Comparison of conventional and directional freezing for the cryopreservation of human umbilical vein endothelial cells

AIM:To compare conventional slow equilibrium cooling and directional freezing(DF) by gauze package for cryopreservation of human umbilical vein endothelial cells(HUVECs).METHODS:HUVECs were randomly assigned to conventional freezing(CF) and DF by gauze package group. The two groups of HUVECs were incubated with a freezing liquid consisting of 10% dimethylsulfoxide(DMSO), 60% fetal bovine serum(FBS) and 30%Dulbecco’s modified Eagle’s medium(DMEM) and then put into cryopreserved tubes. CF group, slow equilibrium cooling was performed with the following program:precool in 4℃ for 30 min,-20℃ for 1h, and then immersion in-80℃ refrigerator. DF group, the tubes were packaged with gauze and then directional freezing in-80℃ refrigerator straightly. One month later, the vitality of HUVECs were calculated between two groups.RESULTS:There was no significant difference in the survival rate and growth curve between CF and DF groups. The DF group was significantly better than CFgroup in adherent rates, morphological changes and proliferative ability.CONCLUSION:In the conventional cryopreserved method, cells are slow equilibrium cooling by steps(4℃,-20℃ and finally-80℃), which is a complicated and time-consuming process. But the improved DF by gauze package method is better than conventional method, for which is convenient and easy to operate.

Extracellular signal regulated kinases 1/2 signal pathway and responses of astrocytes after diffuse brain injury

BACKGROUND： The treatment of diffuse brain injury during an acute period is focused on relieving degrees of secondary brain injury. Generation and development of pathological changes of secondary brain injury depend on signal conduction, so down-regulating over response of astrocyte through interfering a key link of signal conduction pathway may bring a new thinking for the treatment of diffuse brain injury. OBJECTIVE： To observe the effect of over activity of extracellular signal regulated kinases 1/2 （ERK1/2） signal pathway on the response of astrocyte during an acute period of diffuse brain injury. DESIGN： Completely randomized grouping and controlled animal study. SETTINGS： Department of Neurosurgery, the Third Affiliated Hospital, Nanchang University; Department of Neurosurgery, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology. MATERIALS： A total of 158 healthy male SD rats, of 11 weeks old, weighing 320-370 g, were provided by Experimental Animal Faulty, Tongji Medical College, Huazhong University of Science and Technology. Rabbit-anti-phosphorylated ERK1/2 （pERK1/2） polyclonal antibody was provided by R＆D Company; rabbit-anti-glial fibrillary acidic protein （GFAP） polyclonal antibody, SP immunohistochemical kit and horseradish peroxidase （HRP）-labeled goat-anti-rabbit IgG by Santa Cruz Company; specific inhibitor U0126 of ERK1/2 signal pathway by Alexis Company. METHODS： The experiment was carried out in the Laboratory of Neurosurgery, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology from September 2004 to March 2006. ① Detection of pERK1/2 expression： A total of 110 rats were randomly divided into sham operation group （n =5）, model group （n =35）, high-dosage U0126 group （n =35） and low-dosage U0126 group （n =35）. Rats in the sham operation group were only treated with incision of epicranium and fixation of backup plate, but not hit. Rats in the model group were used to establish diffuse brain injury models based on Marmarou free falling body without drug intervention. Rats in the high- and low-dosage U0126 groups were injected into caudal vein with 0.1 and 0.05 mg/kg U0126, respectively, and then, rats were hit to establish injured models. Every 5 rats were collected from model, high- and low-dosage U0126 groups at 5, 30 minutes, 3, 12, 24, 72 hours and 7 days after diffuse brain injury to detect pERK1/2 expression in cortex of parietal lobe based on Western blot technique. ② Distribution of pERK1/2 and positive GFAP cells in brain tissue： Another 48 rats were randomly divided into sham operation group （n =3）, model group （n =15）, high-dosage U0126 group （n =15） and low-dosage U0126 group （n =15）. The intervention and administration were dealt as the same as those mentioned above. Every 3 rats were collected from model, high- and low-dosage U0126 groups at 30 minutes, 3, 12, 24 and 72 hours after model establishment to observe the distribution of pERK1/2 and postive GFAP cells in brain tissue which was cut from coronal section at Bregma -4.8 mm layer with immunohistochemical staining. MAIN OUTCOME MEASURES： pERK1/2 expression in cortex of parietal lobe and distribution of pERK1/2 and positive GFAP cells in brain tissues. RESULTS： ① pERK1/2 expression： After diffuse brain injury, pERK1/2 expression in cortex of parietal lobe was rapidly increased in the model group, reached at peak at 5 minutes and then decreased gradually. But the expression was still in a high level until the 72nd hour and fallen to the basic level on the 7th day. pERK1/2 level was lower in high- and low-dosage U0126 groups than that in model group at various time points （P 〈 0.01）; meanwhile, pERK1/2 level was lower in high-dosage U0126 group than that in low-dosage U0126 group. The results showed that there was a certain dosage dependence on pERK1/2 expression. ② Distribution of pERK1/2 and positive GFAP cells in brain tissue： Positive expression of pERK1/2 lasted inbrain tissue from 30 minutes to 72 hours after diffuse brain injury （P 〈 0.05）. In addition, from 30 minutes to 3 hours, brown-yellow stained cells were mainly distributed in plasma, but rarely in nucleus. A lot of positive cells had tree-like apophysis, which was similar to neurons. With the time passing by, more and more nuclei manifested positive stains; moreover, nuclei mainly manifested positive staining until 24 hours after diffuse brain injury. Immune-positive pERK1/2 cells were widely distributed in brain tissue, especially mainly in binding site between deep cortex and cerebral white matter, and then in hippocampus. In addition, ependymal cell and vascular endothelial cells of choroids plexus also manifested strongly positive staining. As compared with model group, positive cells were decreased gradually in high- and low-dosage U0126 groups. However, number of positive cells was less in high-dosage U0126 group than that in low-dosage U0126 group. CONCLUSION： Diffuse brain injury strongly induces the activity of ERK1/2 signal pathway and response of astrocyte; in addition, U0126 can inhibit response of glial cells during an acute period, and the effect manifests dosage dependence.

Relationship between contrast sensitivity and corneal shape following overnight orthokeratology

AIM: To evaluate the relationship between contrast sensitivity(CS) and corneal shape following overnight orthokeratology(OK). METHODS: We conducted a retrospective clinical study of 80 lens-wearing myopia patients, all of whom had undergone OK and had been evaluated by Orbscan II topography. We measured the surface irregularity index(SIRI) of corneal topography at 3 and 5 mm, the size of the flattened central corneal curvature of OK lens(zone A), the size of the cornea altered by OK lens(zone B), the size of the pupillary area at the corneal level(zone C), the area of crossover between zones A and C(zone AC), the area of crossover between zones B and C(BC), the ratio of BC to B(BC/B), and the ratio of AC to C(AC/C). CS was evaluated using the CSV-1000 with spatial frequencies of 3, 6, 12, and 18 cycles/degree(CPD). RESULTS: Multiple correlation analyses indicated significant negative correlations between CS, zone C, BC/B, and 3-mm SIRI(all P<0.01). There were no significant differences between CS, zone B, AC/A, or 5-mm SIRI(P=0.60, 0.94 and 0.11, respectively). Zone C was negatively correlated with 3, 6, 12, and 18 CPD. 5-mm SIRI were negatively correlated with 6, 12, and 18 CPD. BC/C was negatively correlated with 6 and 18 CPD. AC/C was positively correlated with 3 CPD. CONCLUSION: Zone C, 3-mm SIRI and BC/B affect the CS following overnight OK.

Exosomes in Sepsis Diagnosis and Treatment

Sepsis has been redefined as a disorder of host response to infection, systemic circulation and cell/metabolic abnormalities. Exosomes are small (30 - 150 nm) vesicles produced by all cells under physiological and pathological conditions, with the potential to transfer proteins, lipids, small RNAs, messenger RNAs, or DNA between cells. Exosomes are natural cargoes for proteins, carbohydrates, nucleic acids and lipids. Exosomes play a central role in cellular communication and contribute to many pathophysiological processes, including immune responses and tumor progression. Exosomes have made great progress in many subject areas, and their potential role in sepsis is now being explored. In this review, several topics are mentioned. Firstly, we discuss the biological characteristics and functions of exosomes. Next, we focus on the diagnostic and therapeutic potential of exosomes in sepsis. Finally, we discuss some of the problems encountered by the current exosomes research institute. Therefore, the exosomes with combined diagnostic and therapeutic functions play a huge clinical application for the future research in sepsis.

Change in foveal position based on age and axial length in high myopic eyes

AIM：To evaluate the influence of age and axial length（AL） on the position of the fovea in patients with high myopia（HM）.METHODS：In this prospective study,96 patients（186 eyes） with HM were consecutively recruited from the Third Affiliated Hospital of Nanchang University.DRI-OCT Atlantis,fundus imaging,and IOL Master were used in this study.Three indices were measured：the distance between the fovea and the optic nerve head（ONH） center（DFO）,the vertical distance between the fovea and the horizontal line pass of the ONH center（VDFO）,and the horizontal distance between the fovea and the vertical line pass of the ONH center（HDFO）.These measurements were used to analyze the effects of different age groups（A1,A2,A3 groups） and AL（AL1,AL2,AL3,AL4 groups） on these indices.RESULTS：The results showed that there was no statistical significance in DFO among the age and AL groups（F=0.46,0.37;P=0.62,0.76,respectively）.In HDFO,there was also no statistical significance among the age and AL groups（F=0.10,0.48;P=0.90,0.69,respectively）.In VDFO,however,the difference in the age and AL groups was statistically significant（F=3.21,3.12;P=0.04,0.02,respectively）.Thus,VDFO were correlated with age and AL（r=0.21,0.23,all P〈0.01）,while HDFO and DFO were not correlated with age and AL（r=0.30,P〉0.05）.CONCLUSION：In high myopia,the foveal position changes mainly in the vertical direction along with factors of age and AL.

Clinical and neuroradiological features of 15 patients diagnosed with Marchiafava-Bignami disease

We retrospectively reviewed the clinical data of 15 patients with MBD who visited at The Affiliated Hospital of Jiujiang University and The Third Affiliated Hospital of Nanchang University between January 2014 and December 2018.The diagnosis of MBD was made based on the following criteria:(1) a history of chronic alcoholism or malnutrition;(2) acute or sub-acute onset of neuropsychiatric symptoms;(3) symmetrical hyperintensity of the CC on T2-weighted and FLAIR images;(4) exclusion of other diseases including Wernicke encephalopathy (WE),reversible splenial lesion syndrome,intra-cranial infections,cerebrovascular diseases,and demyelinating diseases of the central nervous system.Clinical data,including demographics (gender and onset age),risk factors,types of onset,clinical presentations,MRI findings,treatment and outcome,were collected.At the time of follow-up (time range:1-2 months),the treatment outcomes were classified as complete,partial,and bad according to the objective improvement in neurological findings during drug therapy (death,apathetic state,or vegetative state was considered as a bad outcome).

Snail promotes metastasis of nasopharyngeal carcinoma partly by down-regulating TEL2

Background:Metastasis is the major cause of treatment failure in patients with nasopharyngeal carcinoma(NPC).We previously reported that TEL2,a negative regulator of SERPINE1,could inhibit NPC metastasis to lymph nodes.Method:A series of in vivo and in vitro assays were performed to elucidate the regulation between Snail and TEL2.TEL2 expression was analyzed in three representative NPC cell lines expressing low levels of Snail(S26,6-10B,HK1)and two cell lines expressing high levels of Snail(S18,5-8F).Luciferase and chromatin immunoprecipitation assays were used to analyze the interaction between Snail and TEL2.The roles of the Snail/TEL2 pathway in cell migration and invasion of NPC cells were examined using transwell assays.Metastasis to the lungs was examined using nude mouse receiving NPC cells injection through the tail vein.Results:Ectopic Snail expression down-regulated TEL2 at the mRNA and protein levels,whereas knockdown of Snail using short hairpin RNA up-regulated TEL2.Luciferase and chromatin immunoprecipitation assays indicated that Snail binds directly to the TEL2 promoter.Ectopic Snail expression enhanced migration and invasion of NPC cells,and such effects were mitigated by TEL2 overexpression.TEL2 overexpression also attenuated hypoxia-induced cell migration and invasion,and increased the number of metastatic pulmonary nodules.Snail overexpression reduced the number of metastatic pulmonary nodules.Conclusions:TEL2 is a novel target of Snail and suppresses Snail-induced migration,invasion and metastasis in NPC.

Quantitative comparisons between relaxation enhanced compressed sensing 3D MERGE and conventional 3D MERGE for vessel wall imaging in equal scan time: preliminary studies

In this study, we quantitatively compared relaxation enhanced compressed sensing(RECS-3D MERGE) with conventional 3D MERGE techniques on blood suppression efficiency, wall-lumen contrast and plaque burden measurement for carotid atherosclerotic imaging in equal scan time. Twelve patients were recruited in the study. RECS-3D MERGE and conventional 3D MERGE were implemented. 2D DIR-FSE was carried out as a reference standard. The lumen signal-to-tissue ratio(STR) was used as the quantitative measure of blood suppression efficiency. The contrast-to-tissue ratio(CTR) was used as the quantitative measure of wall-lumen contrast. Vessel lumen area(LA) and wall area(WA) were measured for morphological comparisons. The lumen STR of RECS-3D MERGE was significantly lower than that of 3D MERGE while the wall-lumen CTR of RECS-3D MERGE was significantly higher. There were no significant differences in plaque burden measurements between RECS-3D MERGE and 2D DIR-FSE. For comparison between conventional 3D MERGE and 2D DIR-FSE, there were no significant differences in LA measurement. However, the WA of 3D MERGE was significantly larger. The RECS-3D MERGE sequence achieved more sufficient blood suppression and higher image contrast without prolonging the scan time. These improvements lead to more accurate morphological measurements of carotid atherosclerotic imaging.