Effect of supplementation of cryoprotectant solution with hydroxypropyl cellulose for vitrification of bovine oocytes.

BACKGROUND: Successful cryopreservation of bovine oocytes is very important for research and commercial applications. However, the survival and development rate of vitrified-thawed (VT) oocytes are lower than those of non-vitrified-thawed (non-VT) oocytes. OBJECTIVE: To investigate the effect of adding hydroxypropyl cellulose (HPC) to the vitrification solution for bovine oocytes. MATERIALS AND METHODS: For vitrification, bovine metaphase II oocytes were pretreated with a solution containing 10% ethylene glycol supplemented with 0, 10, 50, or 100 ug/mL HPC for 5 min, exposed to a solution containing 30% ethylene glycol supplemented with 0, 10, 50, or 100 ug/mL HPC for 30 s, and then directly plunged into liquid nitrogen. RESULTS: The survival rate of oocytes was significantly higher in the 50 HPC group than in the 0, 10, and 100 HPC groups. The reactive oxygen species level was lower in the non-VT and 50 HPC groups than in the other groups. The mRNA levels of proapoptotic genes (Bax) were lower in the non-VT, 0, and 50 HPC groups than in the other groups. The mRNA levels of antiapoptotic genes (BCl2) were higher in the non-VT than in the other groups. The development rates of embryos (day 8) obtained via parthenogenetic activation (PA) were determined in the non-VT, 0 HPC, and 50 HPC groups. The cleavage rate was significantly higher in the non-VT group. CONCLUSION: Supplementation of vitrification solution with HPC improves the survival of VT bovine oocytes and the development capacity of embryos derived from these oocytes via PA. doi.org/10.54680/fr23110110212.

Impact of time-to-reperfusion on outcome in patients with poor collaterals.

BACKGROUND AND PURPOSE: The relationship between reperfusion and clinical outcome is time-dependent, and the effect of reperfusion on outcome can vary on the basis of the extent of collateral flow. We aimed to identify the impact of time-to-reperfusion on outcome relative to baseline angiographic collateral grade in patients successfully treated with endovascular revascularization for acute large-vessel anterior circulation stroke. MATERIALS AND METHODS: Two hundred seven patients were selected for analysis from our prospectively maintained registry. Inclusion criteria were M1 MCA ± ICA occlusions, onset-to-puncture time within 8 hours, and successful endovascular reperfusion. Baseline angiographic collateral grades were independently evaluated and dichotomized into poor (0-1) versus good (2-4). Multivariable analyses were performed to identify the effect of collateral-flow adequacy on favorable outcome on the basis of onset-to-reperfusion time and puncture-to-reperfusion time. RESULTS: In the poor collateral group, the odds of favorable outcome significantly dropped for patients with onset-to-reperfusion time of >300 minutes or puncture-to-reperfusion time of >60 minutes (onset-to-puncture time: ≤300, 59% versus >300, 32%; OR, 0.24; P = .011; puncture-to-reperfusion time: ≤60, 73% versus >60, 32%; OR, 0.21, P = .011), whereas the probability of favorable outcome in the good collateral group was not significantly influenced by onset-to-reperfusion time or puncture-to-reperfusion time. In the subgroup lesion-volume growth analysis by using DWI, the effect of puncture-to-reperfusion time of >60 minutes was significantly greater compared with the effect of puncture-to-reperfusion time of <60 minutes in the poor collateral group (ß = 41.6 cm(3), P = .001). CONCLUSIONS: Time-to-reperfusion including onset-to-reperfusion time and puncture-to-reperfusion time in patients with poor collaterals is an important limiting factor for favorable outcome in a time-dependent fashion. Future trials may benefit from a noninvasive imaging technique to detect poor collaterals along with a strategy for rapid reperfusion.

Predictors for the progression of geographic atrophy in patients with age-related macular degeneration: fundus autofluorescence study with modified fundus camera.

PURPOSE: We examined the association between abnormal fundus autofluorescence (FAF) features on images obtained by a modified fundus camera (mFC) and geographic atrophy (GA) progression in patients with age-related macular degeneration (AMD). METHODS: Serial FAF images of 131 eyes from 131 patients with GA were included in the study. All FAF images were obtained with an mFC (excitation, ∼ 500-610 nm; emission, ∼ 675-715 nm). The GA area was quantified at baseline and 1 year later using a customized segmentation program. The yearly GA enlargement rate was then calculated. Abnormal FAF patterns in the junctional zone of GA were classified as None or Minimal change, Focal, Patchy, Banded, or Diffuse according to previously published classification based on confocal scanning laser ophthalmoscopy (cSLO). The relationship between GA enlargement and abnormal FAF was evaluated. RESULTS: The mean rate of GA enlargement was the fastest in eyes with Diffuse pattern (1.74 mm(2) per year), followed by eyes with the Banded pattern (1.69 mm(2) per year). Binary logistic regression analysis revealed that eyes with the Banded and Diffuse pattern had significantly higher risk for GA enlargement compared with eyes with the other patterns. CONCLUSIONS: FAF image obtained by mFC appears to be acceptable for evaluating GA in accordance with an established cSLO-based classification. Eyes with the Banded or the Diffuse patterns of abnormal FAF at baseline indicate a high risk for GA progression. Identifying patients at high risk for GA progression using an mFC is broadly available method that can provide additional information to help predict disease course.

Diagnostic efficacy of total homocysteine and C-reactive protein for ocular ischemic syndrome.

BACKGROUND: To assess diagnostic efficacy of plasma total homocysteine (tHcy) and C-reactive protein (CRP) levels for ocular ischemic syndrome (OIS). METHODS: In all, 87 patients with retinal vein occlusion (RVO), 955 patients with a stenosis of internal carotid artery (ICA) <90% and 159 patients with a stenosis of ICA >90% were included between 2003 and 2009. A total of 43 patients with a stenosis ICA >90% were diagnosed as OIS. Fasting tHcy, CRP, lipid profiles, creatinine were measured, and diagnostic values of hyperhomocysteinemia or elevated CRP for OIS were evaluated. RESULT: The mean plasma levels of tHcy (18.8 µmol/l) and CRP (1.1 mmol/l) were the highest in patients with OIS among the groups. The prevalences of hyperhomocysteinemia (72%) and elevated CRP (77%) were the highest in OIS among the groups. In patients with stenosis of ICA, the diagnostic sensitivity/specificity for OIS was 70/79% in hyperhomocysteinemia and 73/73% in elevated CRP. The diagnostic sensitivity and specificity for OIS were 53 and 86% in both hyperhomocysteinemia and elevated CRP. The lipid profiles and creatinine levels were similar among the groups. CONCLUSION: Our results suggest that hyperhomocysteinemia and elevated CRP may be associated with the development of OIS. The measurements of tHcy and CRP in blood may help to assist the diagnosis of OIS in a stenosis of ICA.

EBV specific antibody-based and DNA-based assays in serologic diagnosis of nasopharyngeal carcinoma.

We assessed 5 EBV specific assays for their capacity to effect serologic diagnosis of suspected NPC. The assays were the immunofluorescent assays, VCA IgA and EA IgA, the enzyme-linked immunosorbent assays specific for EBNA 1 IgA or zta IgG and an EBV DNA assay. Serum samples were taken from 218 symptomatic NPC patients presenting consecutively at a public hospital in Hong Kong, 51 of whom were subsequently diagnosed as having NPC; 4 had EBV-associated lung cancer with similar serology as NPC. The remaining patients included 23 who had other cancers and 140 who had other diseases. Objectives of serodiagnosis under such clinical settings, therefore, are to both exclude and predict a diagnosis of NPC. None of the assays individually can meet both requirements adequately, however. The difficulty was best overcome by combining EBNA 1 IgA and zta IgG. It was shown that 68.3% of the patients gave a confirmed test results, negative or positive, by both tests. A confirmed negative result was associated with a negative predictive value of 99.1%, providing a clear indication to exclude a diagnosis of NPC; a confirmed positive result was associated with a positive predictive value of 86.8%, providing a clear indication to proceed with diagnostic work-up of NPC. The remaining patients gave equivocal test results, being positive for one or the other test, which were associated with a positive predictive value of 43.3% and 24.2%, respectively.

Pregnancies resulting from in vitro matured oocytes collected from women with regular menstrual cycle.

PURPOSE: To demonstrate that human immature oocytes retrieved from women with regular menstrual cycles can undergo maturation and fertilization, and that the resulting embryos can establish pregnancies. METHODS: Immature oocytes (n = 568) were retrieved from women with regular menstrual cycle. The intact immature oocytes (n = 506) were allowed to mature in YS medium supplemented with 70% human follicular fluid (hFF); the matured oocytes were fertilized with husband sperm. Two pronuclei oocytes were cocultured with cumulus cells in YS medium supplemented with 10% hFF until 2 or 3 days after insemination. The cleaved embryos were transferred in uteri. RESULTS: Follicles were aspirated on Day 9.2 +/- 5.3 of 63 natural cycles from 51 patients (mean age = 34.8 +/- 4.0 years). The average number of retrieved immature oocytes was 9.0. The maturation rate was 74.3% (376/506). The two PN and cleavage rates were 72.6% (273/376) and 89.0% (243/273), respectively. Embryo transfer was achieved in 51 cycles and clinical pregnancy rate was 17.6% (9/51). CONCLUSIONS: The results suggest that in vitro matured oocytes can undergo fertilization and the resulting embryos may successfully lead to pregnancies. However, further research is needed to improve IVM technique to achieve success rate comparable to gonadotrophin stimulated cycles.

Atherothrombotic middle cerebral artery territory infarction: topographic diversity with common occurrence of concomitant small cortical and subcortical infarcts.

BACKGROUND AND PURPOSE: MRI has superior capabilities for the detection of cerebral infarcts compared with CT. CT was used to locate infarcts in most previous studies of atherothrombotic middle cerebral artery (MCA) territory infarcts. Thus, there was a possibility of missing concomitant small infarcts. More accurate identification of topographic lesions in MCA territory with MRI may help to establish the pathogenesis of stroke. The present study determines topographic patterns, distribution of vascular lesions, and probable mechanisms. METHODS: Forty-two patients with MCA territory infarcts on routine MRI and no major cause of cardioembolism were studied with conventional angiography or MR angiography. RESULTS: The topographic patterns seen on MRI were subdivided into 4 groups: cortical border-zone infarcts (n=6), pial territory infarcts without insular infarct (n=3), pial territory infarcts with insular infarct (n=14), and large subcortical infarcts (n=19). Of 6 patients with cortical border-zone infarcts, 4 had concomitant small cortical or subcortical multiple lesions. Angiography showed intrinsic MCA disease in 4 patients. Of 3 patients with pial territory infarcts without insular infarct, 2 also had small multiple centrum ovale lesions. All had intrinsic MCA disease. Pial territory infarcts with partial or whole insular lesions were present in 10 and 4 patients, respectively. Five patients had additional multiple cortical or subcortical lesions. Ten patients had intrinsic MCA disease. Of the 19 patients with large subcortical infarcts, 12 had centrum ovale infarcts, and 4 had both basal ganglia and centrum ovale lesions. Ten had concomitant small cortical or subcortical lesions. Six patients had intrinsic MCA disease. CONCLUSIONS: Similar vascular lesions induce different topographic patterns in MCA territory infarction, which are related to individual vascular variability, degree of primary and secondary collateralization, and pathogenesis of infarcts. Our study indicates that concomitant small cortical or subcortical lesions are also commonly associated findings in diverse patterns of MCA territory infarction, which can mostly be explained by probable embolic mechanism.

Ultra-rapid freezing of human multipronuclear zygotes using electron microscope grids.

Developmental capacity of human multipronuclear (PN) zygotes cryopreserved using an ultra-rapid freezing method and electron microscope (EM) grids was studied. Multipronuclear zygotes obtained from a human IVF programme were used as an alternative to normal 2PN zygotes; they were divided into 3PN or >or =4PN zygotes and their in-vitro development and cryo-injury were compared according to PN number. EFS30, which consisted of 30% ethylene glycol, 18% Ficoll, 0. 5 mol/l sucrose and 10% fetal bovine serum with added modified Dulbecco's phosphate buffered saline was used as the freezing solution. After ultra-rapid freezing and thawing 85.5% of multipronuclear zygotes survived. A comparison of cleavage rates between the control and cryopreserved groups showed no significant difference (3PN; 81.3 and 85.4% and > or =4PN; 90.0 and 95.7% respectively). Comparing the in-vitro development after thawing up to blastocyst formation on day 5 after IVF, the outcome of the frozen 3PN group (22.0%) was not different from that of control 3PN group (38.5%), while the outcome of the frozen > or =4PN group (4.5%) was significantly lower than that of control > or =4PN group (44.4%) (P < 0.05).

The distribution and requirements of microtubules and microfilaments in bovine oocytes during in vitro maturation.

Microtubules and microfilaments are major cytoskeletal components and important modulators for chromosomal movement and cellular division in mammalian oocytes. In this study we observed microtubule and microfilament organisation in bovine oocytes by laser scanning confocal microscopy, and determined requirements of their assembly during in vitro maturation. After germinal vesicle breakdown, small microtubular asters were observed near the condensed chromatin. The asters appeared to elongate and encompass condensed chromatin particles. At the metaphase stage, microtubules were observed in the second meiotic spindle at the metaphase stage. The meiotic spindle was a symmetrical, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Treatment with nocodazole did not inhibit germinal vesicle breakdown. However, progression to metaphase failed to occur in oocytes treated with nocodazole. In contrast, microfilaments were observed as a relatively thick uniform area around the cell cortex and overlying chromatin following germinal vesicle breakdown. Treatment with cytochalasin B inhibited microfilament polymerisation but did not prevent either germinal vesicle breakdown or metaphase formation. However, movement of chromatin to the proper position was inhibited in oocytes treated with cytochalasin B. These results suggest that both microtubules and microfilaments are closely associated with reconstruction and proper positioning of chromatin during meiotic maturation in bovine oocytes.

Five cases of ocular toxocariasis confirmed by serology.

We report 5 cases of ocular toxocariasis in Korean adults complaining of visual impairment along with floating or bubbling sensation. Fundoscopic examination revealed a retinal detachment along with exudate in 4 cases. They all showed typical reaction by ELISA and immunoblot against Toxocra excretory-secretory antigen. One case showed high level of anti-Toxocara IgE antibodies (34,000 Toxocara units/L) as well as increased level of serum total IgE antibodies and the specific IgE antibodies for 3 inhalant antigens, suggesting that high level of anti-Toxocara IgE antibodies was associated with an atopic status. Clinical manifestations were improved after the sequential use of steroids then mebendazole. We also suggest that ocular toxocariasis should be thoroughly investigated even when an evocative uniocular inflammatory lesion is encountered in peripheral retina without a systematic disease.

Simple, efficient and successful vitrification of bovine blastocysts using electron microscope grids.

This study demonstrates that higher survival of vitrified-thawed bovine blastocysts can be obtained using electron microscope (EM) grids as embryo containers at freezing, rather than plastic straws. In-vitro produced day 7 bovine blastocysts after in-vitro fertilization (IVF) were vitrified on grids or in straws with EFS40 freezing solution and their survival after thawing was compared. Embryo survival was assessed as re-expanded and hatched rates at 24 and 48 h after thawing respectively. When the effects of exposure to vitrification solution and chilling injury from the freezing procedure were examined, embryo survival in the exposure group (24 h: 100, 48 h: 73.3%) was not different compared with that in the control group (100, 84.4%). After vitrification, the hatched rate of the EM grid group 48 h after thawing (67.8%) was significantly higher than that of the straw group (53.3%) (P < 0.05). Fast developing embryos (expanded blastocyst and early hatching blastocyst stage) showed better resistance to freezing than delayed ones (early blastocyst stage), irrespective of embryo containers (early: 24 h, 57.1 and 48 h, 24.4%; expanded: 84.7 and 60.6%; early hatching: 91.7 and 80.0%) (P < 0.001). When using expanded and early hatching blastocysts, embryo survival rates in the vitrification-EM grid group (67.8, 95.0% respectively) were significantly higher than that of the vitrification-straw group (53.0, 65.0%) at 48 h.

Atherothrombotic cerebellar infarction: vascular lesion-MRI correlation of 31 cases.

BACKGROUND AND PURPOSE: Correlation of MRI findings with atherosclerotic vascular lesions has rarely been attempted in patients with cerebellar infarction. The aim of this study was to correlate the MRI lesions with the vascular lesions seen on conventional cerebral angiography in cerebellar infarction. METHODS: The subjects included 31 patients with cerebellar infarcts who underwent both MRI and conventional cerebral angiography. We analyzed the risk factors, clinical findings, imaging study, and angiography results. We attempted to correlate MRI lesions with the vascular lesions shown in the angiograms. RESULTS: The vascular lesions seen on angiograms were subdivided into 3 groups: large-artery disease (n=22), in situ branch artery disease (n=6), and no angiographic disease with hypertension (n=3). The proximal segment (V1) lesions of vertebral artery were the most common angiographic features in patients with large-artery disease in which stroke most commonly involved the posterior inferior cerebellar artery (PICA) cerebellum. The V1 lesions with coexistent occlusive lesions of the intracranial vertebral and basilar arteries were correlated with cerebellar infarcts, which had no predilection for certain cerebellar territory. The intracranial occlusive disease without V1 lesion was usually correlated with small cerebellar lesions in PICA and superior cerebellar artery (SCA) cerebellum. The subclavian artery or brachiocephalic trunk lesion was associated with small cerebellar infarcts. The in situ branch artery disease was correlated with the PICA cerebellum lesions, which were territorial or nonterritorial infarct. No angiographic disease with hypertension was associated with small-sized cerebellar infarcts within the SCA, anterior inferior cerebellar artery, or SCA cerebellum. CONCLUSIONS: Our study indicates that the topographic heterogeneity of cerebellar infarcts are correlated with diverse angiographic findings. The result that large-artery disease, in which nonterritorial infarcts are more common than territorial infarcts, is more prevalent than in situ branch artery disease or small-artery disease, suggest that even a small cerebellar infarct can be a clue to the presence of large-artery disease.

In vivo microdialysis measures of extracellular serotonin in the rat hippocampus during sleep-wakefulness.

We investigated extracellular 5-hydroxytryptamine (5-HT) levels in rat hippocampus during different stages of the sleep-waking cycle using in vivo microdialysis. The extracellular 5-HT level was highest in active waking (AW) and, when compared to AW, 5-HT level was progressively lower in quiet waking (QW; 78%), quiet sleep (QS; 50%) and REM (which we termed active sleep (AS); 40%). Functional implications of AS related-decreased 5-HT in the hippocampus are discussed.

A case of presumed ocular toxocariasis in a 28-year old woman.

This is a case of presumed ocular toxocariasis in a 28-year old woman complaining of a sudden onset of nasal side field defect of the right eye. The patient had been suffering from uveitis for ten months. Fundoscopic examination of the right eye showed a rhegmatogenous retinal detachment. Furthermore, a retinochoroidal granulomatous lesion was observed nearby the tear site. Scleral buckling, cryotherapy, and gas injection(SF6, pure gas, 0.7 cc) were conducted. Mebendazole was prescribed for one month at 25 mg/kg per body weight daily. Even though the interventions resulted in the recovery of the field defect, anti-Toxocara IgG and IgE titer levels did not decrease when checked three months after the treatment ended. This is the first confirmed serological ocular toxocariasis case in Korea. Uveitis may be a clinical presentation prior to retinal detachment of a person with toxocariasis.

Promoter analysis of the Drosophila genes encoding TFIIB and TATA box-binding protein.

We have analyzed the 5'-flanking regions of the Drosophila genes encoding the TFIIB and TATA box-binding proteins (TBP) and mapped the regulatory regions required for their efficient expression. We found that the 500 bp long region (-439 to +60) and the 401 bp long region (-261 to +138) are required for the efficient expression of TFIIB and TBP genes, respectively. In the TFIIB promoter region, the upstream DNA between -439 and -280 and the downstream DNA between +8 and +60 are necessary for the stimulation of promoter activity. The upstream DNA between -439 and -280 stimulates transcription in an orientation dependent manner. In the TBP promoter region, the upstream DNA between -261 and -207, and the downstream DNA between +15 and +138 are necessary for the stimulation of promoter activity. The upstream DNA (-261 to -207) required for TBP promoter activity contains a 11 bp long palindromic sequence and a DNA replication-related element sequence. Particularly, we could find that the downstream promoter regions of TFIIB and TBP genes contain the conserved nucleotide sequences, suggesting the presence of a common regulatory mechanism for the expression of these two genes.

Systems for production of calves from cultured bovine embryonic cells.

The development of totipotent bovine embryonic cell cultures has great value in cattle breeding. They provide: (1) a mechanism for making large numbers of clonal offspring by nuclear transfer; (2) an efficient gene transfer system through the use of selectable markers to select transgenic cells; and (3) a mechanism for site-specific gene transfer or deletion by homologous DNA sequence recombination. Bovine embryonic cell cultures have been established from blastocyst inner cell mass (ICM) cells, morulae and the precompaction 16-20-cell stage. All have exhibited similar morphology to mouse embryonic stem (ES) cells, pluripotency on differentiation and proliferation in culture. Culture systems have consisted of microdrop loose suspension short-term cultures or long-term cultures on bovine or murine fibroblast feeder layers, in either a microdrop or a culture dish. The relative merit of culture systems or media requirements for mitosis and prevention of differentiation have not been determined. At present, totipotency is also unknown for cultured cells of the 16-20-cell stage. For cultured ICM cells, totipotency was demonstrated by the birth of four calves from ICM cells cultured 27 days or less in a loose suspension microdrop. Advanced pluripotency and perhaps totipotency was demonstrated in one fetus in a recently reported study where morulae cells cultured in vitro were chimaerized with non-cultured cells. DNA fingerprinting to associate cell lines with offspring and karyotyping to ascertain chromatin normalcy is important in ES cell research. Data pertaining to the use of each are presented.