[Predictive value of plasma exosomal miR-124-3p for the risk of chronic cerebral hypoperfusion].

Objective: To investigate the predictive value of plasma exosomal microRNA (miR)-124-3p in the risk of chronic cerebral hypoperfusion (CCH). Methods: A case-control study. Thirty patients who were diagnosed with CCH (CCH group) based on cranial artery spin labeling (ASL) in the neurology outpatient clinic of Sichuan Provincial People's Hospital from March 2022 to June 2022 and 30 healthy volunteers (control group) were included. Age, gender, smoking history, alcohol consumption history, diabetes history, hypertension, hyperlipidemia history, uric acid, fasting blood glucose, homocysteine and plasma exosomal miR-124-3p expression level were compared between the two groups. Comparisons of categorical variables were analyzed by either χ2 test or Fisher's exact test. If the data of continuous variables followed a normal distribution, they were expressed as mean±standard deviation (SD) and compared by t-test for two independent samples; otherwise, the data were expressed as M(Q1, Q3), and analyzed by Mann-Whitney U test for comparison between two groups. The correlation between cerebral blood flow and exosomal miR-124-3p levels was analyzed by Pearson's correlation. Binary multifactorial logistic regression analysis was used to determine the risk factors associated with CCH, and corresponding odds ratios (OR) and 95% confidence intervals (CI) were calculated. P<0.05 was considered significant. Results: There was no significant difference in age (64±8 vs. 60±8 years old), gender (33.3% vs. 30.0%), history of smoking (20.0% vs. 3.3%), alcohol consumption (20.0% vs. 6.7%), diabetes mellitus (13.3% vs. 13.3%), hypertension (53.3% vs. 30.0%), history of hyperlipidemia (46.7% vs. 36.7%), uric acid (288±60 vs.319±67 µmol/L), and fasting glucose [4.99(4.63, 5.91) vs. 5.28(5.09, 6.05) mmol/L] and homocysteine [11.35(10.18, 13.08) vs.11.00(9.78, 13.03) µmol/L] between the CCH and control groups (P>0.05). Plasma exosomal miR-124-3p expression was significantly higher in the CCH group than in the control group [13.08 (8.59, 21.55) vs. 2.85 (1.44, 5.10), respectively; U=169.50, P<0.001]. Pearson's correlation test showed that the level of exosomal miR-124-3p was negatively correlated with cerebral blood flow in the hypoperfused region in patients with CCH (r=-0.932, P<0.001). Multi-factor logistic regression analysis showed that plasma exosomal miR-124-3p was independently associated with the risk of CCH (OR=1.169,95%CI 1.063-1.286,P=0.001). Conclusions: The expression of plasma exosomal miR-124-3p is negatively correlated with cerebral blood flow in areas of low perfusion and is an independent risk factor for CCH. Plasma exosomal miR-124-3p may thus serve as a valid biomarker for CCH risk prediction.

Gonadal transformation in male Rana catesbeiana tadpoles intraperitoneally implanted with estradiol capsules.

Tadpoles of Rana catesbeiana at TK stages X-XII, about 9 months old, were laparotomized, and the males were implanted intraperitoneally with silastic tubes containing estradiol (E2) for various periods. Male tadpoles implanted with empty tubes served as the controls. Histology, secretions of E2, and testosterone (T) in the gonads were investigated. A rough estimate of estradiol released from silastic tubes suggested that about 90 micrograms per tadpole in 6 months. Histological observation revealed various degrees of transformation from testes toward ovaries in E2-treated testes. Ten in thirteen (77%), the testes were transformed into ovaries 6 months after the treatment. The testes of the controls, however, displayed normal histology. Radioimmunoassay showed that E2 level was increased while T level was decreased in E2-treated testes. These results indicate that a low dose of exogenous E2 may induce transformation of the testes into ovaries.

Gonadal differentiation and secretions of estradiol and testosterone of the ovaries of Rana catesbeiana tadpoles treated with 4-hydroxyandrostenedione.

Laparotomized female tadpoles of Rana catesbeiana at TK stages X-XII, about 9 months old, were implanted intraperitoneally with empty capsules or capsules containing 4-hydroxyandrostenedione (4-OHA), known as an aromatase inhibitor in vertebrates. Histology, gonosomatic index, and secretions of estradiol (E2) and testosterone (T) of the ovaries were investigated. Three months after the treatment, histological examination revealed various degrees of sex reversal in the ovaries treated with 4-OHA and 79% (57 in 72) were transformed into testes. The ovaries of control tadpoles, however, displayed normal histological appearance. Radioimmunoassay showed that secretion of E2 was decreased while that of T was increased in 4-OHA treated ovaries. The gonosomatic index displayed a decline tendency from control females through experimental animals to untreated control males. These results indicated that activity of aromatase in the ovaries was inhibited by 4-OHA, resulting in accumulation of T which induced transformation of the ovaries into testes.

Estradiol secretion by the ovarian tissue, in response to hypophyseal stimulation, during ontogenesis of the bullfrog.

Ovaries of tadpoles, froglets, young frogs, and mature frogs of Rana catesbeiana were cut into small pieces. They were incubated for 6 hr in Krebs-Ringer bicarbonate buffer as controls. Another series of ovaries of the same developmental stages were incubated with pituitary extracts in the buffer as experimentals. Media were then analyzed for estradiol secretion by radioimmunoassay. The results showed that estradiol secretion by tadpole ovaries during development was not affected by the addition of pituitary extracts of mature frogs in the media at any stage while those of young and mature frogs with pituitary extracts secreted more estradiol than those without. These findings indicate that tadpole ovaries are unresponsive to pituitary agents to produce estradiol while frog ovaries are dependent on some pituitary hormones to synthesize estradiol. Thus frog ovaries acquire dependence on the pituitary agent only after metamorphosis.

Estrogen suppression of preincubated tadpole ovaries in vitro by cyanoketone.

In a previous RIA study we found that cyanoketone (CK) inhibited ovarian E2 secretion of tadpoles in vitro and that this inhibition effect was through inactivation of delta 5-3 beta-HSD activity. A complete 100% inhibition was expected at a CK dosage of 0.1 microgram/ml of the medium, but, instead, it was 85%. The discrepancy might be due to the fact that the previous experiments did not preincubate the ovaries with CK in order to get rid of the residual E2. To this end, the present study was designed. Tadpole ovaries of Rana catesbeiana were preincubated with CK of a dosage of 0, 0.001, 0.01, 0.1, 1, or 10 micrograms/ml of the KRbb medium for 30 min. The media were discarded. Fresh media with the same series of CK doses were added to the ovaries and were incubated again for 6 hr. The media were collected for RIA of estrogen. The results showed the same tendency of estrogen inhibition as the previous study. However, a maximal inhibition effect of 95% was obtained at the dose of 0.1 microgram/ml. Therefore, the difference between non-preincubation of the previous experiments and preincubation of the present study does exist as we predicted.

In vitro inhibition of estradiol secretion of tadpole ovaries by cyanoketone.

We have demonstrated the presence of delta 5-3 beta-hydroxysteroid dehydrogenase (HSD) activity in tadpole ovaries of Rana catesbeiana. In the present study, we wish to determine whether estradiol (E2) secretion of tadpole ovaries could be influenced by cyanoketone (CK), a specific inhibitor of delta 5-3 beta-HSD. R catesbeina tadpoles at the premetamorphic climax were used, and pooled ovaries were incubated, 30 mg/tube, with CK at dosages of 0, 0.001, 0.01, 0.1, 1, and 10 micrograms/ml of Krebs-Ringer bicarbonate buffer for 6 hr. Media were collected for assay of E2 by radioimmunoassay (RIA). Results showed an inhibition of E2 secretion by CK that was positively correlated with CK dosage, but plateaued at doses of 0.1 microgram/ml and higher. This finding was comparable to that of G.F. Young, H. Kagawa, and Y. Nagahama (1982, Gen. Comp. Endocrinol. 47, 357-360) on adult fish ovaries. However, adult vertebrates depend on gonadotropins to regulate secretion of E2 while tadpoles, being immature, might secrete E2 independently of pituitaries. When the histochemical test for delta 5-3 beta-HSD activity was performed on in vitro CK-treated ovaries, there was a decrease of enzyme activity by CK. The RIA and histochemical findings may contribute to the concept of sex transformation in which a disturbance of steroidogenesis may induce sex reversal from females to males, at least in tadpoles.

Contrasting effects of ACTH and cyanoketone on delta 5-3 beta-hydroxysteroid dehydrogenase activity in interrenal glands of tadpoles of Rana catesbeiana in vitro.

The present communication describes an investigation of stimulation and inhibition of delta 5-3 beta-hydroxysteroid dehydrogenase in interrenal glands of tadpoles of Rana catesbeiana. Frozen sections of interrenal glands, together with kidneys, were prepared histochemically for assay of delta 5-3 beta-HSD activity. Concentrations of 0.01, 0.1, 1, and 10 IU/ml of ACTH or of 0.01, 0.1, 1, and 10 micrograms/ml of cyanoketone were added to the incubation media. The reaction products of the histochemically prepared slides, in terms of absorbance, were scanned at a defined area with a computerized microscope spectrophotometer. The results indicate that ACTH causes a significant dose-response stimulation of delta 5-3 beta-HSD activity in tadpole interrenals; cyanoketone, on the other hand, causes significant dose-dependent inhibition.

The development of ACTH-like substance during tadpole metamorphosis.

The interrenals of tadpoles of Rana catesbeiana at various developmental TK stages were frozen, sectioned, and prepared histochemically for assessment of delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-HSD) activity. The interrenals of hypophysectomized (HX) tadpoles from the same batch of eggs as control tadpoles were prepared similarly for delta 5-3 beta-HSD activity following a series of intraperitoneal injections of different concentrations of mammalian ACTH. The histochemically stained sections of control and HX tadpoles were scanned with a computerized microscope spectrophotometer for diformazan absorbance. Two regression equations were obtained: Y = 0.1460 + 0.0015X and Y' = 0.1494 + 0.0893X', where Y or Y' = delta 5-3 beta-HSD activity absorbance (for the control and HX tadpoles, respectively), X = developmental stages of control, and X' = exogenous ACTH dosage administered to the HX tadpoles. Taking absorbance of delta 5-3 beta-HSD activity as the common factor, a curve of concentrations of ACTH-like substance against TK stages was made. The developing concentration of ACTH-like substance was very low at first and then rose during metamorphic climax. The trend of the development of ACTH-like substance was discussed from the view point of thyroid and delta 5-3 beta-HSD activities.