OBJECTIVETo research the animal model with mimetic aging effect in the inner ear predispose to the ototoxicity of kanamycin.

METHODSFifty wistar rats were randomly divided into four groups: group A (D-galactose group, n = 14) were treated with hypodermic 5% D-galactose (150 mg x kg(-1) x d(-1)) for 8 weeks and then with intraperitoneal saline for 10 days; group B (D-galactose and kanamycin group, n = 14) were given the same dose of D-galactose but kanamycin (500 mg x kg(-1) x d(-1)) instead of saline; group C (kanamycin group, n = 12) were treated with saline for 8 weeks and then with intraperitoneal kanamycin for 10 days;group D (control group, n = 10) were given saline only. Auditory brainstem response (ABR) was used to detect the hearing threshold of rats and colorimetry was used to analyze the activity of the GSH-PX. The inner ear tissue was harvested and the mitochondrial DNA was amplified to identify the 4834 bp deletion mutation by nested primer polymerase chain reaction (nested PCR) technique.

RESULTSThe incidence of mitochondrial DNA 4834 bp deletion mutation was 100% (28/28) in group A, 92.86% (26/28) in group B and 0% in group C or group D. The activity of GSHPX in group A was (59.07 +/- 8.70)U, (63.29 +/- 12. 40)U in group B, (136.67 +/- 9.53)U in group C and (142.10 +/- 7.02)U in group D. The difference between group A and D was significant (P = 0.000) while the difference between group A and B was not significant (P = 0.307), which was similarly as between group C and group D (P = 0.151). ABR threshold was (5.36 +/- 3.08) dB peSPL in group A, (61.79 +/- 11.20) dB peSPL in group B, (34.17 +/- 4.69) dB peSPL in group C and (6.50 +/- 3.37) dB peSPL in group D. No difference was found between group A and D (P = 0.398) while the difference in shift of ABR threshold between group B and group C (or group D) was significant (P = 0.000).

CONCLUSIONSThe mimetic aging effect in the inner ear of the rat can be induced by D-galactose, and these rats present high incidence of mtDNA4834 deletion which can greatly enhance the sensitivity of the inner ear to the kanamycin.

Aging, Premature ; chemically induced ; Animals ; DNA, Mitochondrial ; genetics ; Disease Models, Animal ; Disease Susceptibility ; Ear, Inner ; drug effects ; Galactose ; toxicity ; Kanamycin ; toxicity ; Rats ; Rats, Wistar ; Sequence Deletion

BACKGROUNDMitochondrial DNA mutations have been found in sensorineural deafness. The aim of this study was to compare three methods for extraction of nucleic acid from membranate inner ear tissue of rats.

METHODSAlkaline denaturation, a conventional phenol-chloroform method and Trizol reagent were respectively used to extract the slight nucleic acid from membranate inner ear tissue of rats. We assessed the amount and quality of nucleic acid using a UV-spectrometer and polymerase chain reaction (PCR).

RESULTSThe yield and purity (OD260/OD280) of DNA from inner ear tissue using the phenol-chloroform method was the highest of the three methods. Mitochondrial DNA (mtDNA) fragment can be amplified by PCR from nucleic acid prepared by all methods, while no nuclear DNA (nDNA) fragment can be amplified by method of alkaline denaturation. Both nuclear and mitochondrial genes could be amplified by reverse transcriptional PCR from the RNA prepared by Trizol reagent.

CONCLUSIONAdequate amount and high-quality of mtDNA, nDNA and RNA were obtained from unilateral membranate inner ear tissue of rats. Method of alkaline denaturation could be chosen when mtDNA without nDNA was needed, while phenol-chloroform method was suitable for extracting total DNA (including nDNA and mtDNA); method with Trizol reagent was suitable for extracting total RNA and total DNA.

Animals ; DNA ; isolation & purification ; DNA, Mitochondrial ; isolation & purification ; Ear, Inner ; chemistry ; Polymerase Chain Reaction ; RNA ; isolation & purification ; Rats ; Rats, Wistar

Leigh syndrome is a genetically heterogeneous disease caused by defects in enzymes involved in aerobic energy metabolism and the Krebs', cycle. Mitonchondrial complex I deficiency is a main cause of Leigh syndrome. In this study, a Chinese child with Leigh syndrome caused by 13513G>A mutation was reported. The proband was the first child of his parents. The previously healthy boy presented with generalized epilepsy at 12 years of age. When he visited Peking University First Hospital at 13 years of age, he had subacute loss of vision in two eyes and temporal defect of visual field in the left eye. He walked with a spastic gait. His blood lactate and pyruvate levels were elevated. Muscle biopsy showed mild lipid accumulation in muscle fiber. An electrocardiogram showed incomplete right bundle branch block. Brain magnetic resonance imaging showed bilateral, symmetrical lesions in the basal ganglia, supporting the diagnosis of Leigh syndrome. 13513G>A mutation was identified by gene analysis in the patient, which led to mitochondrial respiratory chain complex I deficiency. Multivitamins and L-carnitine were administered. At present, the patient is 16 years old and has progressive deterioration with significant muscle weakness and body weight loss. He is absent from school. He has no obvious retardation in intelligence. 13513G>A mutation was first identified by gene analysis in Chinese population with Leigh syndrome. This may be helpful in genetic counseling.
Adolescent ; DNA, Mitochondrial ; genetics ; Electron Transport Complex I ; deficiency ; Humans ; Leigh Disease ; genetics ; Male ; Mutation

This study reviews a case of mitochondrial respiratory chain complex I deficiency due to the 10191T>C mutation in mitochondrial ND3 gene. The previously healthy boy progressively presented with blepharoptosis, weakness, epilepsy and motor regression at age 6 years. Elevated blood lactate and pyruvate were observed. Brain magnetic resonance imaging showed symmetrical lesions in the basal ganglia. Leigh syndrome was thus confirmed. The protein from the mitochondria and genomic DNA of the boy and his parents was collected from peripheral blood leucocytes for the activity test for mitochondrial complex I to V and genetic analysis. The results showed the activity of complex I (33.1 nmol /min in 1 milligram mitochondrial protein) was lower than normal reference value (44.0±5.4 nmol /min in 1 milligram mitochondrial protein). The ratio of complex I to citrate synthase (19.8%) was also lower than normal reference value (48%±11%). The activities of complexes II to V were normal. 10191T>C mutation in ND3 gene of mitochondria was identified in the boy. 10191T>C mutation and complex I deficiency were not detected in his parents. At present, he is 16 years old, and of normal intelligence with spastic paralysis in both lower extremities after treatment. It is concluded that a Chinese boy with isolated complex I deficiency due to 10191T>C mutation in ND3 gene was firstly diagnosed by peripheral leukocytes mitochondrial respiratory chain enzyme assay and gene analysis. This study can provide clinical data for the nosogenesis of Leigh syndrome.
Adolescent ; Brain ; pathology ; Electron Transport Complex I ; deficiency ; genetics ; Humans ; Leigh Disease ; genetics ; Magnetic Resonance Imaging ; Male ; Mitochondrial Diseases ; genetics ; Mutation

Dry eye is a multifactorial disease, which seriously affect the work and life of patients. Since the pathogenesis of dry eye has not been clarified so far, there is no standard animal model available now. Non-Sjögren syndrome dry eye is one of the main types of dry eye, which requires further research. Studies on dry eye mechanisms have led to the development and improvement of animal models. In this paper, the methods of animal models of non-Sjögren syndrome dry eye, including drug treatments, surgery, exogenous injury, behavioral patterns, changes in diet, are reviewed. In addition,different models are described, the problems are analyzed and perspectives and reflections on future research are offered.

Objective: To explore effect of high glucose on expression of osteoprotegerin (OPG) and receptor activator of NF- κ B ligand (RANKE) in rat aortic vascular smooth muscle cells. Methods: SD rats were intraperitoneally injected with streptozotocin, OPG and RANKL expression in rat thoracic aortas were detected by immunohistochemical staining. In cultured vascular smooth muscle cells (VSMCs) (A7r5), qRT-PCR and Western blot analysis were used to examine the mRNA and protein levels of OPG and RANKL. Results: Our results demonstrated that OPG expression was increased in hyperglycemic rat aortic VSMCs, while RANKL expression was decreased. Besides, in vitro experiments high glucose induced OPG expression, but depressed RANKL expression by dose- and time-dependent manner in cultured A7r5. Conclusions: Our findings suggested that high glucose could promote the expression of OPG, and inhibit the expression of RANKL in VSMCs, which may be partly be the molecular mechanism of diabetic vascular calcification.

Platelets are reprogrammed by cancer via a process called education, which favors cancer development. The transcriptional profile of tumor-educated platelets (TEPs) is skewed and therefore practicable for cancer detection. This intercontinental, hospital-based, diagnostic study included 761 treatment-naïve inpatients with histologically confirmed adnexal masses and 167 healthy controls from nine medical centers (China, n = 3; Netherlands, n = 5; Poland, n = 1) between September 2016 and May 2019. The main outcomes were the performance of TEPs and their combination with CA125 in two Chinese (VC1 and VC2) and the European (VC3) validation cohorts collectively and independently. Exploratory outcome was the value of TEPs in public pan-cancer platelet transcriptome datasets. The AUCs for TEPs in the combined validation cohort, VC1, VC2, and VC3 were 0.918 (95% CI 0.889-0.948), 0.923 (0.855-0.990), 0.918 (0.872-0.963), and 0.887 (0.813-0.960), respectively. Combination of TEPs and CA125 demonstrated an AUC of 0.922 (0.889-0.955) in the combined validation cohort; 0.955 (0.912-0.997) in VC1; 0.939 (0.901-0.977) in VC2; 0.917 (0.824-1.000) in VC3. For subgroup analysis, TEPs exhibited an AUC of 0.858, 0.859, and 0.920 to detect early-stage, borderline, non-epithelial diseases and 0.899 to discriminate ovarian cancer from endometriosis. TEPs had robustness, compatibility, and universality for preoperative diagnosis of ovarian cancer since it withstood validations in populations of different ethnicities, heterogeneous histological subtypes, and early-stage ovarian cancer. However, these observations warrant prospective validations in a larger population before clinical utilities.
Humans ; Female ; Blood Platelets/pathology* ; Biomarkers, Tumor/genetics* ; Ovarian Neoplasms/pathology* ; China