Mechanism study of laser cochleostomy-induced early hearing loss in a rat model.

Hearing loss following laser-assisted ear surgery has been reported. However, the mechanism responsible for the hearing loss remains largely speculative. The aim of this study was to investigate the correlation between laser-induced hearing loss and changes in the number of hair cell ribbon synapses and ultrastructure in the cochlea. Laser cochleostomy was performed with a superpulsed carbon dioxide (CO2) laser at 2 and 5 W in Sprague-Dawley rats. Auditory brainstem responses (ABRs) were measured preoperatively and 2 days after surgery. The synapse numbers in apical and middle cochlear turns were quantified. Transmission electron microscopy was employed to further examine the subcellular changes in the cochlea. Click and tonal ABR threshold shifts in both 2 and 5-W groups displayed a frequency-dependent loss within the frequency range measured. Laser cochleostomy induced a significant decrease of synapse numbers in the middle turn in both groups (p < 0.05). Electron microscopy data indicated varying degrees of auditory nerve degeneration in both groups. Auditory nerve degeneration might contribute to laser-caused hearing loss even under low-energy laser cochleostomy. The high-energy laser-induced hearing loss was associated with more reduction of synapse number.

Experimental study of millimeter wave-induced differentiation of bone marrow mesenchymal stem cells into chondrocytes.

Low power millimeter wave irradiation is widely used in clinical medicine. We describe the effects of this treatment on cultured mesenchymal stem cells (MSCs) and attempted to identify the underlying mechanism. Cells cultured using the whole marrow attachment culture method proliferated dispersedly or in clones. Flow cytometric analyses showed that the MSCs were CD90 positive, but negative for CD45. The negative control group (A) did not express detectable levels of Cbfa1 or Sox9 mRNA at any time point, while cells in the millimeter wave-induced groups (B and C) increasingly expressed both genes after the fourth day post-induction. Statistical analysis showed that starting on the fourth day post-induction, there were very significant differences in the expression of Cbfa1 and Sox9 mRNA between groups A and B as well as A and C at any given time point, between treated groups B and C after identical periods of induction, and within each treated group at different induction times. Transition electron microscopy analysis showed that the rough endoplasmic reticulum of cells in the induced groups was richer and more developed than in cells of the negative control group, and that the shape of cells shifted from long-spindle to near ellipse. Toluidine blue staining revealed heterochromia in the cytoplasm and extracellular matrix of cells in the induced groups, whereas no obvious heterochromia was observed in negative control cells. Induced cells also exhibited positive immunohistochemical staining of collagen II, in contrast to the negative controls. These results show that millimeter wave treatment successfully induced MSCs to differentiate as chondrocytes and the extent of differentiation increased with treatment duration. Our findings suggest that millimeter wave irradiation can be employed as a novel non-drug inducing method for the differentiation of MSCs into chondrocytes.

Electroacupuncture Delays Cartilage Degeneration by Modulating Nuclear Factor-κB Signaling Pathway.

OBJECTIVE: To illustrate the molecular mechanisms underlying the therapeutic effects of electroacupuncture (EA) on knee osteoarthritis (OA). METHODS: Twenty-seven six-month-old New Zealand white rabbits were allocated into three groups in accordance with a random number table: normal group (no surgery-induced OA; without treatment), model group (surgery-induced OA; without treatment) and EA group [surgery-induced OA; received treatment with EA at acupoints Dubi (ST 35) and Neixiyan (EX-LE 5), 30 min twice a day]. After eight consecutive weeks of treatment, the histopathological alterations in cartilage were observed using optical microscopy and transmission electron microscopy, cartilage degeneration was evaluated by modified Mankin's score principles, the synovial fluid concentration of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-3 (MMP-3) were evaluated by enzyme-linked immunosorbent assay, and the protein expression levels of IL-1ß, IL-6, TNF-α, MMP-3, IκB kinase-ß (IKK-ß), nuclear factor of α light polypeptide gene enhancer in B-cells inhibitor α (IκB-α) and nuclear factor-κB (NF-κB) p65 were quantified by Western blot analysis. RESULTS: EA treatment significantly improved cartilage structure arrangement and reduced cellular degeneration. The IL-1ß, IL-6, TNF-α and MMP-3 of synovial fluid in the EA-treated group were significantly decreased compared with the model group (all P<0.01). Compared with the model group, the IL-1ß, IL-6, TNF-α, MMP-3, IKK-ß and NF-κB p65 protein expressions in cartilage of EA-treated group were significantly decreased (all P<0.01), whereas IκB-α expression was significantly up-regulated (P<0.01). CONCLUSION: EA treatment may delay cartilage degeneration by down-regulating inflammatory factors through NF-κB signaling pathway, which may, in part, explain its clinical efficacy in the treatment of knee OA.

Ultrastructural and immunohistochemical studies on Trichomonas vaginalis adhering to and phagocytizing genitourinary epithelial cells.

BACKGROUND: Trichomonas vaginalis (T. vaginalis) belongs to a common sexually transmitted disease pathogen causing genitourinary trichomoniasis in both sexes. We investigated the pathogenetic mechanism of genitourinary trichomoniasis. METHODS: Cultured T. vaginalis bodies were injected into the vaginas of rats, or incubated with genitourinary epithelial cells of female subjects, male subjects, and sperm. The ultrastructural and microscopic changes were observed via transmission and scanning electron microscopy and through microscopic histochemistry. RESULTS: Groups of T. vaginalis adhered to PAS positive columnar cells at the surface of stratified epithelium in the middle and upper portions of the vaginas. They also traversed under these cells. The parasites were shown to be PAS, cathepsin D, and actin positive, and they could release hydrolase into the cytoplasm of adhered epithelial cells. In the amebiform T. vaginalis, microfilaments were arranged into reticular formation. Similar phenomena were found during the interaction of T. vaginalis with host cells, both in vitro and in vivo. Usually several protozoa adhered to an epithelial cell and formed polymorphic pseudopodia or surface invaginations to surround and phagocytize the microvilli or other parts of the epithelial cytoplasm. Adhesion and phagocytosis of sperm by the protozoa occurred at 15 - 30 minutes of incubation. Digestion of sperm was found at 45 - 75 minutes and was complete at 90 - 105 minutes. CONCLUSIONS: T. vaginalis tends to parasitize at the fornix of the vagina, because this is the site where columnar cells are rich in mucinogen granules and their microvilli are helpful for adhesion and nibbling. T. vaginalis possesses some invading and attacking abilities. Shape change, canalization, encystation, phagocytosis, digestion, the cell coat, cytoskeleton, and lysosome all play important roles in the process of adhesion. They have two methods of phagocytosis: nibbling and ingestion. Genitourinary epithelium may be injured directly by the digestive action of hydrolases, phagocytosis, and the mechanical action of pseudopodia.

[Research propress of co-culture system of osteoblast with osteoclast and its applications].

Osteoclasts and osteoblasts are not exist alone,while communicating with each other through direct contact, diffusible paracrine factors and cell-bone matrix interaction. Co-culture system of osteoblast with osteoclast,including direct co-culture and indirect co-culture. It should be according to the ratio of osteoclasts and osteoblasts under the pathology, choosing the same species. Compared with lonely culture of osteoblasts or osteoclasts,co-culture system is much closer to the microenvironment in vivo. It benefits to explain the interactions between osteoblasts and osteoclasts, exploring molecular communication in bone diseases. It was mainly used to investigate the pharmacological mechanism of herbal and western medicine in bone remodeling. Some osteoporosis drugs (such as epimedium,sanchi, fructus psoraleae, ranelate strontium) not only promoted osteoblastic bone formation, but also inhibited osteoclastic bone resorption in the system,so as to balance bone homeostasis. At the same time,it has been used to study medical physics and assess biomedical materials in recent years. Considerably,the co-cultrue system will be used to study the subchondral bone remodeling and its pharmacological mechanism of herbal and western medicine in osteoarthritis.

Experimental study on the suppression of sodium nitroprussiate-induced chondrocyte apoptosis by Tougu Xiaotong Capsule (透骨消痛胶囊)-containing serum.

OBJECTIVE: To study the mechanism of action of Tougu Xiaotong Capsule (透骨消痛胶囊, TGXTC) ex vivo in suppressing chondrocyte (CD) apoptosis induced by sodium nitroprussiate (SNP). METHODS: Thirty New Zealand rabbits, 2 months old, were randomized by lottery into five groups, six in each: the blank group treated with saline, the positive control group treated with Zhuanggu Guanjie Pill (壮骨关节丸, 70 mg/kg), and the three experimental groups, EGA, EGB, and EGC, treated with low dose (35 mg/kg), moderate dose (70 mg/kg), and high dose (140 mg/kg) of TGXTC, respectively. All treatments were administered via gastrogavage twice a day for 3 days. Arterial blood was collected from the abdominal aorta and drug or drug metabolites-containing serum was prepared. CDs obtained from knee joints of 16 four-week-old New Zealand rabbits were cultured to the third passage and confirmed by toluidine blue staining. SNP of various final concentrations (0, 0.5, 1.0, and 2.0 mmol/L) was used to induce CD apoptosis, and the dosage-effect relationship of SNP in inducing CD apoptosis was determined. Serum samples from the blank, control, and three dosages of TGXTC-treated rabbits were tested in the CD culture in the presence of SNP. Cell apoptosis was determined by Hoechst 33342 staining, viability of CDs was quantified by MTT, CD apoptosis rate was determined by annexin V-FITC/PI staining, levels of p53 and Bcl-2 mRNA expression in CDs were determined with RT-PCR, and contents of caspase-3 and caspase-9 proteins were determined by colorimetry. RESULTS: CD apoptosis was induced by SNP at all concentrations tested and in a dose-dependent manner. The SNP concentration of 1 mmol/L and treatment duration of 24 h appeared to be optimal and were selected for the study. Serum samples from the positive control rabbits and from the two higher doses of TGXTC-treated rabbits showed reduction of SNP-induced CD apoptosis, decrease in p53 mRNA expression, inhibition of catalytic activities of caspase-3 and caspase-9, and increase in Bcl-2 mRNA expression when compared with the serum from the blank group (P<0.05). CONCLUSION: TGXTC-containing sera antagonized SNP-induced CD apoptosis and the molecular basis for the action was associated with up-regulation of Bcl-2, down-regulation of p53 expression, and inhibition of caspase-3 and caspase-9 catalytic activities.

Anticolchicine cytotoxicity enhanced by Dan Gua-Fang, a Chinese herb prescription in ECV304 in mediums.

OBJECTIVE: To study the effect of anticolchicine cytotoxicity of Dan Gua-Fang, a Chinesea Chinese), a Chinese herbal compound prescription on endothelial cells of vein (ECV304) cultivated in mediums of different glucose concentrations as well as the proliferation of those cells in the same conditions, in order to reveal the value of Dan Gua-Fang in preventing and treating endothelial damage caused by hyperglycemia in diabetes mellitus. METHODS: The research was designed as three stages. The growing state and morphological changes were observed when ECV304 were cultivated in the culture mediums, which have different glucose concentrations with or without Dan Gua-Fang and at the same time with or without colchicine. RESULTS: (1) Dan Gua-Fang at all concentrations reduced the floating cell population of ECV304 cultivated in hyperglycemia mediums. (2) Dan Gua-Fang at all concentrations and hyperglycemia both had a function of promoting "pseudopod-like" structure formation in cultivated ECV304, but the function was not superimposed in mediums containing both hyperglycemia and Dan Gua-Fang. (3) Colchicine reduced and even vanished the "pseudopod-like" structure of the endotheliocyte apparently cultivated in mediums of hyperglycemia or with Dan Gua-Fang. The "pseudopod-like" structure of the endotheliocyte emerged quickly in Dan Gua-Fang groups after colchicine was removed, but it was not the case in hyperglycemia only without Dan Gua-Fang groups. (4) Dan Gua-Fang reduced the mortality of cells cultivated in mediums containing colchicine. The cell revived to its normal state fast after colchicine was removed. CONCLUSION: Dan Gua-Fang has the functions of promoting the formation of cytoskeleton and fighting against colchicine cytotoxicity.

[A three-dimension in vitro model for angiogenesis of hemangioma].

OBJECTIVE: To create a three dimension (3D) in vitro model for angiogenesis of hemangioma. METHOD: The fragment of hemangioma specimen was embedded in fibrin gel to set up the three-dimension (3D) in vitro model for angiogenesis of hemangioma. RESULT: In the model, microvessels grew out from the tissue fragments at the 2nd to 3rd day after culture, and at the 8th to 9th day a compact network of microvessels come into being, then tending to be stationary. The compact network around the tissue fragment was confirmed to be blood vessels by immunohistochemistry and electron microscopy. CONCLUSION: This model helps to study the mechanism of hemangioma angiogenesis and investigate the drugs of anti-angiogenesis.