Objective:To provide a standard protocol for the rapid quantitative analysis of neutrophils in inflamed corneas with flow cytometry.Methods:The corneal epithelium layer of 15 C57BL/6 mice (6-8 weeks old) was mechanically scraped off using a golf-like knife to generate a 2 mm wound region.The mouse corneas with intact limbus were cut out at 18 hours after abrasion.After mechanical shredding, the single cell suspension was obtained by collagenase I and DNase digestion.Then, the number of neutrophils in the corneal cells was sorted under the FACSCanto flow cytometer using the gate technique.Another 6 mice were taken and randomized into wounded group and normal group according to a random number table method, with 3 mice in each group.Corneal cell staining was performed using fluorescent-conjugated anti-mouse CD45, Ly6G, and CD11b antibodies.The number of neutrophils in the corneas of the two groups were enumerated and compared.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO). The study protocol was approved by the Animal Ethics Committee of Medical College of Jinan University (No.JN-A-2002-01).Results:A standard procedure for detecting neutrophils in the cornea by flow cytometry was established.The ratio of CD45 + cells in the total corneal tissue cell population was (20.93±1.72)%.The Ly6G + and CD11b + double positive neutrophil population was sorted in the wounded corneal cell population.The ratios of Ly6G + and CD116 + cells in the CD45 + cells were (75.50±3.25)% and (93.40±4.53)%, respectively, and the ratio of the Ly6G + and CD11b + double positive neutrophils in the total number of CD45 + cells was (67.33±2.80)%.In addition, the number of neutrophils recruited to the cornea at 18 hours after corneal abrasion was (151.47±10.82)%, which was higher than (15.36±1.02)% in the normal cornea ( t=21.689, P<0.01). Conclusions:Flow cytometry can quickly and accurately quantitatively analyze the neutrophil population in the wounded cornea.It provides a rapid quantitative analysis method to further evaluate the changes of neutrophils in corneal inflammation caused by different reasons.

Background To understand the distribution and development of corneal nerve in animal or human has an important significance for clinical and basic research of corneal diseases.At present,some studies on cornea nerve development and location have been performed.However,the quantified study on innervation and distribution of corneal nerve fibers as embryonic development has not been reported.Objective This study attempted to understand the distribution of corneal nerve fibers in the development of chick embryo,and to evaluate the changes of the length and density of corneal nerve fibers with aging of chick embryo.Methods Whole chick corneas with limbus were obtained from chick embryo aged 6-20 days (E6-E20),and corneal nerve fiber was labeled using immunofluorescence technique by anti-neuron-specific β-Ⅲ tubulin antibody.The corneas were radially cut into 4 parts,and the integrate corneal flat mounts were prepared with the upward epithelium and mounting with anti-fade fluorescent quenching buffer glycerin containing DAPI.Fluorescence microscope was used to capture the nerve fiber images in cornea,and cornea area and the number of nerve fiber bundles were exhibited by using Photoshop CS4.Cornea nerve fiber density and total length were measured by Imaris x64 7.4.2 software.Results Total cornea flat mounts showed that the nerve bundles grew from temporal scleral forward cornea limbus at E6-E8,and the nerve fibers formed the ring surrounding by limbus during E9-E10.Then the fibers extended forward the central cornea in E11 to E15 and developed into nerve fiber plexus on the whole cornea in E16 to E20.During the period of E6-E20,the corneal surface area,the length and density of corneal nerve fibers were gradually increased with the aging of chick embryo,showing statistically significant differences among different time points (F =127.007,227.051,67.748,all at P＜0.01).The increase of the corneal area of the chick embryo presented a strong positive correlation with the extending of length of the corneal nerve fiber (r =0.863,P＜0.01).Chick corneal nerve fiber bundle appeared at E13,with a number of (59.00 ± 1.14)/mm2 and then increased to a peak of (576.75 ±29.16)/mm2 at E 18 and reduced to (299.67± 25.46)/mm2 at E20,with a significant difference among them (F =13.759,P=0.000).Conclusions Corneal nerve starts to develop in E9 of chick embryo,and the corneal surface area,the total length of the corneal nerve fibers and the density rapidly increase concurrently with the development of chick embryo.

Objective To explore the clinical features and risk factors of children with severe hand foot and mouth disease (HFMD).Methods The clinical data of 68 children with HFMD associated with brainstem encephalitis were analysed retrospectively from Apr to Sep 2010.Results Among the 68 cases,63cases(92.6％ ) were younger than 3 years.The major symptoms and physical signs of 68 cases included rash (68 cases,100％ ),fever (68 cases,100％ ),fatigue (68 cases,100％ ),irritability (65 cases,95.6％ ),somnolence (42 cases,61.8％ ),vomiting (28 cases,41.2％ ),limb tremor (23 cases,33.8％ ),tendon reflexe hyperactivity (60 cases,88.2％ ),tachypnea or respiratory rhythm abnormality (63 cases,92.6％ ),tachycardia (65 cases,95.6％ ),and hypertension (54 cases,79.4％ ).Twenty-five cases (36.8％,25/68 ) had leucocytosis ( ＞ 12 × 109/L ),and 19 cases ( 27.9％,19/68 ) had hyperglycaemia.X-ray appearances:patchy and pathy shadows in single or bilateral pulmonary were seen in 46 cases( 67.6％,46/68 ).Forty-eight cases were examined by MRI,eight cases displayed ischemic lesions or demyelination.Spinal cord MRI was performed in 3 cases with flaccid paralysis,which showed demyelination.The etiology indicated that 38 cases ( 55.9％,38/68 ) were infected by enterovirus 71,25 cases( 36.8％,25/68 ) were infected by other enterovirus,5 cases (7.3％,5/68) were negative.All the cases were treated by mechanical ventilation with tracheal intubation,in whom 63 cases recovered well,4 cases improved,and 1 case gave up to die.Conclusion Ages ＜ 3 years,enterovirus 71 infection,continual fever,fatigue,somnolence,irdtability,vomiting,limb tremor,tendon reflexes hyperactivity,tachypnea or respiratory rhythm abnormality,tachycardia and hypertension are the high risks of critically ill children associated with severe HFMD.To reduce the fatality rate of HFMD,it is crucial to early judge the high risk factors,and take mechanical ventilations earlier.

[ ABSTRACT] AIM: To observe the effect of circadian rhythms on wound healing of mouse corneal epithelium. METHODS:The C57BL/6 male mice were used in the study.A part of corneal epithelium (2 mm in diameter) was struck off by a golf-like knife to form a round wound area.The dynamics of epithelial healing in the wound area were ob-served under microscope with fluorescein staining.In addition, with related antibodies and DAPI, the dynamic changes of the neutrophils, platelets and dividing cells were also investigated.RESULTS:The healing rates in LL group (12 h light/12 h light) and DD group (12 h dark/12 h dark) were obviously slower than that in LD group (12 h light/12 h dark), mainly showing delayed re-epithelialization, decreased epithelial cells, increased diameter of blood vessel, and delayed re-cruitment of neutrophils and platelets, but more cell number.CONCLUSION:Disruption of circadian rhythms significant-ly inhibits the wound healing of corneal epithelium, mainly through delaying the inflammation and re-epithelialization, but aggravating the inflammatory responses.

OBJECTIVE:To study the effects of butin in Vernohia anthelmintica(VW)on proliferation of human immortal ke-ratinocyte cell strain HaCaT and cell secretory factors,and explore the mechanism of butin in VW in the treatment of vitiligo. METHODS:MTT method was used to determine the survival rate of HaCaT cells cultured by 0 (blank control),0.1,0.5,1.0, 5.0,10.0 μg/mL of butin for 48 h. Enzyme-linked immunosorbent assay was used to determine the contents of cell secretory factors as endothelin 1 (ET-1),ET-3,melanocyte stimulating hormone (MSH),stem cell factor (SCF),basic fibroblast growth factor (bFGF)in culture medium after HaCaT cells were cultured by 0.5,1.0,5.0 μg/mL of butin for 48 h. RESULTS:Compared with blank control,cell survival rate was increased to varying degrees after cultured by 0.1-5.0μg/mL of butin for 48 h,while decreased after cultured by 10.0 μg/mL of butin. Contents of ET-1,SCF,bFGF in culture medium were significantly increased after cultured by 0.5,1.0,5.0μg/mL of butin for 48 h(P<0.01);and contents of ET-3,MSH in culture medium were significantly increased af-ter cultured by 1.0,5.0 μg/mL of butin for 48 h(P<0.01). CONCLUSIONS:Butin can promote the proliferation of HaCaT cells, the mechanism may be associated with promoting the secretion of cell secretory factors of ET-1,ET-3,MSH,SCF,bFGF.

Skin wounds are global public problems,especially in terms of the difficult healing of chronic wounds,which may seriously affect patients'lives.Most skin-wound animal models are currently established by physical method,and different animal models have different biological characteristics.This review therefore classifies mouse,rat,and other animal models of skin wounds according to the literature,summarizes and analyzes the construction of skin-wound animal models based on physical method and evaluation indicators,and considers the advantages and disadvantages of different animal models,to provide a basis for the rational construction of skin-wound animal models and drug research and development.

Objective To investigate the prevalence of thyroid nodules and thyroid dysfunction in southern mountainouss areas of Ningxia.Methods A cross-sectional study was conducted among a representative sample of 10 639 adults in Jingyuan county with a population proportionate sampling method.High-resolution ultrasound was used to examine the thyroid and fasting blood specimens were collected in the morning for measurement of TSH,FT4,FT3.Chi-square test and spearman rank correlation analysis were used for statistical analysis.Results The prevalence of thyroid nodules was 29.08％,the sex-and age-adjusted rate was 27.17％.The prevalence of thyroid nodules was higher in women than in men (32.68％ vs.24.88％,x2=76.029 2,P＜0.001) and age was positively associated with thyroid nodules (r=0.272,P＜0.001).The rate of thyroid dysfunetion,subclinical hypothyroidism,subclinical hyperthyroidism,hypothyroidism,hyperthyroidism were 17.39％,13.00％,0.42％,0.96％,3.01％,respectively.The prevalence of thyroid nodules was higher in abnormal TSH group than in normal TSH group (39.44％ vs.27.24％,x2=95.624 0,P＜0.001).The level of THS,FT3,FT4 in thyroid nodules group differed fromn control group (Z=-9.144,P＜0.001;Z=-6.140,P＜0.001;Z=-1.997,P=0.046).Conclusion The prevalence of thyroid nodules and thyroid dysfunction were higher in southern mountainous areas of Ningxia.The relationship between thyroid nodules and thyroid function needs further research.We should pay attention to the early screening and diagnosis of thyroid nodules in mountainous areas.

Objective:To investigate the effect of circadian rhythm changes on the expression of retinoic acid-related orphan receptors (RORs) and the RORs agonist SR1078 on corneal epithelial wound repair.Methods:A total of 228 SPF C57BL/6 female mice aged 6-8 weeks old were selected, and 180 mice were divided into the normal circadian rhythm group, full-day group, full-night group, 12-hour reversed circadian rhythm group and 3-week reversed circadian rhythm group, with 36 mice in each group.The remaining 48 mice were randomly divided into phosphate buffered saline (PBS) control group and SR1078 group by random number table method, with 24 mice in each group.According to grouping, the mice were placed in a light box where the light (light intensity of 300 lx) and dark time could be controlled.The light time of the normal circadian rhythm group, the PBS control group and the SR1078 group in the light box was from 7: 00 to 19: 00, and the dark time was from 19: 00 to 7: 00 the next day.According to the Zeitgeber Time method, the starting time of light at 7: 00 was recorded as ZT0, and the time of closing light at 19: 00 was recorded as ZT12.Real-time fluorescence quantitative polymerase chain reaction was used to detect the relative expression levels of RORα and RORγ mRNA at ZT1, ZT5, ZT9, ZT13, ZT17, ZT21 in the five groups.In the PBS control group and SR1078 group, a golf-like knife was used to establish the mouse corneal epithelial injury model, and the model eyes were administered with drugs once every 6 hours according to the grouping.The corneal epithelial defect area was measured with Adobe Photoshop CC2019 software, and the corneal epithelial defect rate was calculated and compared between the two groups.The correlation between the relative expression levels of RORα and RORγ mRNA in mice corneal epithelium of the five groups and corneal epithelial defect rate in the PBS control group and SR1078 group was analyzed.The corneal epithelium repair was observed by whole cornea spreading and immunofluorescence staining, and the number of corneal epithelial dividing cells in the PBS control group and the SR1078 group was calculated and compared.The use and care of animals complied with the ARVO statement.This study protocol was approved by the Laboratory Animal Ethics Committee of Jinan University (No.JN-A-2002-01).Results:Compared with the normal circadian rhythm group, the relative expression levels of RORα/RORγ mRNA in the full-day group, full-night group, 12-hour reversed cirdian rhythym group and 3-week reversed cirdian rhythym group showed an overall decreasing trend.There was a statistically significant difference in the corneal epithelial defect rate between the PBS control group and the SR1078 group at different time points after modeling ( Fgroup=74.01, P<0.001; Ftime=5 171.48, P<0.001). Twelve hours after modeling, the corneal epithelial defect rate in the SR1078 group was significantly lower than that in the PBS control group, and the difference was statistically significant ( P<0.05). The relative expression levels of RORα and RORγ mRNA in corneal tissue was moderately positively correlated with the corneal epithelial defect rate in mice ( r=0.614, 0.537; both at P<0.01); The regression equation of the straight line between the relative expression level of RORα mRNA and the change in corneal epithelial defect rate was Y=33.153X-43.052 ( F=20.58, P<0.001), and the linear regression equation between the relative expression level of RORγ mRNA and the change of corneal epithelial defect rate was Y=2.764X-1.364 ( F=13.11, P<0.001). There was a significant overall difference in the number of corneal epithelial dividing cells at various time points following modeling between the PBS control group and the SR1078 group ( Fgroup=160.55, P<0.001; Ftime=83.57, P<0.001). The number of dividing cells in the SR1078 group was significantly less than that in the PBS control group at 24, 30, and 36 hours following modeling, and the differences were statistically significant (all at P<0.05). Conclusions:Circadian rhythm changes reduce the expression of RORα and RORγ mRNA in the mouse cornea.SR1078 can promote the expression of RORα and RORγ mRNA in corneal epithelium to decrease the number of mouse corneal epithelial dividing cells, and inhibit the repair after corneal trauma.

Objective:To investigate the effect of alprostadil combined with metformin in the treatment of diabetic nephropathy.Methods:50 cases of diabetic nephropathy patients were enrolled and then divided equally into the observation group and the control group. The patients in the control group were treated with conventional therapy and metformin, and the patients in the observation group were treated with alprostadil on the basis of the treatment of the control group. Compare the glycemic index, lipid index, renal function index, inflammatory response index, and oxidative stress response index of the two groups of patients before and after the 4-week treatment. The ratio of the number of effective cases (significant + effective) to the total number of cases, i.e., the total effective rate, was used to characterize the treatment effect.Results:The total effective rate in the observation group was higher than that in the control group (88.00% vs. 60.00%, P<0.05). After the 4-week treatment, no adverse effects occurred in either group. Compared with the control group, patients in the observation group had higher fasting blood glucose (FBG), glycosylated hemoglobin(HbA1c), mean blood glucose(MBG), blood glucose fluctuation rate(BGFR), standard deviation of blood glucose(SDBG), triglyceride(TC), total cholesterol(TG), high-density lipoprotein(HDL), low-density lipoprotein(LDL), serum creatinine(Scr), urinary microalbumin(UmALB), glomerular filtration rate(eGFR), albumin/creatinine ratio(ACR), cyclic adenosine monophosphate(cAMP), renin, protein kinase(PKA), epinephrine (E), angiotensin-converting enzyme inhibitor Ⅱ(ACEI Ⅱ), and norepinephrine(NE) were improved(all P<0.05). Conclusions:The effect of alprostadil combined with metformin in the treatment of diabetic nephropathy is accurate, safe, and reliable.

Objective To observe the effect of reversed circadian rhythms on wound healing of mouse corneal epithelium.Methods Ninety male C57BL/6 mice were divided into LD group (12 hours light/12 hours dark) and DL group (12 hours dark/12 hours light) by random number table,and then were placed in circadian rhythm box for 12 days.The circular area was scarped and marked as 2 mm diameter area in the center of the mouse's cornea with a golf-like knife.The dynamics of epithelial healing in the wound area were observed under microscope by fluorescein staining and hematoxylin-eosin staining.Besides,being marked antibodies of anti-Ly6G-FITC,anti-γδT-PE and DAPl,dynamic changes of the dividing cells,neutrophils and γδT cells were also investigated for every 6 hours until 42 hours.All mice were treated in accordance with the Association for Research in Vision and Ophthalmology's Statement for the Use of Animals in Ophthalmology and Vision Research and the guidelines of the Animal Experimental Committee at Jinan University (JN-A-2002-01).Results In LD group,percentage of corneal epithelial defective area were (100.000 ± 0.000) ％,(37.677 ± 5.243) ％,(14.959 ± 1.739) ％ and (0.000 ± 0.000) ％ after wounding 0 hour,6,12,18 and 24 hours.In DL group,percentage of the corneal epithelial defective area were (100.000±0.000) ％,(10.967 ± 1.065 ％) ％,(1.985 ±0.106) ％ and (0.000±0.000) ％ after wounding 0 hour,6,12,18 and 24 hours.The healing rate in DL group was higher than that in LD group,with a significant difference between them (P＜0.05).As with the uninjured corneal,thickness of corneal epithelium was (33.983 ± 1.074)μm in DL group and (33.993±0.904)μm LD group,with no statistically significant difference between them (P＞0.05).After 24 hours,thickness of corneal epithelium in DL group was (19.473 ±0.856) μm,and was more than that in LD group [(17.485±0.718)μm],with a significant difference between them (P＜0.05).Paraffin section of wounded corneal epithelium after 24 hours by hematoxylin and eosin staining showed that corneal epithelium cells arranged loosely and disorderly and were in irregular shape in both groups.The epithelium were mainly basal cells in LD group,while epithelium included basal cell and a few pinacocytes in DL group.After corneal epithelium wounded,the number of cell division,neutrophils and corneal limbus γδT cells in two groups were statistically significant difference,respectively(P＜0.05).Conclusions Reversed circadian rhythms can significantly regulate the wound healing of corneal epithelium.