Background To optimize the culture method of human retinal microvascular endothelial cells is very important for the study of retinal angiogenesis disease.Human retinal microvascular endothelial cells have been successfully cultured in previous studies,but further improvement of the culture method to harvest higher yields and purity cells is still needed.Objective This study was to design a modified method to isolate and purify human retinal microvascular endothelial cells much easily and quickly,and to compare the expression of specific markers of vascular endothelial cells,factor Ⅷ and CD31/CD34 in the cells.Methods The use of human donor eyeballs was approved by the Ethic Commission of Zhongshan Ophthalmic Center of Sun Yat-sen University.The retina tissue from healthy donor was isolated and digested by the two-step digestion method with 2％ trypsin and 0.133％ collagenase Ⅳ.Human retinal microvascular endothelial cells were collected and plated in 60 mm dishes coated by 0.1％ fibronectin and cultured in endothelial cell-specialized medium supplemented with 10％ fetal bovine serum,0.3 mg/L β-endothelial cell growth factor (ECGF) and 100 ng/L sodium heparin.During the culturing,the growth situation of the cells was monitored by morphological observation,and immunohistochemical staining was performed to probe vascular endothelial cell-specific membrane protein CD31,CD34 and factor Ⅷ for identification of the cell purity.Results Human retinal microvascular endothelial cells were isolated successfully from the retina by the twostep digestion method.The primary cultured cells adhered to well 72 hours later and achieved confluence with the typical cobblestone appearance 9 to 10 days after cultured.The cells exhibited the blue nuclei and reddish cytoplasm by regular haematoxylin and eosin stain and showed a strong positive response for CD31,CD34 and factor Ⅷ by immunohistochemistry.The positive dye of CD31 and CD34 was lower than Ⅷ factor in both endothelial cells.Conclusions Modified culture method of human retinal microvascular endothelial cells can improve cell culture result and purify target cells.

BackgroundHuman retinal gliocytes play an important role in proliferative diseases,which are the basis of in vitro studies.Researchers have cultured human retinal gliocytes in the past.In our study,we found that the cells we cultured presented a unique shape different from those by other researchers.ObjectiveThis study was to design to produce a new culture and purification method for retinal gliocyte in vitro.Methods Retinal tissue was isolated from human eyeballs and digested using the two-step digestion method (2％ pancreatin and 0.133％collagenase Ⅵ) to harvest the retinal glio cytes.The cells were collected and cultured in endothelial cell-targeted nutrient culture containing 10％ fetal calf serum and supplemented with β-endothelial cell growth factor (ECGF) and sodium heparin,and the culture dishes were coated with fibronectin(FN) to promote the attachment of retinal gliocyte.During the culturing process,the gliocytes were identified by the observation of morphological characteristic and regular histological examination.The identification of the cells also was performed by immunochemistry targeting glial fibrillary acidic protein (GFAP),Vimentin,neuron specific enolase ( NSE ),S-100,CD34,and Ⅷ factor.Results Retinal gliocytes were isolated successfully from the human retina by the two-step digestion method.Primary cultured cells attached after 72 hours and achieved confluency between day 9 and 10 that were aligned petaliform in shape.Regular histological examination after H&E staining showed blue cell nuclei and light red cytoplasm.The target cells presented with strong responses for GFAP and Vimentin and no response for NSE,S-100,CD34 and Ⅷ factor.ConclusionsLarge amount of purified human retinal gliocytes can be obtained by two-step digestion and cultured in endothelial cells-targeted culture medium supplemented with β-ECGF and sodium heparin in plates coated with FN.The cultured cells expressed markers for retinal gliocytes.However,specific features of these cells remain to be further elucidated.

AIMTo explore an accurate neurophysiological technique that demonstrates small functional differences after spinal cord injury and assesses therapeutic interventions.

METHODSA modified weight drop (WD) technique was used at T8 in rats to build graded spinal cord injury model. Rubrospinal MEPs were recorded at T13 epidurally to monitor spinal cord function in end week 4 after graded spinal cord injury. The efficacy of this techniques to monitor spinal cord function was compared to BBB locomotor rating scale and histologic evaluation.

RESULTSA characteristic peak complex of rubrospinal MEPs in sham-operated group consisted of 5-7 positive waves and 4-5 negative waves emerging after red nucleus stimulation. The summed peak to peak amplitude (for practical reasons, called peak amplitude) was (195.25 +/- 34.35) microV and decreased following spinal cord injury. The latency of the first peak (positive wave) was (1.57 +/- 0.15) ms and prolonged following spinal cord injury. Significant Linear relationship existed between the peak amplitude and the BBB scores (r = 0.79) and between the peak amplitude and the residual matter obtained from the section with maximum tissue damage( r = 0.87). The close relationship between the latency of the first peak and the BBB scores (r = -0.88) and between the latency of the first peak and residual matter (r = -0.86) were observed.

CONCLUSIONAmplitudes and latencies of rubrospinal MEPs are very valuable parameters to demonstrate small function differences. Rubrospinal MEPs can be used as a reliable measure for motor function prognosis after spinal cord injury.

Animals ; Evoked Potentials, Motor ; Male ; Rats ; Rats, Wistar ; Red Nucleus ; physiopathology ; Spinal Cord Injuries ; pathology ; physiopathology

Objective To explore inhibition of amlodipine on myocar-dial cell injury induced by ischemia reperfusion .Methods Thirty Wistar rats were randomly into three groups: sham operation group ( n =10 ) , model group group ( n =10 ) , test group ( 2 mg? kg -1 amlodipine , n=10).The model group and test group were made by ligation of left anterior descending coronary artery to make the model of myocardial ischemia reperfusion.Rats in each group were administered 7 d before ligation.Cell apoptosis was examed by flow dual staining method .The activity of cysteinyl aspartate specific proteinase 3 ( Caspase 3 ) was measured by spectrophotometry .The activation of phosphatidylinositol 3 kinase ( PI3K)/protein kinase B ( AKT) signalling pathway, B-cell lymphoma-2 ( Bcl-2 ) , Bcl-2 associated X protein ( Bax ) were assayed by Western blot .Results Compared to sham operation group on early and late myocardial cell apoptosis , myocardial cell apoptosis with ( 2.34 ±0.35 )%, (3.58 ±0.39 )%, that on early and late myocardial cell apoptosis were increased with ( 15.69 ±1.14 )%, (24.74 ±2.56)%in model group ( P <0.05 ) .Compared to sham operation group on the activity of Bax with (0.18 ±0.01) and Caspase 3 activity with (1.00 ±0.10), the expression of Bax express with (0.62 ±0.06) and Caspase 3 activity with (3.98 ±0.18) in model group were increased (P<0.05).Compared to sham operation group on the expre-ssion of Bcl-2 with (0.99 ±0.10 ) and expression of PI3K with (0.89 ±0.06 ), on the expression of Bcl-2 with (0.14 ±0.01) and expression of PI3K with (0.18 ±0.01) in model group were decreased (P<0.05). Compared to sham operation group on phosphorylation of AKT with ( 0.95 ±0.10 ) , the phosphorylation of AKT in model group with (0.13 ±0.01 ) was decreased ( P<0.05 ).Compared with model group , test group could change the variation on the early and late myocardial cell apoptosis with ( 5.23 ±0.13 )%, ( 8.09 ±0.35 )% while on the Caspase 3 activity with ( 1.47 ±0.14 ) ( P<0.05 ) .Conclusion These results suggested that amlodipine inhibited myocardial ischemia reperfusion injury, which was related to activition PI3K/AKT signal pathway.

OBJECTIVETo determine whether tanycytes be able to support the regeneration of completely transected spinal cord in adult rats.

METHODSSubcultured tanycytes was transplanted into completely T8 transected spinal cord using the untranslated completely transected rats as control. After transplantation the rubrospinal motor evoked potentials were recorded below the injury level at the end of 12th week, assistant by Basso-Beatie-Bresnahan locomotor rating scale and histology method.

RESULTSAt the end of 12th week the total peak amplitude of rubrospinal motor evoked potentials (MD = 133.2 microV, P < 0.01) and BBB locomotor rating scale (MD = 5.0000, P < 0.01) were both significantly improved in cell transplanted group compared with that in the untranslated control group, while the latency of the first peak was shortened (MD = 0.061 ms, P = 0.040). HE staining showed more integrity in transected spinal cords in cells transplanted groups.

CONCLUSIONTransplanted tanycytes can support the regeneration of transected spinal cords in rats.

Animals ; Cell Transplantation ; Cells, Cultured ; Evoked Potentials, Motor ; Male ; Neuroglia ; cytology ; transplantation ; Rats ; Rats, Wistar ; Recovery of Function ; Spinal Cord Injuries ; physiopathology ; surgery

Objective: Several COVID-19 patients have overlapping comorbidities. The independent role of each component contributing to the risk of COVID-19 is unknown, and how some non-cardiometabolic comorbidities affect the risk of COVID-19 remains unclear. Methods: A retrospective follow-up design was adopted. A total of 1,160 laboratory-confirmed patients were enrolled from nine provinces in China. Data on comorbidities were obtained from the patients' medical records. Multivariable logistic regression models were used to estimate the odds ratio ( Results: Overall, 158 (13.6%) patients were diagnosed with severe illness and 32 (2.7%) had unfavorable outcomes. Hypertension (2.87, 1.30-6.32), type 2 diabetes (T2DM) (3.57, 2.32-5.49), cardiovascular disease (CVD) (3.78, 1.81-7.89), fatty liver disease (7.53, 1.96-28.96), hyperlipidemia (2.15, 1.26-3.67), other lung diseases (6.00, 3.01-11.96), and electrolyte imbalance (10.40, 3.00-26.10) were independently linked to increased odds of being severely ill. T2DM (6.07, 2.89-12.75), CVD (8.47, 6.03-11.89), and electrolyte imbalance (19.44, 11.47-32.96) were also strong predictors of unfavorable outcomes. Women with comorbidities were more likely to have severe disease on admission (5.46, 3.25-9.19), while men with comorbidities were more likely to have unfavorable treatment outcomes (6.58, 1.46-29.64) within two weeks. Conclusion Besides hypertension, diabetes, and CVD, fatty liver disease, hyperlipidemia, other lung diseases, and electrolyte imbalance were independent risk factors for COVID-19 severity and poor treatment outcome. Women with comorbidities were more likely to have severe disease, while men with comorbidities were more likely to have unfavorable treatment outcomes.
Adult ; Aged ; COVID-19/virology* ; China/epidemiology* ; Comorbidity ; Female ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Severity of Illness Index ; Treatment Outcome