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@#AIM:To investigate the application value of fluorescent staining technique in the detection of amoebic pathogens in corneal tissue biopsy, and to apply fluorescent staining technique in the histopathological diagnosis of Acanthamoeba keratitis(AK), comparing the results with those of hemotoxyiln-eosin staining(HE staining)and periodic acid-schiff staining(PAS staining), and analyzing the sensitivity and specificity of these three staining methods.<p>METHODS:Specimens of infected corneal tissue were collected from 74 cases(75 eyes), and then they were divided into an AK group and a non-Acanthamoeba keratitis(NAK)group based on the results of corneal scraping, culture and histopathological diagnosis. The tissues of consecutive sections were stained with HE staining, PAS staining and fluorescence respectively, and the sensitivity and specificity of the three staining methods for the diagnosis of AK were analyzed. Area under the curve(AUC)was calculated using the receiver operating characteristic(ROC)curve. Further analysis was performed to count the number of Acanthamoeba pathogens found by the three staining methods under the same magnification field of view at the same site, and to clarify the diagnostic value of fluorescent staining technique for AK.<p>RESULTS: The sensitivity of HE staining was 69%(27/39)with a specificity of 92%; the sensitivity of PAS staining was 62%(24/39)with a specificity of 97%, and the sensitivity of fluorescent staining was 95%(37/39)with a specificity of 97%. There were differences in the sensitivity of the three staining methods for the diagnosis of AK(χ2=19.857, <i>P</i><0.001), and pairwise comparison revealed that the differences between HE staining and fluorescent staining, PAS staining and fluorescent staining for the diagnosis of AK were statistically significant(<i>P</i>=0.003,<0.001), while the difference in sensitivity between HE staining and PAS staining for the diagnosis of AK was not statistically significant(<i>P</i>=0.978). The maximum AUC was 0.960 for fluorescence staining, followed by 0.804 for HE staining and 0.794 for PAS staining, respectively. The median number of amoeba cysts detected by HE staining, PAS staining and fluorescent staining at the same site under the same magnification field of view was 4(0, 11), 2(0, 9)and 12(3, 33), respectively(χ2=56.561, <i>P</i><0.001). Pairwise comparison revealed that the differences in the number of amoeba cysts found by HE staining and fluorescence staining, PAS staining and fluorescence staining were statistically significant(<i>P</i><0.001), while the difference in the number of amoeba cysts found by HE staining and PAS staining was not statistically significant(<i>P</i>=0.210). Fluorescently stained histopathological sections make it easier to identify amoebic pathogens.<p>CONCLUSION:Fluorescent staining technique is more sensitive to histopathological diagnosis of AK than HE staining and PAS staining, which can significantly improve the positive rate of detection of amoebic pathogens.
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BACKGROUND: Currently, studies have focused on the role and mechanism of nuclear factor-kappa B pathway in the pathological process of acute lung injury in burned rats, such as the targeting inhibition of kB kinase by miR-155, which further weakens the activity of nuclear factor-KB and plays a role in acute lung injury in burned rats. However, there are still some pathological mechanisms to be studied and confirmed. OBJECTIVE: To investigate the effect of miR-155 on acute lung injury in burned rats through nuclear factor-KB pathway. METHODS: The rat models of acute lung injury were established by warm water bath simulating bum injury. The burned rats were divided into acute lung injury, miR-155-mimics and miR-155-inhibitor groups. After fluid resuscitation, the rats in the miR-155-mimics and miR-155-inhibitor groups were injected into the tail vein of 5 |_iL of miR-155-mimics and miR-155-inhibitions, respectively. The expression levels of tumor necrosis factor-a and interleukin-1 p in bronchoalveolar lavage fluid were detected by ELISA. The lung morphology in the three groups was observed by hematoxylin-eosin staining. The protein expression levels of nuclear factor-KB and cyclooxygenase 2 were evaluated by western blot assay. The nuclear factor-KB protein in lung tissues was detected by immunohistochemistry. RESULTS AND CONCLUSION: (1) The results of hematoxylin-eosin staining showed that the severity of lung injury in the miR-155-inhibitor group, acute lung injury group and the miR-155-mimics group was increased gradually (P < 0.05). (2) ELISA results showed that compared with the acute lung injury group, the expression levels of tumor necrosis factor-a and interleukin-1 p were increased in the miR-155-mimics group (P < 0.05), and decreased in the miR-155-inhibitor group (P < 0.05). (3) Western blot assay results showed that compared with the acute lung injury group, the expression levels of nuclear factor-KB and cyclooxygenase 2 proteins were increased in the miR-155-mimics group (P < 0.05), and decreased in the miR-155-inhibitor group (P < 0.05). (4) Immunohistochemical results showed that the expression level of nuclear factor-KB was increased in the miR-155-inhibitor group, which was dark brown. The expression of nuclear factor-KB in cytoplasm and nucleus of neutrophils, mononuclear macrophages, alveolar epithelial cells was the most obvious. (5) These results indicate that in lung tissue cells, decreased miR-155 can down-regulate nuclear factor-KB activity, which reduces the inflammatory response of the lung between the damaged tissue. The study was approved by the Laboratory Animal Ethics Committee of the First People’s Hospital of Neijiang, approval No. 1801270.
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BACKGROUND: Studies have shown that Lycium barbarum polysaccharide (LBP) has the functions of anti-aging, nerve protection, anti-fatigue, blood sugar control, anti-oxidation, and anti-tumor. It may have some protective effects against osteoarthritis of the knee, but have been rarely reported. CD151 and matrix metalloproteinase 3 (MMP-3) are two common cytokines for assessing knee osteoarthritis. OBJECTIVE: To observe the effect of LBP on the expression of CD151 and MMP-3 in rabbit osteoarthritis. METHODS: Sixty-four healthy 6-month-old white rabbits were randomly divided into four groups: blank group, model group, LBP group and normal saline group. Animal models of knee osteoarthritis were made using Hulth method in the rabbits except those in the blank group. The rats in the LBP and normal saline groups were fed with normal dose of LBP and normal saline for 4 weeks, and then the articular cartilage tissues were taken from the affected side at 12 weeks after modeling. The morphological changes of the articular cartilage were observed by hematoxylin-eosin staining. The expression levels and spatial distribution of CD151 and MMP-3 in articular cartilage was observed by immunohistochemical staining and western blot. Ethic approval was given by the People’s Hospital of Ningxia Hui Autonomous Region (approval No. 2014-30817). RESULTS AND CONCLUSION: immunohistochemistry staining and western blot results showed that the absorbance values and protein expression of MMP-3 and CD-151 were significantly lower in the LBP group than the normal saline and model groups (P < 0.05). Therefore, the expression of CD151 and MMP-3 in the articular cartilage of osteoarthritis was increased, and LBP could inhibit the expression of CD151 and MMP-3 in osteoarthritis, so as to slow down the occurrence of osteoarthritis.
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OBJECTIVE@#To explore the treatment conditions of acid decalcified specimens and improve the poor quality of sections and unclear structure of hematoxylin-eosin (HE) staining caused by the change in pH in tooth and hard tissue after acid decalcification.@*METHODS@#A total of 20 cases of oral pathological specimens that contain hard tissues were decalcified and treated with routine treatment, concentrated ammonia water immersion treatment, and saturated lithium carbonate solution immersion treatment. The quality and HE staining effects of hard tissue sections treated with different methods were compared.@*RESULTS@#Compared with routine treatment, lithium carbonate saturated solution treatment showed complete sections. Hematoxylin is strongly stained, the nucleus is clear, and the cytoplasm is bright.@*CONCLUSIONS@#Soaking acid decalcified specimens in lithium carbonate saturated solution before embedding in dehydration can neutralize the acidic environment of the tissue. The quality of sections and HE staining effect are improved and are suitable for the pretreatment of acid decalcified tissue samples of oral pathology.
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Eosine Yellowish-(YS) , Hematoxylin , Staining and Labeling , ToothABSTRACT
@#AIM:To explore the diagnostic effect of hematoxylin-eosin staining(HE)and Giemsa staining in the diagnosis of bacterial and allergic conjunctivitis in children. <p>METHODS:Totally 422 children with conjunctivitis diagnosed by conjunctivitis from the ophthalmology department of our hospital during 2016-10/2019-10 as the research objects. HE and Giemsa staining methods were used to stain the conjunctival scratches, and the staining results were used to diagnose bacterial/allergic conjunctivitis. Observe the positive detection rate of the two staining results for bacterial/allergic conjunctivitis and the staining situation. <p>RESULTS: The positive rate(33.0%)and coincidence rate(63.6%)of HE staining for the diagnosis of bacterial conjunctivitis were significantly lower than Giemsa staining(90.7% and 88.8%, <i>P</i><0.001), while the positive rate of allergic conjunctivitis was not significantly different 90.8% <i>vs </i>87.2%, <i>P</i>>0.05).<p>CONCLUSION: The Giemsa staining method can accurately diagnose bacterial conjunctivitis in children and the method is simple. Both HE and Giemsa staining methods have good diagnostic effects on allergic conjunctivitis, which can provide a basis for improving the clinical diagnosis efficiency and early treatment options.
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OBJECTIVE: To observe the effect of electroacupuncture (EA) on ischemic electrocardiogram (ECG), histopathological changes and serum metabolite profile in chronic myocardial ischemia (CMI) rats, so as to reveal its mechanisms underlying protecting ischemic myocardium. METHODS: A total of 45 male Wistar rats were randomly divided into normal control, CMI model and EA groups, with 15 rats being in each group. The rats in the control group received subcutaneous injection of 0.9% normal saline (5 mg•mg－1•d－1, for 7 days), and those in the model and EA groups received subcutaneous injection of isopropylarterenol hydrochloride (5 mg•mg－1•d－1, for 7 days) to establish CMI model. EA (2 Hz/100 Hz, 1 mA) was applied to bilateral "Zusanli" (ST 36), "Guanyuan" (CV 4) and "Neiguan" (PC 6) for 10 min, once daily for 21 days. The ECG-ST segment of the standard limb lead II was used for evaluating the severity of myocardial ischemia, and the histopathological changes of myocardium were observed under microscope after H．E. staining. The profile of serum metabolites was analyzed by nuclear magnetic resonance mass spectrometry combined with principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) in 21 rats (n＝7 in each group)．. RESULTS: After modeling, the amplitude of ECG-ST was significantly increased in comparison with the normal control group (P 1). The PLS-DA analysis revealed deviations in 51 differential biomarkers in serum，among which, Glucose, Lactate, Creatine, Acetate and 3-Hydroybutyrate may contribute to the effect of EA in improving CMI. CONCLUSION: EA stimulation of acupoints can ameliorate ischemic myocardial injury in CMI rats, which may be related to its effect in regulating serum sugar, lipid metabolism and energy metabolism.
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This study aimed to investigate the anti-apoptotic effects of Tangnaikang (TNK) on islet β cells in Zucker diabetic fatty (ZDF) rats.Six male fa/+ ZDF rats were took as the control group,while other thirty male fa/fa ZDF rats were divided into five groups at random:the model group,the metformin group,the high-,mediumand low-dose TNK groups,depending on their body weight and random blood glucose.Prior to the administration,fasting blood glucose and fasting insulin were measured by drawing blood with inner canthusl.Materials were prepared when administered for six weeks.Fasting blood glucose and fasting insulin were detected again.When the sections of the rat pancreatic tissue were embedded,the morphological changes of the islet were observed via HE staining,and the apoptosis of islet β cell were observed using TUNEL.Positive expression of Caspase-3,the transduction enzyme of cell death signal,was tested by immunohistochemical method.It was found that the fasting blood glucose of the (fa/fa) ZDF rats in the high-,medium-and low-dose TNK group was significantly improved after administration (P ＜ 0.01).The serum insulin of rats in the high-,medium-and low-dose TNK group arised compared with the model group,while the high-and low-dose TNK group showed differences in a statistical sense.Compared with the model group,the HOMA-IR of all the treatment group decreased,while significant difference was presented between the high-dose TNK group and the metformin group.HE staining showed that the morphology of the islet β cell of the rats in all the treatment group was improved.The results of TUNEL showed significantly apoptotic changes on islet β cell of the fa/fa ZDF rats.Compared with the model group,the positive expression of TUNEL in the metformin group and the high-dose TNK group were significantly reduced (P ＜ 0.05).The result of immunohistochemistry method showed that the protein levels of Caspase-3 in the metformin group and the high-dose TNK group decreased (P ＜ 0.05).In conclusion,it was demonstrated that TNK effectively reduced the apoptosis of islet β cells in fa/fa ZDF rats,which presented a protective effect.
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Background and purpose: Adequate tissue ifxation, transparent dewaxing is an important step of hematoxylin eosin (HE) staining and lfuorescence in situ hybridization (FISH) in detection of breast cancerHER-2 gene. The purpose of this study was to make a comparison between poly hydroxyl acrylic acid which is an environmen-tally friendly ifxation liquid and 4% neutral buffered formaldehyde in tissue ifxation for HE staining and FISH to detect theHER-2 gene in the breast cancer tissue sections. The study aimed to evaluate the feasibility of replacing 4% buffered formaldehyde, a traditional ifxation liquid, with the poly hydroxyl acrylic acid, an environmentally friendly ifxation lfuid.Methods:This project was performed on tissue samples collected from 69 cases of breast cancer, 41 cases of breast ifbroadenoma, 40 cases of uterine leiomyoma, 25 cases of cervical tissue, 25 cases of placenta obtained from the outpatient and inpatient departments of Zhongshan Boai Hospital from Mar. 2011 to Jan. 2015, from each of which two samples were drawn and two blocks of each specimen were divided into two groups randomly. Then one group was ifxed with 4% neutral buffered formaldehyde and made into 200 sections by HE while the other group was ifxed with poly hydroxyl acrylic acid and made into another 200 sections. The slice level of the two groups was determined by the staining condition of the sections, and SPSS 19.0 was employed to compare the excellent and good rate of HE staining. Additional 69 sections were produced with two groups of breast invasive ductal carcinoma tissues, and SPSS 19.0 was used to detect the ampliifcation ofHER-2 gene by FISH.Results:First, the number of best-quality slices stained with HE ifxed separately by poly hydroxyl acrylic acid and 4% neutral buffered formaldehyde was 155 and 166, respectively. The number of excellent pieces was 41 and 33, respectively, while the number of mediocre pieces was 3 and 1 with bad pieces being 1 and 0, respectively. The excellent and good rates of HE staining were 98% and 99.5%, respectively. There was no significant difference between the two groups (χ2=1.33,P>0.05).Second, the positive rates of the tis-sue slices by FISH ifxed separately by poly hydroxyl acrylic acid and 4% neutral buffered were 26.09% and 23.19%, respectively. There was no signiifcant difference between the two groups (χ2=0.50,P>0.05).Conclusion:The results obtained with HE staining and FISH using poly hydroxyl acrylic acid as a ifxation liquid are not signiifcantly different from those using 10% neutral buffered formaldehyde. Therefore, poly hydroxyl acrylic acid meets the requirements of environmental protection, and thus has the potential to be promoted and widely used.
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Background The filtration surgery is the main method of treating glaucoma,which usually fails due to postoperative scarring.The study about application of anti-scarring agents in filtration surgery is a hotspot.Objective This study was to investigate whether topical administration of hydroxycamptothecin (HCPT) could be used to prevent postoperative scarring in after experimental glaucoma filtration surgery,and explore its optimal dose.Metbods Trabeculectomy was performed on the right eyes of 40 New Zealand white rabbits to establish the trabeculectomy animal models.The rabbits were then randomized into normal saline solution group,0.3 g/L mitomycin C(MMC) group,0.3 g/L HCPT group and 1.0 g/L HCPT group based on the intraoperative topical drugs using randomized number table method.The different drugs above-mentioned were put beneath the conjunctival flap and scleral flap for 5 minutes during the surgery.The intraocular pressure (IOP) was measured before and day 1,4,7,14,21 and 28 after the surgery with Icare tonometer,and the filtering area and height were measured under the slit lamp microscope to assess the efficacy of various drugs.The adverse effects were evaluated by examining the responses of the ocular anterior segment and retinal change.The specimens at operative zone were obtained in 7,14 and 28 days after surgery with the size 5 mm×5 mm for the hematoxylin-eosin staining and Masson trichrome staining to estimate the anti-fibrosis effect of various drugs,and to evaluate the survival time of functional bleb.Kaplan-Meier analysis was used to compare the survival time of functional bleb of different groups.The use and care of the animals complied with the Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Committee.Results The IOP was significantly different in the rabbits from different groups among various time points (Fgroup =20.79,P =0.00 ; Ftime =85.34,P =0.00 ; Fiion =2.13,P =0.01).From 1 day through 28 days after operation,the IOPs in MMC group and 1.0 g/L HCPT group were significantly lower than those before operation (all at P＜0.05).The survival time of functional bleb of different groups was (11.3 ±2.8),(19.5 ±2.4),(13.3 ±2.2) and (20.2 ± 4.5) days,respectively,showing a significant difference (log rank =11.92,P ＜ 0.01),with a considerable prolong in the 1.0 g/L HCPT group.No significant change was found in the bleb area and height among the four groups within 7 days after operation,but postoperative 7,14,28 days,the area and height values of bleb were significantly smaller in the normal saline solution group and 0.3 g/L HCPT group compared with the MMC group and 1.0 g/L HCPT group (all at P ＜ 0.05).Histopathological examination showed loosen subconjunctival tissue,less inflammatory cells and weaker collagenous fibrillary staining in the MMC group and 1.0 g/L HCPT group in comparison with the normal saline solution group and 0.3 g/L HCPT group.Conclusions The topical administration of 1.0 g/L HCPT inhibit the inflammatory response and collagen fibrosis and therefore prolong the survival time of functional bleb after glaucomatous filtering surgery.
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OBJECTIVES: Although biofilms have been implicated in poor prognosis after endoscopic sinus surgery (ESS), traditional methods detecting biofilm such as scanning electron microscope and confocal scanning laser microscope were rarely used in the practice. The aims of this study was to determine whether the presence or absence of a biofilm detected by hematoxylin and eosin (H&E) staining followed by light microscopy (LM) that is widely used in daily practice, predicts surgical outcomes after ESS. METHODS: Retrospective analysis of prospectively collected data. Fifty-five consecutive adult patients (>18 years) who underwent ESS for chronic rhinosinusitis with a minimum of 12-months of follow-up were enrolled in this study. Random sinonasal mucosal samples were assessed for biofilm presence using H&E staining with LM. Three independent observers scored whether a biofilm was present or absent based on H&E staining/LM, and the interrater variability was calculated. Pre- and postoperative sinus symptoms and sinonasal mucosal grading were assessed. RESULTS: Biofilms were present in 28 patients (51%), and the intraclass correlation coefficient according to H&E staining/LM was 0.731. The presence of a biofilm was associated with a higher preoperative Lund-MacKay computed tomography score (22.3 for biofilm-positive patients vs. 18.6 for biofilm-negative patients; P=0.021) and persistent inflammation (mucosal edema and discharge) after ESS (P<0.05). CONCLUSIONS: The presence or absence of a biofilm based on H&E staining/LM is correlated with disease severity and surgical outcomes after ESS. H&E staining/LM for detecting biofilm could be practical and cost-effective methods for predicting prognosis of ESS.
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Adult , Humans , Biofilms , Edema , Eosine Yellowish-(YS) , Follow-Up Studies , Hematoxylin , Inflammation , Microscopy , Prognosis , Prospective Studies , Retrospective StudiesABSTRACT
BACKGROUND: It is difficult to in vitro isolate and culture the ovarian surface epithelium with high purity, strong vitality and stable biological characteristics. Tissue adherence and enzymatic digestion are commonly used for primary culture, but there are certain problems in cel col ection, cel viability and cel purity. OBJECTIVE: To establish a method for primary isolating, culturing and identifying human ovarian epithelium. METHODS: The ovarian surface epithelium was obtained with cel brush innovatively, and then the cells were isolated and purified with erythrocyte spal ation and differential adherence. The epidermal growth factor was added into the serum-free Dulbecco’s modified Eagle’s medium-F12 medium for cel culture. The cel morphology was observed under inverted microscope, and hematoxylin-eosin staining and immunocytochemical staining were used to identify the cells, then the growth curve was draw. RESULTS AND CONCLUSION: The ovarian surface epithelium became adherent after cultured for 24 hours, and reached fusion basical y after cultured for 6-12 days. The cells were polygonal or flat with strong transparency and refraction. The morphological characteristics of the cells were in line with those of the normal epithelial cells, and almost al the isolated cells could express the epithelial cells surface marker CK19. The cells could be passaged for 6-8 generations with wel growth and the cel growth curve was in “S” shape. The purity of the cells was more than 95%. The results suggest that cel brush method is simple to operate and can obtain a large amount of ovarian surface epithelium rapidly. The purity of the isolated cells can reach to 95% after treated with erythrocyte spal ation and differential adherence method and the cells are in stable growth.