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Background Congenital stationary night blindness (CSNB) is a common genetic eye disease.Retinal angiogenesis is rarely obtained in retinal degeneration animal.The effects of CSNB on retinal angiogenesis require further study.Objective This study was to improve Chinese ink perfusion technology and to explore the effect of CSNB on oxygen induced neovascularization.Methods Eighteen clean 7-day old SD rats and 18 clean 7-day old CSNB rats were included,twelve SD rats and twelve CSNB rats were chosen randomly for oxygen induced retinopathy (OIR) modeling,and served as OIR group,six SD rats and six CSNB rats were chosen as normal control.Nine rats were chosen randomly from both SD rats and CSNB rats in OIR group,respectively.The rats were separated into 1 ∶ 1 ratio ink group,2 ∶ 1 ratio ink group and conventional ink group,which were perfused with 1 ∶ 1 ratio ink perfusate,2 ∶ 1 ratio ink perfusate and conventional ink,respectively.The unilateral eyes of the rats were prepared for whole-mount retina,the other eyes were performed for paraffin imbedding.The quality of retinal vascular imaging were compared among different ink perfusate groups.The normal control rats,three SD rats in OIR group and three CSNB rats in OIR group were perfused with 2 ∶ 1 ratio ink perfusate.Histopathology examination was performed on the paraffin section,and the number of nuclei breakthrough the inner limiting membrane were counted.Immunocytochemistry were performed on the paraffin section for detecting the expression of von Wilebrand factor (vWF).Results Compared with 1 ∶ 1 ratio ink perfusion and conventional ink perfusion,2 ∶ 1 ratio ink perfusion showed the full vascular net clearly.Histopathology showed that the structure of retina was normal in the normal control group,and there were no endothelial nuclei breakthrough the inner limiting membrane.A large number of endothelial nuclei breakthrough the inner limiting membrane in the OIR group,the number of endothelial nuclei breaking through the inner limiting membrane were (23.08±2.99)/slide and (41.12±9.36)/slide for SD rats and CSNB rats,respectively.The number of endothelial nuclei breakthrough inner limiting membrane was higher in the CSNB rats than that in the SD rats,with no difference between the two group (q =1.70,P =0.50).Immunocytochemistry results showed that v-WF was positive expressed in the cells breakthrough inner limiting membrane.Conclusions Improved ink perfusion method was an easy-to-use whole-mount retina method with good repeatability.Hyperoxia can induce retinal neovascularization of CSNB rats.
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Background Retina fixed flat-mount perfused by Evans blue (EB) is a common method for the evaluation of blood-retinal barrier (BRB).However,previous method is inconvenient for some laboratories because the retinal specimen can not be observed by gereral microscope rather than confocal laser scanning microscope after the fixation.Objective This study was to modify the preparing way of flat-mounted retina in order to obtain transparent specimen for the observation of rat retinal vessels and the evaluation of leakage under the ordinary fluorescence microscope.Methods Forty male SD rats were divided into the control group,diabetes mellitus (DM) 1-month group,DM 3-month group and DM 6-month group according to the random number table.Streptozotocinum (STZ) of 2％ dissolved in 0.05 mmol/L sodium citrate-hydrochloric acid buffer was intraperitoneally injected in SD rats to establish DM models,and the equal volume of solvent was injected in the same way in the control rats.One month,three months and six months after injection,EB of 30 g/L was injected via rat femoral vein in the dose of 45 mg/kg.Fifteen minutes after injection of EB,the rats were sacrificed and the retinas were isolated and cut radially to prepare the flat-mounted retinas in PBS immediately and then were dried till the specimens were transparent.The specimens were examined under the fluorescence microscope.The percentage of EB leakage was quantitatively calculated by IPP 6.0 software.All procedures were performed following approval of the institutional animal care and use committee of Tianjin Medical University.Results The retina morphology was normal in the control group,and EB filled the vessels,exhibiting the red fluorescence under the fluorescence microscope.Compared with the control group,retinal background fluorescence was enhanced slightly in the DM 1-month group,and focal leakage of the EB from capillaries and focal dilated vessels were found in the DM 3-month group,further,vascular caliber inequality,retinal hypoperfusion area and a larger number of hyperfluorescence areas were seen in the DM 6-month group.The percentage of leakage area was (0.05 ±0.02) ％,(0.27 ±0.06) ％,(1.17 ±0.18)％ and (4.77 ±0.66)％ in the control group,DM 1-month group,DM 3-month group and DM 6-month group,respectively,showing a significant difference among the four groups (F =795.800,P＜0.001),and the leakage area was obviously larger in the DM 3-month group and DM 6-month group than that in thecontrol group (q'=10.338,q'=43.475,both at P＜0.001).Conclusions Modified EB-perfused retinal wholemount method is easy and helpful for clear visualization of retinal vessel leakage induced by BRB breakdown in the diabetic rats under the common fluorescence microscope.
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Background The retinal degeneration 11 (rd11) mouse is a newly discovered naturally occurring recessive animal model with lysophosphatidylcholine acyltransferase 1 (Lpcatl) mutation.Previous studies showed that the photoreceptor cells are characterized by typical rod-cone degeneration pattern in rd1 1 mice,while cone degeneration pattern in rd11 mice is unclcar.Objective Using immunofluorescence staining techniques with retinal wholemount,we aim to clarify the degeneration patterns of cone-function related M-opsin or S-opsin in different ages of rd1 1 mice.Methods A total of thirty rd1 1 and C57BL/6J mice at postnatal (P) day 14,28,42 (five in each age group) were sacrificed and retinal wholemounts were prepared.Immunohistochemistry was performed to identify the expression of M-opsin or S-opsin in retinal wholemounts,which were photographed with a fluorescent microscope.Cone opsins were compared between rd1 1 retinas and age-matched normal C57BL/6J retinas by manually counting the opsin positive cone cells in different quadrants of the retinas.Results The number of M-opsin or S-opsin positive fluorescent dots in each quadrant was similar at all ages of normal C57BL/6J retina.M-opsin positive fluorescent dots in dorsal/temporal,ventral/temporal,dorsal/nasal and ventral/nasal quadrants of rdl 1 retina at P28 were (414±32),(300± 8),(324 ± 22) and (250± 20)/0.037 mm2,which were lower than the age-matched normal C57BL/6J mice (t =4.114,15.225,7.505,17.990,all at P＜0.05).At the same time the S-opsin positive fluorescent dots in P28 rd11 were (8 ±4),(175 ± 16),(74 ± 13) and (315 ±20)/0.037 mm2,with significant decrease in comparison with those in the age-matched normal C57BL/6J mice (t =8.555,17.076,21.637,13.498,all at P＜0.05).With the development of retinal degeneration in rd11 mice,the M-opsin degeneration spread from central to ventral,nasal and then to temporal and dorsal peripheral retina;and the S-opsin loss started from dorsal/temporal to ventral/nasal retina.Conclusions Most of the M-opsin and S-opsins,especially the S-opsins in rd11 mice,degenerate in 6 weeks.Retinal wholemount and cone opsin immunofluorescent staining provide a useful tool to show the cone degeneration pattern and to evaluate the therapeutic efficiency in ongoing gene therapy study.
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ObjectiveTo evaluate the value of MRI diffusion weighted imaging in localization of prostate cancer with whole-mount step section pathology.MethodsWe treated 36 patients using laparoscopic radical prostatectomy from Oct. 2009 to Jun. 2010. Patients who did not have an MRI/DWI examination or a surgical history of prostate and previous hormonal therapy were excluded, leaving 19 patients in our study. We analyzed the MRI and DWI collected before radical prostetectomy surgey and the post-surgery step section pathology made by the whole mount technique. The prostatic gland was divided into six sections. Two doctors read the MRI/DWI without knowing the patients' information and scored, using a 5 point scale, for each section. The tissue was graded according to the following scale: 1-definitely no cancer, 2-probably no cancer, 3-possible cancer, 4-probable cancer and 5-definite cancer. When the average score ≥4 the region was assumed to be the prostate cancer region by MRI or MRI/DWI.ResultsIn 19 patients with 114 regions, there were 48 (42％) prostate cancer regions approved by whole-mount step section pathologically.The number of prostate cancer regions was 15 (39％), 21 (55％) and 12 (32％) in base, mid and apex parts respectively. The sensitivity and specificity of the MRI was 62.5％ and 69.7％. When considering DWI, the specificity and sensitivity was elevated to 79.1％ and 83.3％. As for the apex and mid parts, the sensitivities of MRI were a little bit low (46.7％ and 66.7％ ). But the sensitivities of localization of prostate cancer for the apex and mid of prostate were elevate to 73.3％ and 85.7％ respectively when DWI was also used.ConclusionsWith whole-mount step-section pathology, the combination of MRI and DWI can improve the sensitivity of MRI for localized diagnosis in prostate cancer, especially in apex and mid parts of the prostate.
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Objective: To explore the molecular evolution of zebrafish rnf141 gene and its expression in the embryo and adult zebrafish. Methods: The molecular characteristics of zebrafish rnf141 gene were analyzed through a bioinformatics approach. Expression of rnf141 gene in the embryos of different developmental stages and tissues of adult fish were examined by whole-mount in situ hybridization and multiple-tissue RT-PCR technique. Results: We noticed that zebrafish RNF141 protein shared a 79.8% and 78.5% homologies to its homologues in human and mouse (ZNF230, Znf230), respectively, especially that the C3HC4-type domain and the same conserved cysteine and histidine residues shared a 84.8% and 100% homologies to its homologues in human or mouse. Moreover, the secondary structure of C3HC4-type domain in zebrafish RNF141 protein was the same as that in its human homologue. Whole-mount in situ hybridization showed that zebrafish rnf141 gene expressed ubiquitously during embryogenesis, with higher expression found in the telencephalon, cerebellum and hindbrain. RT-PCR revealed rnf141 gene expression in all the adult tissues detected, and abundance was observed in the testicular tissue. Conclusion: These data suggest that zebrafish rnf141 gene is conservative in the vertebrate and might play important roles in embryogenesis and spermatogenesis.
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PURPOSE: Recently, magnetic resonance imaging (MRI) has become the preferred diagnostic tool for preoperative assessment of TNM staging and circumferential resection margin (CRM) in patients with rectal cancer. The aim of this study is to evaluate the accuracy of preoperative MR imaging in the prediction of T, N stage and CRM compared with pathologic results on whole- mount sections. METHODS: Thirty-five consecutive patients with rectal cancer were enrolled between Dec. 2005 and Apr. 2006. 1.5-T MR imaging, was performed, and pathologic results were investigated on whole-mount sections. The agreement between MR imaging and pathologic examination for the assessment of T, N stage and status of CRM were analyzed using kappa statistics. RESULTS: The accuracy of MR imaging compared with pathologic assessment of T stage was 82.9% (kappa=0.56), and that of N stage was 74.3% (kappa= 0.31). Of the MR imaging planes, the oblique axial plane showed the most accurate prediction of CRM, regardless of tumor position within the circumference of the rectum. The accuracy of MR imaging in the oblique axial plane for predicting the CRM was 81.0% (kappa=0.62) in anterior and posterior rectal tumors and 71.4% (kappa=0.43) in laterally located rectal tumors. With a different CRM criteria for the measured distance in MR imaging, the accuracy of the 2-mm CRM criterion was 77.1% (kappa=0.53). CONCLUSIONS: MR imaging in predicting T stage showed fair agreement according to kappa statistics. Of the MR imaging planes, the oblique axial plane provided the most accurate CRM information compared with pathologic examination. The actual measured distance of the CRM in MR imaging can be applied to the pathologic CRM.
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Humans , Magnetic Resonance Imaging , Neoplasm Staging , Rectal Neoplasms , RectumABSTRACT
PURPOSE: There were many studies on the distributions of the retinal ganglion cells(RGC) in the experimental model of the retinal ischemia. RGC was known to be more sensitive to the ischemic injury than the other types of the retinal cells. So, we would identify the changes of the retinal ganglion cell morphologies and distribution after the iatrogenic retinal ischemia induced by intraocular pressure(IOP) elevation. METHODS: Eight pigmented and six white rabbits were used and retinal ischemia was induced by increasing IOP higher than 120 mmHg for 60 minutes. Electroretinogram were recorded at 6 days or 13 days, and histologic findings were observed at 7 or 14 days. RESULTS: After 7 days, RGC densities decreased, cytoplasmic staining disappeared, and the intranuclear hyperpigmentation was noted. RGC densities decreased significantly at 14 days. In the vertical retinal section, some flattening of retinal ganglion cell layer and inner plexiform layer was observed. Changes in the cellular morphologies were prominent. CONCLUSIONS: It may be more appropriate to examine both the retinal whole-mount and the vertical tissue section for the estimatation of the changes of retinal ganglion cell layer in the pressure-induced retinal ischemia.
Subject(s)
Rabbits , Cytoplasm , Ganglion Cysts , Hyperpigmentation , Ischemia , Models, Theoretical , Retina , Retinal Ganglion Cells , RetinaldehydeABSTRACT
Acute anterior uveitis was induced by footpad injection of bacterial lipopolysaccharide(LPS) in the Lewis rats. To evaluate the distribution and density of MHC class II positive cell, macrophages, B lymphocytes infiltration in the iris and ciliary body 24 hours after injection of bacterial LPS, immunohistochemical study was performed in the wholemount tissues with monoclonal antibodies. Quantitative analysis reveals that the density of MHC class II positive dendritic cells in the iris was 348.5+/-55cells/mm2 in the control group and 448.0+/-176cells/mm2 in the LPS injected group(p<0.05). The density of ED2 positive resident tissue macrophages was higher in the LPS injected group(761.9+/-82cells/mm2) than the control group(620.8+/-57cells/mm2)(p<0.05). ED1 positive macrophages infiltrated significantly in the LPS results in increased group(3225.0+/-522cells/mm2) than in the control group(590.5+/-52cells/mm2).OX33 positive cells were not observed in both control and LPS injected eyes. In conclusion, destruction of blood ocular barrier by infection of LPS results in increased infiltration of OX6 positive cells and macrophages, particularly massive influx of blood monocytes into the iris and ciliary body in acute phase of inflammation. This study confirmed that dendritic cells and macrophages play important roles in acute phase of endotoxin induced uveitis.