procedural diagnosis of pheochromocytoma, multidisciplinary management, and intensive care setting for monitoring hemodynamic response to initiation and titration of a and b-adrenergic blockade appeared to be key factors in achieving uncomplicated completion of cardiac interventions prior to surgical resection of pheochromocytoma and critical AS. Successful TAVR was associated with marked improvement in hemodynamic status. Maintenance of b-adrenergic blockade to reduce myocardial contractility was critical to preservation of the prosthetic aortic valve and in preparation for planned robotic resection of the pheochromocytoma.

Hipertensão refratária em paciente jovem: Um relato de caso

Introduction: Pheochromocytoma, a rare tumor originating from the chromaffin tissue of the adrenal medulla, has a prevalence of about 0.1-0.6% in patients with hypertension. Usually benign, its surgical removal can be curative. However, delays in diagnosis and inadequate treatment can lead to high mortality and morbidity rates. Case Report: A 23-year-old man with a history of resistant hypertension since December 2022 presented with symptoms of headache, palpitation, sweating, and anxiety. Tests revealed elevated levels of metanephrines and catecholamines, leading to the suspicion of pheochromocytoma. An MRI of the abdomen identified an expansive lesion in the right adrenal gland. Treatment included clinical management with alpha- and beta-blockers, followed by successful adrenalectomy, resulting in improvement of symptoms and hypertension. Discussion: The case illustrates the complexity inherent in the diagnosis of pheochromocytoma, a condition that often manifests surreptitiously in hypertensive patients. Resistance to conventional antihypertensive therapy, combined with symptoms such as headache, palpitations, and sweating, should raise suspicions of secondary hypertension and, consequently, pheochromocytoma. This case underlines the importance of a meticulous diagnostic evaluation in patients with refractory hypertension. Conclusion: Pheochromocytoma, despite being a rare cause of hypertension, represents a crucial differential diagnosis in secondary hypertension. Early diagnosis and appropriate surgical intervention are essential to prevent associated morbidity and mortality. This case reinforces the need for clinical surveillance for suggestive symptoms and the importance of appropriate diagnostic investigations in patients with resistant hypertension, particularly in young people.

Comparison of intraoperative hemodynamics in classical and single-port adrenalectomy

BACKGROUND: Pheochromocytoma (PCC) is a tumor from the chromaffin tissue of the adrenal medulla, capable of hyperproduction of catecholamines. An important feature of pheochromocytoma, due to its hormonal activity, is the threat of sudden changes in hemodynamic parameters during surgical treatment. Retroperitoneoscopic access is currently becoming increasingly popular in the surgical treatment of chromaffin tumors of the adrenal glands. In clinical practice, two variants of the mentioned access are used: classic (three-port) and single-port. In this study, the influence of these options of retroperitoneoscopic access on intraoperative hemodynamic control was evaluated.AIM: Comparison of treatment results and intraoperative hemodynamic parameters of patients with adrenal pheochromocytomas operated with single-port and three-port retroperitonescopic access options.MATERIALS AND METHODS: A retrospective single-center cohort study was conducted on a sample of patients initially operated for pheochromocytoma at the Pirogov St. Petersburg State University High Medical Technology Clinic from October 2015 to February 2020 with intraoperative invasive hemodynamic control using PiCCO+ systems.RESULTS: 47 patients were examined, 24 of whom underwent single–port adrenalectomy (included in the first group), 23 — three-port (second group). The average tumor size in the second group was larger (40.12±9.3 and 56.7±21.5, p<0.05), presumably, in this regard, the average duration of surgery was statistically significantly higher (73±21.5 and 111.7±36.1, p<0.05). The body mass index (BMI) of patients in the two groups did not differ statistically significantly. Intraoperatively measured values of maximum and minimum mean systolic and diastolic blood pressure, the number of episodes of increased diastolic blood pressure above 120 mmHg and their duration did not differ statistically in both groups (p>0.05). Episodes of increased systolic blood pressure above 180 mm Hg were more frequent in the second group (p=0.03), but their total duration in the two groups was without significant differences (p>0.05). Intraoperative consumption of antihypertensive drugs during the release of the adrenal gland and vasopressor drugs after compression of the central vein of the adrenal gland were similar. Blood pressure after compression of the central vein in the first and second groups did not differ significantly (p>0.05). No deaths and postoperative complications were registered in both groups.CONCLUSION: Single-port modification of retroperitoneoscopic access with sufficient experience of the anesthesiological team and the operating surgeon does not lead to deterioration of hemodynamic control and operation results, being safe and effective in the surgical treatment of pheochromocytoma.

Neurotrophin-4 promotes in vitro development and maturation of human secondary follicles yielding metaphase II oocytes and successful blastocyst formation.

Does a matrix-free culture system supplemented with neurotrophic factor 4 (NT4) improve human in vitro follicular development and meiotic maturation, ultimately resulting in fertilizable oocytes? NT4 supplementation of in vitro culture significantly enhances the growth, steroid hormone production, and maturity potential of human secondary follicles derived from fresh ovarian medulla (from post- and pre-pubertal patients), thereby yielding fertilizable oocytes. Reconstituting folliculogenesis in vitro is of paramount importance in the realms of fertility preservation, reproductive biology research, and reproductive toxicity assessments. However, the efficiency of in vitro culture systems remains suboptimal, as the attainment of fertilizable oocytes from in vitro growth (IVG) of human follicles remains unachieved, with the data being particularly scant regarding follicles from prepubertal girls. We have previously found that mouse oocytes from secondary follicles derived from IVG are deficient in neuroendocrine regulation. NT4 and its corresponding receptor have been identified in human follicles. Significantly, the addition of NT4 during the IVG process markedly enhances both follicle growth and oocyte maturation rates in mice. Fresh medulla tissue obtained during tissue preparation for ovarian tissue cryopreservation (OTC) were collected from 10 patients aged from 6 to 21 years old, all of whom had undergone unilateral oophorectomy as a means of fertility preservation. Isolated secondary follicles were individually cultured in vitro with or without NT4 in a matrix-free system. Secondary follicles, extracted via enzymatic digestion and mechanical disruption from each patient, were randomly allocated to either a control group or an NT4-supplemented group (100 ng/ml), followed by individual culture on an ultra-low attachment plate. Follicle growth and viability were assessed by microscopy. Levels of anti-Müllerian hormone (AMH), estradiol, and progesterone in the medium were quantified. An oocyte-specific marker was identified using confocal fluorescence microscopy following DEAD box polypeptide 4 (DDX4) staining. The competence of individual oocytes for maturation and fertilization were assessed after IVM and ICSI with donated sperm samples. Overall, isolated follicles from both groups survived up to 6 weeks with increasing diameters over the duration (P < 0.05), reaching terminal diameters of almost 1 mm with confirmed steroidogenesis and expression of oocyte marker (DDX4), and producing morphologically normal MII oocytes. When compared with the control group, the NT4 group had a similar initial follicular diameter (206 ± 61.3 vs 184 ± 93.4 μm) but exhibited a significant increase in follicular diameter from the ninth day of culture onwards (P < 0.05). From Week 3, estradiol and progesterone production were significantly increased in the NT4 group, while no significant difference was observed in AMH production between groups. The proportion of 'fast-growth' follicles in the NT4 group was significantly higher than that in the control group (13/23 vs 6/24, P < 0.05). An increased efficiency of MII oocyte maturation per live follicle in the NT4 group was also observed (control group vs NT4 group, 4/24 vs 10/23, P < 0.05). It is noteworthy that an MII oocyte obtained from the control group exhibited abnormal fertilization after ICSI. In contrast, an MII oocyte acquired from the NT4 group progressed to the blastocyst stage and showed potential for transfer. N/A. The cohort examined in this study was all patients diagnosed with beta-thalassemia major. Whether this culture system is effective for patients with other diseases remains unknown. Since the chosen dose of NT4 was established based on dose finding in mice, the optimal dose for use in a human IVG system needs further confirmation. The oocytes and embryos procured from this study have not been quantified for ploidy status or epigenetic signatures. Fresh medulla tissue obtained during tissue preparation for OTC may serve as a precious source of fertilizable oocytes for female fertility preservation, even for pre-pubertal girls, without the threat of tumour reintroduction. After further characterization and optimization of the system, this culture system holds the potential to provide a powerful future research tool, for the comprehensive exploration of human follicular development mechanisms and for conducting reproductive toxicity evaluations. This work was supported by the National Key R&D Program of China (grant number 2022YFC2703000) and National Natural Science Foundation of China (grant numbers 82271651 and 81871214). The medium used in human follicle in vitro culture in this study has been applied for a national invention patent in China (No. 202211330660.7). The inventors of the patent, in order, are: Y.G., C.F., and X.L.

Short-term Administration of Naringin Improves Renal Function in Renal Ischemia-reperfusion by Increasing Aquaporin-1 and Aquaporin-2 Levels

Background:: Since renal ischemia-reperfusion (I/R) can lead to a serious health problem, aquaporins have important roles in preventing negative changes in electrolyte-water balance. This study aimed to determine the effect of naringin treatment on renal function and AQP1 and AQP2 levels in the kidney cortex and medulla tissues in experimental renal I/R in rats. Method and Material: The study was carried out on 40 male Wistar-type rats, 8-12 weeks old. Experimental groups were formed as follows: 1) Control, 2) Sham+vehicle, 3) Renal (I/R)+vehicle, 4) Renal I/R+ Naringin (50mg/kg/day) (3 days of administration), and 5) Renal I/R+ Naringin( 100mg/kg/day) (3 days supplementation) group. First, the left kidney was removed by nephrectomy under general anesthesia, and then the right kidney was subjected to 45 minutes of ischemia and then 72 hours of reperfusion. Naringin was given to the experimental animals by an intraperitoneal route at the beginning of the reperfusion, after 24 and 48 hours. At the end of the experiments, first of all, blood samples were taken from the heart in animals under general anesthesia, and then the animals were killed by cervical dislocation, and kidney tissue samples were taken. Osmolarity in plasma and urine and plasma creatinine levels were evaluated. AQP1 and AQP2 levels were analyzed in the kidney cortex and medulla tissues by ELISA and PCR methods. Result:: In kidney tissues, I/R led to a decrease in plasma and urinary osmolality, AQP1 and AQP2 levels in the cortex and medulla, and an increase in urea and creatinine levels (p &lt; 0.001). However, naringin supplementation corrected the deterioration to a certain extent. Conclusion:: The results of the study show that naringin supplementation at different doses, such as 50 or 100 mg/kg, may have protective effects on the deterioration of renal function caused by unilateral nephrectomy and I/R in rats.

The role of thymus graft on the onset of puberty in juvenile male rats

There exists a bidirectional relation between the thymus gland and the hypothalamo- hypophysial-gonadal axis. The thymus secretes various hormones, some of which can regulate the reproductive axis. So, it is worth to explore the possible role of an additional thymus on the onset of puberty in male albino rats. Two groups of male rats aged 5 and 10 days were used for the study. Each subgroup consisted of 4 pups each in control and experimental group. Thymus glands for graft were obtained from newborn male rat pups. Depending on the age group, 5 day or 10-day-old male rat pup were anaesthetized, 2-3 mm incision was made, and a thymus gland was grafted to the axilla. Sham operations were performed to the control pups. Without handling, animals were observed daily for the onset of puberty. On the day of descent of testes, body weight of the animal was noted, blood was collected and used for radio immunoassay. All morphometric measurements were done using an occular micrometer. Volume fraction of seminiferous tubules, intertubular connective tissue of testes, cortex and medulla of the thymus were estimated by point count method. In both the age groups thymus graft advanced the age of descent of testes, and increased body weight and organ weight. It also increased the serum hormone levels. In conclusion, this study indicates that an additional thymus gland has a positive effect on the male reproductive system towards pro-gonadal action, and this effect is more pronounced in older-age groups of rats.

Comparison of the effectiveness of various methods for determining the level of metanephrines in the diagnosis of pheochromocytomas

Pheochromocytoma (PHEO) is a tumor from the chromaffin tissue of the adrenal medulla, capable of hyperproduction of catecholamines. The increased production of hormones by the tumor leads to catecholamine crises, which have a pathological effect on all organs and systems. In the primary diagnosis of pheochromocytomas, it is important to determine the level of the metabolite of catecholamines - metanephrines. Currently, in clinical practice, various methods are used to determine the level of this metabolite: in blood plasma or in urine, total or only free form, fractionated analysis or unfractionated. Comparison of the effectiveness of various methods for determining the level of metanephrines for the diagnosis of pheochromocytomas. A retrospective single-center cohort study was conducted on a sample of patients who were initially operated on for adrenal neoplasm at the Pirogov St. Petersburg State University High Medical Technology Clinic from November 2007 to December 2022 and who passed analysis to determine the level of blood or urine metanephrins before surgical treatment. The results of tests for metanephrine and tumor size were evaluated. 1088 patients with adrenal neoplasms who underwent surgical treatment were examined, of which 348 had histologically confirmed the presence of pheochromocytoma. Four types of metanephrine assays were compared: free fractionated plasma metanephrines (232 patients), unfractionated daily urine metanephrines (431 patients), fractionated total daily urine metanephrines (427 patients) and fractionated free daily urine metanephrines (178 patients). The greatest sensitivity was demonstrated by the analysis of free fractionated plasma methanephrines (95.4%). Unlike others, the sensitivity of this analysis did not decrease in the group of patients with small pheochromocytomas (3 cm or less). The greatest specificity was demonstrated by the analysis of unfractionated metanephrines in daily urine (97.8%), with the lowest sensitivity among all tests (67.6%). The study of fractionated total daily urine metanephrins showed good results of sensitivity and specificity, only slightly inferior to the best indicators, and the analysis of free daily urine metanephrins demonstrated unexpectedly low efficiency. There is a positive correlation between the level of metanephrine in the blood and the size of the tumor. Based on the data obtained, the preferred assays for the primary diagnosis of pheochromocytoma can be considered the determination of fractionated free plasma metanephrines and fractionated total daily urine metanephrines, which is consistent with relevant clinical recommendations. It was found that the size of the tumor correlates with the severity of an increase in the level of metanephrins determined by any of the described methods.

Midkine characterization in human ovaries: potential new variants in follicles

To characterize the growth factor midkine (MDK) in the human ovary to determine whether MDK is produced locally within the ovary, examine whether different ovarian cell types are more likely to produce MDK, and determine whether there are any stage-specific variations during follicle growth. Previous studies have revealed that MDK potentially affects human follicle growth and oocyte maturation. Proteomic analyses in follicular fluid (FF) have identified MDK to functionally cluster together and follow a similar expression profile to that of well-known proteins involved in ovarian follicle development. Midkine has not yet been characterized in the human ovary. Descriptive study. University Hospital. The study included samples from 121 patients: 71 patients (aged 17-37 years) who underwent ovarian tissue cryopreservation provided granulosa cells (GC), cumulus cells, ovarian cortex, medulla tissue, and FF from small antral follicles (SAF); and 50 patients (aged 20-35 years) receiving invitro fertilization treatment provided FF from preovulatory follicles before and after induction of final follicle maturation. None. MDK relative gene expression was quantified using a real-time quantitative polymerase chain reaction in cumulus cells, GC, and medulla tissue. Additionally, immunostaining and western blotting assays were used to detect MDK protein in the ovarian cortex, which contains preantral follicles, SAF, and medulla tissue. Furthermore, enzyme-linked immunosorbent assay analyses were performed to measure the concentration of MDK in FF aspirated from SAF and preovulatory follicles both before and 36 hours after inducing the final maturation of follicles. Immunostaining and reverse transcription-quantitative polymerase chain reaction revealed a more prominent expression of MDK in GC compared with other ovarian cell types. Intrafollicular MDK concentration was significantly higher in SAF compared with preovulatory follicles. In addition, different molecular weight species of MDK were detected using western blotting in various ovarian sample types: GC and FF samples presented primarily one band of approximately 15 kDa and an additional band of approximately 13 kDa, although other bands with higher molecular weight (between 30 and 38 kDa) were detected in medulla tissue. This is the first time that MDK has been immunolocalized in human ovarian cells at the protein level and that potentially different MDK variants have been detected in human FF, GC, and ovarian medulla tissue. Future studies are needed to sequence and identify the different potential MDK variants found to determine their functional importance for ovary and oocyte competence.

Expression of endocan and vascular endothelial growth factor and their correlation with histopathological prognostic parameters in pheochromocytoma.

Endocan and vascular endothelial growth factor (VEGF) are markers expressed in various cancer types that are highly vascular, and they have prognostic significance for these cancers. In this study, we aimed to show the expression of endocan and VEGF in pheochromocytoma tumor tissues and to evaluate their correlations with histopathological parameters. Thirty-eight patients who had been operated for pheochromocytoma were included in the study. As the control group, 28 subjects whose specimens contained normal adrenal medulla tissue were included. The formalin-fixed paraffin-embedded specimens of pheochromocytoma patients were evaluated for Pheochromocytoma of the Adrenal gland Scaled Score (PASS). Sections were then stained for immunohistochemical analysis. The degree of endocan and VEGF positivity was determined by the proportion of stained cells on a negative to strong scale. Endocan (p < 0.001) and VEGF (p = 0.004) expressions were found to be significantly higher in the pheochromocytoma group than in the control group. In the pheochromocytoma group, total PASS score (r = 0.714; p < 0.001) and most of the PASS score components were positively correlated with the level of endocan expression. Median Ki-67 index (p = 0.010), total PASS score (p < 0.001), tumor cell spindling (p = 0.048), and nuclear pleomorphism (p = 0.030) were higher in pheochromocytoma with VEGF expression than in those without. If our findings are supported by studies with a larger sample size, we think that endocan has the potential to be used both as a tumor marker and in predicting malignancy potential in patients with pheochromocytoma, and that the detection of VEGF expression in these tumors is also associated with an increase in malignancy potential.

PD-L1 Expression is Linked to Tumor-Infiltrating T-Cell Exhaustion and Adverse Pathological Behavior in Pheochromocytoma/Paraganglioma

Pheochromocytoma/paraganglioma (PPGL) is an endocrine-related tumor associated with excessive catecholamine release and has limited treatment options once metastasis occurs. Although recent phase 2 clinical trials of immune checkpoint inhibitors in the treatment of PPGL have preliminarily shown promising results, the fundamentals of immunotherapy for PPGL have not yet been established. In the early research, using bulk RNA sequencing of tumor samples from 7 PPGL patients, we found that PPGL tumor tissues exhibited high PD-L1 mRNA expression compared with adjacent normal adrenal medulla tissues, and this was related to T-cell exhaustion biomarkers. To further validate the association, in this study (n = 60), we first stratified all PPGL samples according to PD-L1 expression as determined by immunohistochemical staining, and then subjected 23 fresh PPGL tumor samples from the cohort to a quantitative polymerase chain reaction (n = 16), flow cytometry (n = 7), and multiplex-immunofluorescence staining. Subsequently, we evaluated the pathological manifestations of all 60 PPGL tumor samples and analyzed the correlation among PD-L1 expression, adverse pathological behavior, various clinicopathological data, and genotypes in PPGL. The results showed that PD-L1-positive expression correlated with the exhaustion of tumor-infiltrating T cells, preoperative abnormal elevation of plasma norepinephrine, high Ki67 index, and adverse pathological behavior in PPGL but not with genetic mutation or metastatic disease, possibly due to the limitation of the small number of patients with metastatic disease (n = 4) in the study cohort. In conclusion, our findings reveal that PD-L1 expression is associated with T-cell exhaustion and adverse pathological behavior in PPGL. These results are expected to provide a new theoretical basis and clinical guidance for the treatment of PPGL.

P-393 The mitochondrial targeted antioxidant Mitoquinol increases survival of isolated human preantral follicles in vitro

Abstract Study question Does Mitoquinol have a protective effect on survival and mitochondrial oxidative stress in cultured human preantral follicles? Summary answer Mitoquinol significantly increased survival of human preantral follicles cultured for 8 days and significantly decreased expression of genes related to the mitochondrial oxidative stress response. What is known already Culturing human ovarian follicles is a promising new source of mature oocytes for fertility preservation, as well as a key model system to investigate fundamental biology. Nevertheless, only a handful of studies have yielded mature human oocytes from preantral stage follicles and optimizing culture systems is challenging due to the scarcity of human material. Mitoquinol is an antioxidant that contains a form of Coenzyme Q10 called ubiquinone that has been modified to selectively target and protect mitochondria from oxidative damage. In human oocytes, the presence of Mitoquinol in the IVM media increased nuclear maturation and protected against chromosomal misalignment. Study design, size, duration Human preantral follicles (n = 213; mean diameter: 71 µm; range: 26-189 µm) were isolated from ovarian medulla tissue. Preantral follicles were mechanically and enzymatically isolated, encapsulated in 0.5% alginate and cultured for 8 days in three experimental groups: Control (n = 68); 50nM Mitoquinol (n = 71), 250nM Mitoquinol (n = 74). The primary endpoints were follicular growth and survival. Secondary endpoints included follicular gene expression and hormone analysis of the spent culture media. Participants/materials, setting, methods Ovarian medulla tissue was donated by 5 patients (aged 30-37 years) undergoing unilateral oophorectomy for ovarian tissue cryopreservation. Follicular growth and survival were assessed every second day during culture by microscopy. For follicles bigger than 95 µm, AMH and Estradiol concentrations were measured by ELISA in the spent media on day 8. Finally surviving follicles were snap-frozen and gene expression was analysed by qPCR; mitochondrial oxidative stress genes: HSP-60, TFAM and apoptosis genes: BAX/BCL-2. Main results and the role of chance After 8 days in culture, the follicular survival rate in the 50nM Mitoquinol group (68%; n = 51/74) was significantly higher than the control group (49%; n = 32/68; p = 0.0344) and the 250nM Mitoquinol (54%; n = 39/71; p = 0.0443). The protective effect of 50nM Mitoquinol was more pronounced in the subset of small follicles (Day0 &amp;lt; 70 µm, n = 135) in which survival rate of the 50nM Mitoquinol group (65%; n = 31/47) was significantly higher than the control (41%; n = 18/43; p = 0.005) and the 250nM group (44%; n = 20/4; p = 0.019). The average growth of surviving follicles was similar in the three experimental groups (Control: 40.7 ± 17.4 µm; Mitoquinol 50nM: 36.9 ± 19 μm; Mitoquinol 250nM: 36.4 ±16.5 μm). A significant positive association between the diameter of the follicle and the concentration of AMH and Estradiol was observed (AMH: p = 0.0215, R = 0.43 and Estradiol: p = 0.0413, R = 0.38) but no differences were found between the experimental groups. The relative expression of genes involved in mitochondrial oxidative stress response were significantly lower in the 50nM Mitoquinol group compared to the 250mM Mitoquinol group (HSP-60 p = 0.018; TFAM p = 0.013) and similar to the control group (HSP-60 p = 0.056; TFAM p = 0.071). Moreover, the gene expression of the ratio BAX/BCL2 was similar in the three groups. Limitations, reasons for caution A larger sample size is required to confirm the statistical analysis and to extrapolate the results to primordial, primary, and secondary stage follicles. Furthermore, long-term culture studies are required to determine the impact of Mitoquinol on antral follicle development and oocyte maturation Wider implications of the findings Our findings show that Mitoquinol can be used as an antioxidant in the culture of human preantral follicles since it increased follicle survival and reduced gene expression of genes related to mitochondrial oxidative stress response. Trial registration number Not appliclable

Metabolic Responses of Normal Rat Kidneys to a High Salt Intake.

In this study, novel methods were developed, which allowed continuous (24/7) measurement of arterial blood pressure and renal blood flow in freely moving rats and the intermittent collection of arterial and renal venous blood to estimate kidney metabolic fluxes of O2 and metabolites. Specifically, the study determined the effects of a high salt (HS; 4.0% NaCl) diet upon whole kidney O2 consumption and arterial and renal venous plasma metabolomic profiles of normal Sprague-Dawley rats. A separate group of rats was studied to determine changes in the cortex and outer medulla tissue metabolomic and mRNAseq profiles before and following the switch from a 0.4% to 4.0% NaCl diet. In addition, targeted mRNA expression analysis of cortical segments was performed. Significant changes in the metabolomic and transcriptomic profiles occurred with feeding of the HS diet. A progressive increase of kidney O2 consumption was found despite a reduction in expression of most of the mRNA encoding enzymes of TCA cycle. A novel finding was the increased expression of glycolysis-related genes in Cx and isolated proximal tubular segments in response to an HS diet, consistent with increased release of pyruvate and lactate from the kidney to the renal venous blood. Data suggests that aerobic glycolysis (eg, Warburg effect) may contribute to energy production under these circumstances. The study provides evidence that kidney metabolism responds to an HS diet enabling enhanced energy production while protecting from oxidative stress and injury. Metabolomic and transcriptomic analysis of kidneys of Sprague-Dawley rats fed a high salt diet.

Effect of SGLT2 Inhibition on the transcriptomic and proteomic profile of non-diabetic Dahl SS Rats

Sodium-Glucose Cotransporter-2 (SGLT2) inhibitors are new, highly effective drugs for treating type 2 diabetes mellitus. SGLT2 inhibitors also substantially reduced the risk of hospitalization for heart failure. Previous studies in rodents and humans have shown that SGLT2 inhibitors lower blood pressure levels, cardiovascular risk events, and kidney diseases in subjects with and without diabetes mellitus. However, the mechanisms behind those effects are still unknown. Our previous study showed that SGLT2 inhibition blunted the development of salt-sensitive (SS) hypertension and caused glucosuria and natriuresis in Dahl SS rats without affecting the expression and activity of Na + channels and changes in the renin-angiotensin-aldosterone system. Here, we aim to investigate the effect of SGLT2 treatment on non-diabetic SS rats using multi-omics analysis, which may provide insights into the underlying mechanisms. 8-week-old male SS rats were treated with or without SGLT2 inhibitor (dapagliflozin, 2 mg/kg/day in drinking water) for 3 weeks. Kidney cortex and medulla tissue were collected for RNA-Seq analysis. Proteomics analysis was performed on kidney cortex tissue and plasma. The data were further processed by Ingenuity Pathway Analysis (IPA).RNA-Seq analysis indicated that despite SLGT2 mainly being expressed in the kidney cortex, dapagliflozin treatment altered the expression of more genes in the medulla (855 in the cortex vs 2244 differentially expressed genes (DEGs) in the medulla). Dapagliflozin treatment also caused significant changes in multiple genes related to inflammation and hypertension, with IPA analysis predicting the downregulation of both functions. In hypertension network Adrb3, Retn, Foxn4 were the most upregulated genes, and Ptgs1, Nos1, Oxtr – downregulated. In addition, disease and biological function enrichment analysis revealed that the altered genes were mainly related to the following functions: lipid metabolism, small molecule biochemistry, amino acid metabolism, cell cycle and morphology, and molecular transport. Interestingly, lipid metabolism was predicted to be downregulated in the cortex but upregulated in the medulla.Proteomic analysis identified 450 differentially expressed proteins (DEPs) in the kidney cortex and 22 in plasma. Disease and biological function enrichment analysis on proteomics data revealed mostly the same biological functions as RNA-Seq data. In addition, canonical pathways analysis based on RNA-Seq and proteomics data revealed that endothelin-1 and oxytocin signaling pathways were the most downregulated pathways in the kidney cortex and medulla under the dapagliflozin treatment. Urea cycle and glucocorticoid receptor signaling were the most changed canonical pathway in proteomic data. In conclusion, our results indicated that inhibition of SGLT2 by dapagliflozin altered various genes and proteins in the kidney of Dahl SS rats, which may provide insights for future mechanistic studies. This research was supported by National Institutes of Health grants R35 HL135749 (to AS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neurogenesis in the regenerating tail of tsinling dwarf skinks (Scincella tsinlingensis)

AbstractSpinal ganglia and peripheral nerves innervate the regenerating tail and muscles of lizards, which provide new opportunities to probe the mechanisms leading to successful functional recovery following spinal cord injury. The regenerated spinal cord and peripheral nerves were detected using immunohistochemistry in original and regenerating tails of Scincella tsinlingensis. Our results showed that positive PCNA, GFAP and SOX2 cells were observed in the ependymal ducts at 15 and 30 days after the autotomy (dpa), with a small number of immunopositive NSE neurons. GFAP and SOX2 positive cells were primarily localized along the regenerated spinal cord after 45 dpa, and the descending nerve of medulla tissue showed positive NSE. Peripheral axons distributed around the muscle and inside the connective tissue and muscle tissue at 15 dpa. The number of axons decreased after 30 dpa. The peripheral axons was mainly distributed between the connective tissue, muscle group and the muscle and epidermis, presenting a radial distribution centred on the cartilage tube at 120 dpa. MBP myelination labelling of the regenerated tail at 30 dpa revealed that the regenerated axons rapidly myelinated along the rostro‐caudal axis. The density of NMJ significantly increased at 120 dpa and 250 dpa. In the second regenerated tails, NMJ density at 250 dpa was still greater than original tails. Overall, these results indicated that neurogenesis was an early event and the ependymal cells were heterogeneous in the regenerated spinal cord of S. tsinlingensis. The regenerated peripheral axons presented a radial distribution centred on the cartilage canal, and the regenerated axons were rapidly myelinated along the rostro‐caudal axis. During the process of regeneration, there were a large number of regenerated neuromuscular joints with high density.

The correlation between nutritional status and CD8+ cell counts in the elderly after the second COVID-19 vaccination in Palembang, Indonesia

Introduction: The SARS-CoV-2 virus is the cause of coronavirus illness (COVID-19). The WHO classified the outbreak a pandemic on March 9, 2020. The COVID-19 vaccine has been widely utilized to manage the pandemic. However, some high-risk conditions that can compromise the vaccine's efficacy require careful consideration. Patients over 60 years old are at a greater risk for COVID-19. Due to the involution of the thymus and the transformation of the cortex and medulla tissue into adipose tissue, CD8+ T cell numbers decline. In addition, undernutrition reduces the number of CD8+ T cells. The purpose of this study is to examine the relationship between nutritional status and CD8+ T cell counts in elderly patients following the second COVID-19 vaccination. Methods: This study is a pilot project employing a cross-sectional design. The sampling employed a technique of sequential sampling. On day 28, following the administration of the second vaccine, the Mini Nutritional Assessment (MNA) and the amount of CD8+ T cells in the blood were utilized to evaluate the nutrition status. Results: Between May and November of 2021, forty subjects were collected. The average number of CD8+ T cells was 517.32±41.67. Therefore, five people (12.5%) were at risk of malnutrition, whereas thirty-five (87.5%) had a normal nutritional status. Conclusion: There is a significant relationship between MNA and CD8+ levels (P&lt;0.05) and a positive correlation between nutritional status and CD8+ T cell counts in the elderly who have received the second covid-19 vaccination in Palembang.

Abstract P108: Compensatory Changes In Metabolomic Profiles In The Kidney Of Sprague-dawley Rats Fed A High-salt Diet.

Goal: The effects of a high salt diet on renal cortical ( Cx ) and outer medulla ( OM ) tissue metabolism of normal age matched male Sprague-Dawley rats (SD; 15-16 wk age; n-5) was determined with rats fed 0.4% NaCl (LS) and after switching to 4.0% NaCl (HS) diet at day HS14 and HS21. Methods: From snap-frozen Cx and OM tissue, novel analytical approaches were developed and applied at the JAX lab to determine metabolic profiles using 4 modes (C18+/- and HILIC+/-) using a Thermo Q-Exactive Orbitrap coupled to a Vanquish UPLC system. Results: Kidney weight was increased (P&lt;0.05) by 0.5 mgkw/gbw at HS14 from from LS level of 3.2±0.1 mgkw/gbw and remained constant at HS21. A principal component analysis (PCA) of each mode found distinct separation of metabolites in both the Cx and OM comparing LS to HS14, and HS21. Of 2085 named compounds identified in Cx and 1886 in OM, the Metaboanalyst database found 968 (Cx) and 861 (OM) with 285 (Cx) and 284 (OM) changed (P&lt;0.05) at HS14. At day HS21, 438 were changed (P&lt;0.05) in Cx and 349 in OM compared to LS. A pathway enrichment analysis (SMPDB) of those metabolites was carried out. Expressed as the enrichment ratio (ER; observed hits/expected hits) found enrichment of Arachidonic acid metabolism pathway at HS13 (ER=2.7), consistent with observed increases (P&lt;0.05) of L-glutamic acid, arachidonic acid and thromboxane B2. Increased levels of phosphoenalpyruvic acid and glucose 1-phosphate were observed (p&lt;0.05), consistent with elevated glycolysis and gluconeogenesis, not observed at HS21. In the OM at HS14, the “Tyrosine metabolism” (ER:1.9), “Lysine degradation” (ER:2.2) and “Beta-alanine metabolism” (ER:1.9) pathways were enriched (P&lt;0.05). Lysine, tyrosine and glutamate levels were reduced (P&lt;0.05) indicating increased amino acid utilization. Also found were reductions of glucose-6-phosphate, pyruvate and alpha-ketoglutarate (P&lt;0.05) indicating reduced glycolysis and TCA activity. At day HS21 these changes were generally not significant in either Cx or OM. Conclusion: Kidney weight increased after 2 wks of HS diet which was associated with increased amino acid metabolism and reduced glycolysis in OM (e.g. anabolic profile). Those metabolites (above) then returned to levels observed on a LS diet as kidney weight stabilized.

The immune cell infiltrate in the tumour microenvironment of phaeochromocytomas and paragangliomas.

Emerging evidence suggests the composition of the tumour microenvironment (TME) correlates with clinical outcome and that each tumour type has a unique TME including a variable population of inflammatory cells. We performed immunohistochemistry on 65 phaeochromocytoma and paraganglioma (PPGL) tumour samples with 20 normal adrenal medulla samples for comparison. The immune cells assessed were macrophages, lymphocytes and neutrophils, and we compared the proportion of infiltration of these immune cells with clinical and histopathological factors. There was a higher proportion of immune cells in tumour tissue compared to non-neoplastic adrenal medulla tissue, with a predominance of macrophages. There was a higher proportion of M2:M1 macrophages and T-helper lymphocytes in aggressive tumours compared to indolent ones. For SDHB-associated tumours, there was a higher proportion of M2 macrophage infiltration, with higher M2:M1 in aggressive SDHB PPGLs compared to indolent tumours. These data demonstrate that immune cells do infiltrate the TME of PPGLs, confirming that PPGLs are immunologically active tumours. Differences in the TME of PPGLs were observed between aggressive and indolent tumours. These differences could potentially be exploited as an aid in predicting tumour behaviour.

Stress and adaptation to repeated stress changed the expression of two isoforms of 11β-hydroxysteroid dehydrogenase (11β-HSD1 and 11β-HSD2) in the adrenal glands of sheep

The aim of this study was to estimate rate of cortisol release from ewe’s adrenal gland cortex and medulla tissue and the participation 11β-HSD1 and 2 in modulating adrenal glucocorticosteroids release, determine the expression size of these two isoforms in both parts of adrenal glands after single and repeated stress (3 times every 24h), when adaptive processes take place. The experiment was carried on 15 ewes, outside the breeding season. The research material consisted of adrenal glands obtained from control sheep (CON), sheep subjected to one-hour individual isolation from the herd (S1) and three times repeated stress factor at one-day intervals (S2). Immediately after slaughter, the adrenal glands from each sheep were placed on ice in 0.9% NaCl, then they were cut into smaller sections of similar weight (approx. 50 mg), covering the cortical and medullary layers of the adrenal glands. Sections of obtained tissues were weighed and placed in incubation wells (Sigma cell culture) containing 1 ml of incubation Eagle's medium with the addition of bovine albumin (BSA, 50 mg / 100 ml). Tissue incubation was carried out in an atmosphere of carbogen - 95% O2 and 5% CO2 at 38oC in a Sanyo incubator (MCO-18AIC). After 10 minutes of stabilization, the sections of core and adrenal cortex tissue were transferred to subsequent wells containing pure Eagle Medium. The same section was transferred to consecutive incubation wells containing clean incubation medium every 30 minutes. The medium harvested from the wells after 30, 60 and 90 minutes of incubation of adrenal tissue was frozen until the scheduled cortisol concentration de-termination by RIA (Orion Diagnostica) was performed. Another pieces of tissue the cortex and adrenal medulla from each sheep immediately placed in the RNAlater. Measurements of the gene expression of 11β-HSD1 and 2 expression in the adrenal cortex and medulla were performed using Real-time qPCR method. Starters and TaqMan MGB probes were designed by Applied Biosystem. In the conducted in vitro experiment, a significant reduction in the amount of cortisol released from adrenal cortex tissue into the medium after single stress factor was found, from 3.27 ± 0.69 pg･mg-1 tissue after first 30 minutes of experiment to 0.70 ± 0.15 pg･mg-1 of tissue after 90 minutes (P &lt; 0.01). A similar profile of cortisol release from the adrenal cortex tissue was found in control sheep (P &gt; 0.05). Experimental Group 2 (S2) had a similar profile of cortisol release during the all-time of incubation, however the values found were always lower than those observed in the control and single stress sheep Experimental Group 1 (S1) (1.34 ± 0.37 to 0.30 ± 0.08 pg･mg-1 of tissue; P &lt; 0.05). After repeated stress, incubation of adrenal cortex tissue showed a similar profile of changes in cortisol release, however the values were significantly lower than those found in the singly stressed animals (from 1.30 ± 0.37 pg･mg-1 after 30 minutes to 0.27 ± 0.08 pg･mg-1 tissue after 90 minutes; P &lt; 0.01). Incubation of adrenal medulla tissue obtained from singly stressed animals showed cortisol release to the medium much lower in comparison to adrenal cortex layer, however constant during the whole incubation time, value 1.04 ± 0.36 pg/mg-1 tissue noted after first 30 minutes to 0.77 ± 0.06 pg･mg-1 after 90 minutes of experiment; P &gt; 0.05). When the stress factor was repeated three times, the amount of cortisol released was like that observed in the control secretion; P &gt; 0.05). Analysis of 11β-HSD 1 expression in adrenal cortex tissue from singly stressed sheep showed an over threefold increase (350 ± 25 RQ), while measurement after stress factor was repeated three times demonstrated increase, however the value turned out to be two times lower than after a single stress (160 ± 35 RQ). The expression value of 11β-HSD 2 in the adrenal cortex tissue showed a significant reduction after both single (15 ± 7 RQ and repeated stress (22 ± 3.0 RQ). No significant differences were found in the expression value of this enzyme in the sheep adrenal medulla in both experimental groups in comparison to control group (P &gt; 0.05). The obtained results indicate the immanent abilities of the adrenal glands, probably to a certain extent, to control the release of cortisol, conditioned by the presence and variable activity of the enzyme 11β-HSD, particularly isoform 1, in adrenal glands without the need to activate all HPA axis structures during stress and adaptation processes.

P-517 Validating reference gene expression stability in human ovarian follicles, oocytes, cumulus cells, ovarian medulla, and ovarian cortex tissue

Abstract Study question How does the expression stability of commonly used reference genes (RG) vary in different human ovarian cells and tissues? Summary answer The RG stability differed among human ovarian cell types and tissues, but an RG with high stability was identified for each cell and tissue type. What is known already The expression of RGs used to normalize RT-qPCR may vary between different tissues, cell types, and experimental conditions. Hence, selecting the most appropriate RGs is critical in any experimental design to interpret data generated by RT-qPCR with the best accuracy. Human ovarian cells are phenotypically very different and often only available in limited amounts. In several animal species RG expression stability has been validated in oocytes and other ovarian cells, however, the suitability of a single universal RG in the different human ovarian cells and tissues has not been determined. Study design, size, duration This is an experimental study performed at a university hospital from January 2021 to September 2021. Participants/materials, setting, methods The freely available NormFinder software was used to analyze the expression stability of five commonly used RGs (GAPDH, B2M, RPLP0, ACTB, and PPIA) in human oocytes (n = 160), preantral follicles (n = 160), cumulus cells (n = 13), ovarian medulla (n = 8), and ovarian cortex tissue (n = 60). Samples were collected from 29 patients (aged 28 years on average; range 14–36) undergoing unilateral oophorectomy and ovarian tissue cryopreservation for fertility preservation. Main results and the role of chance The Normfinder software identified ACTB as the best RG for oocytes and cumulus cells; and B2M for medulla tissue and isolated follicles. Overall, comparisons of the cycle threshold (Ct) values demonstrated a wide variation among the RGs within the same group of samples (P &amp;lt; 0.05), especially for oocytes and preantral follicles with normalized RNA. The genes ACTB and RPLP0 showed the highest levels of expression and PPIA the lowest levels of expression in all types of samples, except for cortex tissue, where PPIA had the highest level of expression and B2M the lowest. These results infer that different results could be obtained when using different RGs for data normalization. The combination of two RGs only marginally increased stability, indicating that using a single validated RG would be sufficient when the available testing material is limited. For cultured ovarian cortex culture, GAPDH or ACTB were found to be the most stable genes depending on culture conditions. Limitations, reasons for caution Since human oocytes are scarce and contain a small amount of RNA, only five RGs were evaluated. Moreover, only germinal vesicle (GV) oocytes and preantral follicles (60-80 µm) were tested. Future studies may validate other genes and investigate the effect of oocyte maturation and follicle growth on RG stability. Wider implications of the findings Our findings highlight the importance of validating RGs for each cell type or tissue and culture condition. Hence, our results can be of use as guidance for future studies involving gene expression analyses in human ovarian cells and tissues, including oocytes and preantral follicles. Trial registration number Not applicable

P-439 Human Platelet Lysate improve the growth and survival of isolated human pre-antral follicles in vitro

Abstract Study question Can human Platelet Lysate (hPL) and umbilical cord plasma (UCP) be used as alternative serum sources in the culture of isolated human pre-antral follicles? Summary answer hPL significantly increased the growth and survival of human follicles cultured for 8 days compared to fetal bovine serum (FBS) and human serum albumin (HSA). What is known already Culture of human ovarian follicles is a potential new source of mature oocytes for fertility preservation and an important model system to study basic biology. However, only a few studies have produced mature human oocytes after culturing pre-antral stage follicles. Moreover, optimising culture systems is challenging due to the scarcity of human material. Platelet-rich plasma solutions have been used extensively in regenerative medicine due to their high platelet content, which upon activation, release a multitude of growth factors, hormones, and cytokines. Clinically cell-based therapies have used platelet-rich solutions, such as hPL or UCP, successfully as an animal-free serum. Study design, size, duration Human pre-antral follicles (n = 378; mean diameter: 78 µm; range: 46-237 µm) were isolated from ovarian medulla tissue. The follicles were encapsulated in 0.5% alginate and cultured for 8 days in media supplemented with one of four sources of serum; 5% FBS (n = 74), 2.5% HSA (n = 74), 5% hPL (n = 140), and 5% UCP (n = 90). The primary endpoints were follicular growth and survival. Secondary endpoints included follicular gene expression analysis and media hormone concentrations. Participants/materials, setting, methods Ovarian surplus tissue was donated by 7 women (aged 19-32 years) undergoing unilateral oophorectomy and ovarian tissue cryopreservation for fertility preservation. Pre-antral follicles were isolated enzymatically and growth and survival were assessed every second day during culture by microscopy. AMH and Estradiol concentrations were measured by ELISA in media collected at day 4 and 8. At day 8, surviving follicles were snap-frozen and the expression of AMH, FSHR, GDF9, and BMP15 was analysed by qPCR. Main results and the role of chance After 8 days in culture, the follicle survival rate in the hPL group (86%; n = 120/140) was significantly higher compared to the FBS group (60%; n = 45/74; p &amp;lt; 0.0003), but comparable to the HSA group (76%; n = 56/74; p = 0.292). On the contrary, the follicle survival rate in the UCP group (33%; n = 30/90) was significantly lower compared to any of the other groups (p &amp;lt; 0.0001 for all three groups). The average diameter of surviving follicles was statistically significantly larger in the hPL and UCP group compared to the FBS and HSA groups (hPL: 149 ±4.6 µm; UCP: 138 ±7.6 μm; FBS: 110 ±3.8 μm; HSA: 115 ±4.2 μm; p &amp;lt; 0.001 for hPL compared to FBS and HSA; p = 0.0129 for UCP compared to HSA). The relative growth of the follicles compared to their initial diameter was 45% and 33% higher in the hPL group compared to the FBS and HSA groups, respectively, which was statically significant in both groups (p &amp;lt; 0.001). In the UCP group, follicle growth was also significantly higher by 25% compared to the FBS group (p &amp;lt; 0.001) and by 14% compared to the HSA group (p = 0.0376). Hormone measurements from the culture media and follicle gene expression analysis are awaited. Limitations, reasons for caution One limitation is the variability in the initial follicle sizes. Extrapolating results according to primordial, primary, and secondary follicles would require more follicles but could provide valuable insight. Moreover, long term culture studies are needed to evaluate the effects of hPL and UCP on antral follicle development and oocyte maturation. Wider implications of the findings Our findings show that hPL can be used as an alternative serum source in the culture of human pre-antral follicles as it increased follicle growth and survival compared to FBS and HSA. UCP also increased follicle growth but had detrimental effects on follicle survival which needs further studies. Trial registration number Not applicable