Neurosurgical intraoperative ultrasonography using contrast enhanced superb microvascular imaging -vessel density and appearance time of the contrast agent.

BACKGROUND: Ultrasonography (US) provides real-time information on structures within the skull during neurosurgical operations. Superb microvascular imaging (SMI) is the latest imaging technique for detecting very low-velocity flow with minimal motion artifacts, and we have reported on this technique for intraoperative US monitoring. We combined SMI with administration of contrast agent to obtain detailed information during neurosurgical operations. MATERIALS AND METHODS: Twenty patients diagnosed with brain tumor (10 meningiomas, 5 glioblastomas, 2 hemangioblastomas, 1 schwannoma, 1 malignant lymphoma, 1 brain abscess) underwent neurosurgery under US with SMI and contrast agent techniques. Vessel density and appearance time following contrast administration were analyzed. RESULTS: Flow in numerous vessels was not visualized by SMI alone, but appeared following injection of contrast agent in all cases. Flow in tumors was drastically enhanced by contrast agent in schwannoma, hemangioblastoma and meningioma, compared to normal brain tissue. Flows in the dilated and bent vessels of glioblastoma were also enhanced, although flow in hypoechoic lymphoma remained inconspicuous. The characteristics of tumor vessels were clearly visualized and tumor borders were demonstrated by the difference between tumor flow and brain flow, by the increased tumor vessel density and decreased appearance time of contrast agent compared to normal brain vessels. CONCLUSIONS: The combination of SMI and contrast agent techniques for intraoperative US monitoring could provide innovative flow images of tumor and normal brain. The neurosurgeon obtains information about tumor flow and tumor borderline before tumor resection.

Myogenetic Oligodeoxynucleotide Induces Myocardial Differentiation of Murine Pluripotent Stem Cells.

An 18-base myogenetic oligodeoxynucleotide (myoDN), iSN04, acts as an anti-nucleolin aptamer and induces myogenic differentiation of skeletal muscle myoblasts. This study investigated the effect of iSN04 on murine embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In the undifferentiated state, iSN04 inhibited the proliferation of ESCs and iPSCs but did not affect the expression of pluripotent markers. In the differentiating condition, iSN04 treatment of ESCs/iPSCs from day 5 onward dramatically induced differentiation into Nkx2-5+ beating cardiomyocytes with upregulation of Gata4, Isl1, and Nkx2-5, whereas iSN04 treatment from earlier stages completely inhibited cardiomyogenesis. RNA sequencing revealed that iSN04 treatment from day 5 onward contributes to the generation of cardiac progenitors by modulating the Wnt signaling pathway. Immunostaining showed that iSN04 suppressed the cytoplasmic translocation of nucleolin and restricted it to the nucleoli. These results demonstrate that nucleolin inhibition by iSN04 facilitates the terminal differentiation of cardiac mesoderm into cardiomyocytes but interferes with the differentiation of early mesoderm into the cardiac lineage. This is the first report on the generation of cardiomyocytes from pluripotent stem cells using a DNA aptamer. Since iSN04 did not induce hypertrophic responses in primary-cultured cardiomyocytes, iSN04 would be useful and safe for the regenerative therapy of heart failure using stem cell-derived cardiomyocytes.

Intralateral and Perilateral Ventricular Arteries on Original Axial Magnetic Resonance Angiography in Adult Moyamoya Disease.

BACKGROUND: Patients with moyamoya disease often develop cerebral infarction and hemorrhage, but the ischemic and hemorrhagic subtypes are difficult to diagnose prior to disease onset. We aimed to differentiate the ischemic and hemorrhagic subtypes of moyamoya disease by analyzing the intralateral and perilateral ventricular arteries on the original axial magnetic resonance angiography (MRA) images. METHODS: We retrospectively analyzed the intralateral and perilateral ventricular arteries on the original axial time-of-flight (TOF)-MRA images of 18 patients with hemorrhagic moyamoya disease, 25 patients with ischemic moyamoya disease, and 22 control patients with unruptured aneurysms. RESULTS: There were significantly more intralateral and perilateral ventricular arteries on the original axial MRA images in the patients with hemorrhagic moyamoya disease (6.3 ± 2.7) than in those with ischemic moyamoya disease (0.8 ± 0.9) and those with unruptured aneurysms (0.4 ± 0.8). CONCLUSION: The intralateral and perilateral ventricular arteries on the original axial TOF-MRA images might suggest the hemorrhagic type of moyamoya disease prior to onset.

Intraoperative real-time near-infrared optical imaging for the identification of metastatic brain tumors via microscope and exoscope.

OBJECTIVE: As chemotherapy and radiotherapy have developed, the role of a neurosurgeon in the treatment of metastatic brain tumors is gradually changing. Real-time intraoperative visualization of brain tumors by near-infrared spectroscopy (NIRS) is feasible. The authors aimed to perform real-time intraoperative visualization of the metastatic tumor in brain surgery using second-window indocyanine green (SWIG) with microscope and exoscope systems. METHODS: Ten patients with intraparenchymal brain metastatic tumors were administered 5 mg/kg indocyanine green (ICG) 1 day before the surgery. In some patients, a microscope was used to help identify the metastases, whereas in the others, an exoscope was used. RESULTS: NIRS with the exoscope and microscope revealed the tumor location from the brain surface and the tumor itself in all 10 patients. The NIR signal could be detected though the normal brain parenchyma up to 20 mm. While the mean signal-to-background ratio (SBR) from the brain surface was 1.82 ± 1.30, it was 3.35 ± 1.76 from the tumor. The SBR of the tumor (p = 0.030) and the ratio of Gd-enhanced T1 tumor signal to normal brain (T1BR) (p = 0.0040) were significantly correlated with the tumor diameter. The SBR of the tumor was also correlated with the T1BR (p = 0.0020). The tumor was completely removed in 9 of the 10 patients, as confirmed by postoperative Gd-enhanced MRI. This was concomitant with the absence of NIR fluorescence at the end of surgery. CONCLUSIONS: SWIG reveals the metastatic tumor location from the brain surface with both the microscope and exoscope systems. The Gd-enhanced T1 tumor signal may predict the NIR signal of the metastatic tumor, thus facilitating tumor resection.

The Diffuse and Severe Traumatic Subarachnoid Hemorrhage Being Hard to Distinguish to Aneurysmal Subarachnoid Hemorrhage.

BACKGROUND: In primary intracerebral hemorrhage, several studies showed that contrast extravasation (CEV) is reported to be an indicator of delayed hematoma expansion, emergent hematoma removal and poor prognosis. On the contrary in head trauma, few researches validated the effectiveness of CEV because of other influences such as subarachnoid hemorrhage (SAH), contusion or brain swelling. The authors experienced a patient showing diffuse SAH caused by traumatic mechanism with acute subdural hematoma (ASDH). In the angiography, a notable rare image of CEV was found and emergency operation mainly to cease the bleeding points was performed. PATIENT PRESENTATION: A 70-year-old man was found being comatose and brought to emergency room. Computed tomography (CT) revealed diffuse SAH with left ASDH. Computed tomography angiography (CTA) also did not clarify any abnormal vessel structure except for slightly dilatation at the part of internal carotid artery, suggesting tiny ruptured aneurysm. The authors performed a cerebral angiography and resulted in no aneurysm or arteriovenous shunt detection but revealed the extravasation from middle cerebral artery and middle meningeal artery. Follow-up CT revealed increased left ASDH though 5 hours have already elapsed. The authors performed surgical intervention to halt the bleeding. CONCLUSION: The authors experienced a notably rare image of the extravasation from middle cerebral artery and middle meningeal artery in the cerebral angiography. When an obscure arterial abnormality is suspected on CTA for the unknown origin SAH with ASDH, the authors may have an affirmative attitude toward performing conventional cerebral angiography, which is most promising modality to detect the source of bleeding including CEV.

Rush progression and fatal result of septic shock related to central line catheter infection in cirrhosis patient with brain stroke.

BACKGROUND: Catheter-related blood stream infection (CRBSI) is one of the most common intractable healthcare-associated infections because catheters can be easily contaminated by resistant bacteria, and is associated with a high mortality. Central lines are currently used for administering medication to patients with severe stroke, and may thus cause CRBSI. CASE PRESENTATION: A 71-year-old woman with cirrhosis presented with subarachnoid hemorrhage (SAH) that was treated by clipping surgery. On postoperative day (POD) 38, sudden high fever (40.3 °C) was detected; the patient died a few hours later. Blood and central line cultures were positive for Klebsiella pneumoniae that may have caused CRBSI and endotoxin shock. In this case, the duration from fever detection to death was notably short. Additionally, inflammatory markers such as white blood cells (WBC) or C-reactive protein (CRP) were almost within normal ranges, even a few hours after fever was detected and before death. Cirrhosis was considered to be the cause of these phenomena. CONCLUSION: The timely diagnosis and complete treatment of patients with liver cirrhosis who develop CRBSI are highly challenging. We suggest that clinicians should rigorously apply preventive measures and strengthen CRBSI monitoring, especially in cirrhosis-associated cases.

Chicken stem cell factor enhances primordial germ cell proliferation cooperatively with fibroblast growth factor 2.

An in vitro culture system of chicken primordial germ cells (PGCs) has been recently developed, but the growth factor involved in the proliferation of PGCs is largely unknown. In the present study, we investigated the growth effects of chicken stem cell factor (chSCF) on the in vitro proliferation of chicken PGCs. We established two feeder cell lines (buffalo rat liver cells; BRL cells) that stably express the putative secreted form of chSCF (chSCF1-BRL) and membrane bound form of chSCF (chSCF2-BRL). Cultured PGC lines were incubated on chSCF1 or chSCF2-BRL feeder cells with fibroblast growth factor 2 (FGF2), and growth effects of each chSCF isoform were investigated. The in vitro proliferation rate of the PGCs cultured on chSCF2-BRL at 20 days of culture was more than threefold higher than those cultured on chSCF1-BRL cells and more than fivefold higher than those cultured on normal BRL cells. Thus, use of chSCF2-BRL feeder layer was effective for in vitro proliferation of chicken PGCs. However, the acceleration of PGC proliferation on chSCF2-BRL was not observed without FGF2, suggesting that chSCF2 would act as a proliferation co-factor of FGF2. We transferred the PGCs cultured on chSCF2-BRL cells to recipient embryos, generated germline chimeric chickens and assessed the germline competency of cultured PGCs by progeny test. Donor-derived progenies were obtained, and the frequency of germline transmission was 3.39%. The results of this study demonstrate that chSCF2 induces hyperproliferation of chicken PGCs retaining germline competency in vitro in cooperation with FGF2.

Executive dysfunction is related with decreased frontal lobe blood flow in patients with subarachnoid haemorrhage.

OBJECTIVE: This study evaluated executive dysfunction in patients with subarachnoid haemorrhage (SAH) using the Behavioural Assessment of the Dysexecutive Syndrome (BADS) and correlated the occurrence of executive dysfunction with cerebral blood flow (CBF) reduction in the frontal lobe as assessed by single photon emission computed tomography (SPECT). DESIGN: Correlational study. SUBJECTS: Twenty-two patients who underwent microsurgical clipping at least 3 months after SAH. METHODS: This study evaluated the BADS and the Wechsler Adult Intelligence Scale-III (WAIS-III). In addition, it assessed activities of daily living (ADL). CBF was evaluated using SPECT. The patients were divided into the following groups according to the results of SPECT: (1) those with reduced CBF in the frontal lobe (reduced CBF group, n = 8) and (2) those with intact CBF (intact CBF group, n = 14). RESULTS: The BADS score was significantly lower in the reduced CBF group compared with that of the intact CBF group, while there was no significant difference in the WAIS-III scores and ADL scale between the two groups. CONCLUSION: Although this result was conducted with a small sample size, executive dysfunction correlates with reduced CBF in the frontal lobes of SAH patients. A detailed evaluation of executive function is suggested in SAH patients, even if the patient's intelligence test and ADL scale reveal no abnormalities.

Development of the 12-Base Short Dimeric Myogenetic Oligodeoxynucleotide That Induces Myogenic Differentiation.

A myogenetic oligodeoxynucleotide (myoDN), iSN04 (5'-AGA TTA GGG TGA GGG TGA-3'), is a single-stranded 18-base telomeric DNA that serves as an anti-nucleolin aptamer and induces myogenic differentiation, which is expected to be a nucleic acid drug for the prevention of disease-associated muscle wasting. To improve the drug efficacy and synthesis cost of myoDN, shortening the sequence while maintaining its structure-based function is a major challenge. Here, we report the novel 12-base non-telomeric myoDN, iMyo01 (5'-TTG GGT GGG GAA-3'), which has comparable myogenic activity to iSN04. iMyo01 as well as iSN04 promoted myotube formation of primary-cultured human myoblasts with upregulation of myogenic gene expression. Both iMyo01 and iSN04 interacted with nucleolin, but iMyo01 did not bind to berberine, the isoquinoline alkaloid that stabilizes iSN04. Nuclear magnetic resonance revealed that iMyo01 forms a G-quadruplex structure despite its short sequence. Native polyacrylamide gel electrophoresis and a computational molecular dynamics simulation indicated that iMyo01 forms a homodimer to generate a G-quadruplex. These results provide new insights into the aptamer truncation technology that preserves aptamer conformation and bioactivity for the development of efficient nucleic acid drugs.

Development, differentiation and manipulation of chicken germ cells.

Germ cells are the only cell type capable of transmitting genetic information to the next generation. During development, they are set aside from all somatic cells of the embryo. In many species, germ cells form at the fringe of the embryo proper and then traverse through several developing somatic tissues on their migration to the emerging gonads. Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. Unlike other species, in avian embryos, PGCs use blood circulation for transport to the future gonadal region. This unique accessibility of avian PGCs during early development provides an opportunity to collect and transplant PGCs. The recent development of methods for production of germline chimeras by transfer of PGCs, and long-term cultivation methods of chicken PGCs without losing their germline transmission ability have provided important breakthroughs for the preservation of germplasm , for the production of transgenic birds and study the germ cell system. This review will describe the development, migration, differentiation and manipulation of germ cells, and discuss the prospects that germ cell technologies offer for agriculture, biotechnology and academic research.

Ischemic stroke patients with low DWI ASPECTS scores require puncture to recanalization within 30 min for large vessel occlusion.

BACKGROUND: The clinical benefits of faster recanalization in acute large vessel occlusion are well recognized, but the optimal procedure time remains uncertain. The aim of this study was to identify patient characteristics that necessitate puncture-to-recanalization (P-R) time within 30 min to achieve favorable outcome. METHODS: We evaluated the patients from a prospective, multicenter, observational registry of acute ischemic stroke patients. The study included patients who underwent endovascular therapy for ICA or MCA M1 occlusion and achieved successful recanalization. Patients were categorized into subgroups based on pre-treatment characteristics and the frequency of favorable outcomes was compared between P-R time < 30 min and ≥ 30 min. Interaction terms were incorporated into the models to assess the correlation between each patient characteristic and P-R time. RESULTS: A total of 1053 patients were included in the study. Univariate analysis within each subgroup revealed a significant association between P-R < 30 min and favorable outcomes in patients with DWI ASPECTS ≤6, age > 85 and NIHSS ≥16. In the multivariable analysis, NIHSS, age, time from symptom recognition to puncture, and DWI ASPECTS were significant independent predictors of favorable outcomes. Notably, only DWI ASPECTS exhibited interaction terms with P-R < 30 min. The multivariable analysis indicated that P-R < 30 min was an independent predictor for favorable outcome in DWI ASPECTS ≤6 group, whereas not in DWI ≥7. CONCLUSIONS: P-R time < 30 min is predictive of favorable outcomes; however, the effect depends on DWI ASPECTS. Target P-R time < 30 min is appropriate for patients with DWI ASPECTS ≤6.

Primary results of mechanical thrombectomy for acute ischemic stroke: The K-NET registry in the Japanese metropolitan area.

BACKGROUND: Endovascular treatment (EVT) for acute large vessel occlusion has proven to be effective in randomized controlled trials. We conducted a prospective cohort study to evaluate the real-world efficacy of EVT in a metropolitan area with a large number of comprehensive stroke centers and to compare it with the results of other registries and randomized controlled trials (RCTs). METHODS: We analyzed the Kanagawa Intravenous and Endovascular Treatment of Acute Ischemic Stroke registry, a prospective, multicenter observational study of patients treated by EVT and/or intravenous tissue-type plasminogen activator (tPA). Of the 2488 patients enrolled from January 2018 to June 2020, 1764 patients treated with EVT were included. The primary outcome was a good outcome, which was defined as a modified Rankin Scale (mRS) of 0-2 at 90 days. Secondary analysis included predicting a good outcome using multivariate logistic regression analysis. RESULTS: The median age was 77 years, and the median National Institute of Health Stroke Scale (NIHSS) score was 18. Pretreatment mRS score 0-2 was 87%, and direct transport was 92%. The rate of occlusion in anterior circulation was 90.3%. Successful recanalization was observed in 88.7%. The median time from onset to recanalization was 193 min. Good outcomes at 90 days were 43.3% in anterior circulation and 41.9% in posterior circulation. Overall mortality was 12.6%. Significant predictors for a good outcome were as follows: age, male, direct transfer, NIHSS score, Alberta Stroke Program Early Computed Tomography Score, intravenous tPA, and successful recanalization. CONCLUSION: EVT in routine clinical use in a metropolitan area showed comparable good outcomes and lower mortality compared to previous studies, despite the high proportion of patients with older age, pretreatment mRS score of >2, posterior circulation occlusion, and higher NIHSS. Those results may have been associated with more direct transport and faster onset-to-recanalization times.

X-irradiation removes endogenous primordial germ cells (PGCs) and increases germline transmission of donor PGCs in chimeric chickens.

Primordial germ cells (PGCs) are embryonic precursors of germline cells with potential applications in genetic conservation, transgenic animal production and germline stem cell research. These lines of research would benefit from improved germline transmission of transplanted PGCs in chimeric chickens. We therefore evaluated the effects of pretransplant X-irradiation of recipient embryos on the efficacy of germline transmission of donor PGCs in chimeric chickens. Intact chicken eggs were exposed to X-ray doses of 3, 6 and 9 Gy (dose rate = 0.12 Gy/min) after 52 h of incubation. There was no significant difference in hatching rate between the 3-Gy-irradiated group and the nonirradiated control group (40.0 vs. 69.6%), but the hatching rate in the 6-Gy-irradiated group (28.6%) was significantly lower than in the control group (P<0.05). No embryos irradiated with 9 Gy of X-rays survived to hatching. X-irradiation significantly reduced the number of endogenous PGCs in the embryonic gonads at stage 27 in a dose-dependent manner compared with nonirradiated controls. The numbers of endogenous PGCs in the 3-, 6- and 9-Gy-irradiated groups were 21.0, 9.6 and 4.6% of the nonirradiated control numbers, respectively. Sets of 100 donor PGCs were subsequently transferred intravascularly into embryos irradiated with 3 Gy X-rays and nonirradiated control embryos. Genetic cross-test analysis revealed that the germline transmission rate in the 3-Gy-irradiated group was significantly higher than in the control group (27.5 vs. 5.6%; P<0.05). In conclusion, X-irradiation reduced the number of endogenous PGCs and increased the germline transmission of transferred PGCs in chimeric chickens.

Epidural anterior petrosectomy with subdural visualization of sphenobasal vein via the anterior transpetrosal approach--technical case report.

The drainage of the superficial middle cerebral vein (SMCV) is classified into four subtypes. The sphenobasal vein (SBV) drains from the SMCV to the pterygoid venous plexus at the temporal skull base. Epidural procedures in the standard anterior transpetrosal approach (ATPA) may damage the route of the SBV. We report a case in which modified surgical procedures via the ATPA were used to preserve the SBV. A 45-year-old man complained of right facial pain. Magnetic resonance images revealed a right cerebellopontine tumor suggestive of an epidermoid cyst. Right carotid angiography revealed that the SMCV drained into the pterygoid venous plexus via the SBV. The convexity dura mater of the temporal lobe was cut and the anterior part of the temporal lobe was retracted subdurally. The SBV was visualized from the subdural side. The basal dura mater of the temporal lobe posterior to the SBV was cut and the posterior part of the temporal lobe was retracted epidurally. After dissecting the dura mater medial to the greater petrosal nerve and to the edge of the petrous apex, the petrous apex was exposed and drilled out without injuring the SBV. The superior petrous sinus and the tentorium were cut. The tumor compressed the root exit zone of the trigeminal nerve. The tumor was grossly totally removed. The modified ATPA (epidural anterior petrosectomy with subdural visualization of the SBV) is effective in preserving the SBV.

Transcription of Endogenous Retrovirus Group K Members and Their Neighboring Genes in Chicken Skeletal Muscle Myoblasts.

Skeletal muscle myoblasts are myogenic precursor cells that generate myofibers during muscle development and growth. We recently reported that broiler myoblasts, compared to layer myoblasts, proliferate and differentiate more actively and promptly into myocytes, which corresponds well with the muscle phenotype of broilers. Furthermore, RNA sequencing (RNA-seq) revealed that numerous genes are differentially expressed between layer and broiler myoblasts during myogenic differentiation. Based on the RNA-seq data, we herein report that chicken myoblasts transcribe endogenous retrovirus group K member (ERVK) genes. In total, 16 ERVKs were highly expressed in layer myoblasts and two (termed BrK1 and BrK2) were significantly induced in broiler myoblasts. These transcribed ERVKs had a total of 182 neighboring genes within ±100 kb on the chromosomes, of which 40% were concentrated within ±10 kb of the ERVKs. We further investigated whether the transcription of ERVKs affects the expression of their neighboring genes. BrK1 had two neighboring genes; LOC107052719 was overlapping with BrK1 and downregulated in the broiler myoblasts, and FAM19A2 was upregulated in the broiler myoblasts as well as BrK1. BrK2 had 14 neighboring genes, and only one gene, LOC772243, was differentially expressed between layer and broiler myoblasts. LOC772243 was overlapping with BrK2 and suppressed in the broiler myoblasts. These data indicate that the transcription of ERVKs may impact the expression of their neighboring genes in chicken myoblasts.

Myogenetic oligodeoxynucleotide complexed with berberine promotes differentiation of chicken myoblasts.

Myoblasts are myogenic precursors that develop into myotubes during muscle formation. Improving efficiency of myoblast differentiation is important for advancing meat production by domestic animals. We recently identified novel oligodeoxynucleotides (ODNs) termed myogenetic ODNs (myoDNs) that promote the differentiation of mammalian myoblasts. An isoquinoline alkaloid, berberine, forms a complex with one of the myoDNs, iSN04, and enhances its activities. This study investigated the effects of myoDNs on chicken myoblasts to elucidate their species-specific actions. Seven myoDNs (iSN01-iSN07) were found to facilitate the differentiation of chicken myoblasts into myosin heavy chain (MHC)-positive myotubes. The iSN04-berberine complex exhibited a higher myogenetic activity than iSN04 alone, which was shown to enhance the differentiation of myoblasts into myotubes and the upregulation of myogenic gene expression (MyoD, myogenin, MHC, and myomaker). These data indicate that myoDNs promoting chicken myoblast differentiation may be used as potential feed additives in broiler diets.

Germline replacement by transfer of primordial germ cells into partially sterilized embryos in the chicken.

We report a novel technique for almost complete replacement of the recipient germline with donor germ cells in the chicken. Busulfan solubilized in a sustained-release emulsion was injected into the yolk of fertile eggs before incubation. A dose of 100 microg was found to provide the best outcome in terms of reducing the number of endogenous primordial germ cells (PGCs) in embryonic gonads (0.6% of control numbers) and hatchability (36.4%). This was applied for preparing partially sterilized embryos to serve as recipients for the transfer of exogenous PGCs. Immunohistochemical analysis showed that the proportion of donor PGCs in busulfan-treated embryos was significantly higher than in controls (98.6% vs. 6.4%). Genetic cross-test analysis revealed that the germline transmission rate in busulfan-treated chickens was significantly higher than in controls (99.5% vs. 6.0%). Of 11 chimeras, 7 produced only donor-derived progenies, suggesting that these produced only donor-derived gametes in the recipient's gonads. This novel germline replacement technique provides a powerful tool for studying germline differentiation, for generating transgenic individuals, and for conserving genetic resources in birds.

Efficient system for preservation and regeneration of genetic resources in chicken: concurrent storage of primordial germ cells and live animals from early embryos of a rare indigenous fowl (Gifujidori).

The unique accessibility of chicken primordial germ cells (PGCs) during early development provides the opportunity to combine the reproduction of live animals with genetic conservation. Male and female Gifujidori fowl (GJ) PGCs were collected from the blood of early embryos, and cryopreserved in liquid nitrogen for >6 months until transfer. Manipulated GJ embryos were cultured until hatching; fertility tests indicated that they had normal reproductive abilities. Embryos from two lines of White Leghorn (24HS, ST) were used as recipients for chimera production following blood removal. The concentration of PGCs in the early embryonic blood of 24HS was significantly higher than in ST (P < 0.05). Frozen-thawed GJ PGCs were microinjected into the bloodstream of same-sex recipients. Offspring originating from GJ PGCs in ST recipients were obtained with a higher efficiency than those originating from GJ PGCs in 24HS recipients (23.3% v. 3.1%). Additionally, GJ progeny were successfully regenerated by crossing germline chimeras of the ST group. In conclusion, the cryogenic preservation of PGCs from early chicken embryos was combined with the conservation of live animals.

Ultrasonographic superb microvascular imaging for emergency surgery of intracerebral hemorrhage.

Ultrasonography (US) has been used as a reliable imaging modality, providing real-time information during neurosurgical operations. One recent innovative US technique, superb microvascular imaging (SMI), visualizes small vessels and flow, which are not detected with standard US with doppler. We apply SMI to intraoperative US monitoring in emergency surgery for intracerebral hemorrhage (ICH). Eleven consecutive patients with ICH underwent endoscopic emergency surgery under US monitoring with SMI. After performing a small craniotomy, US images were obtained using SMI, a fusion technique, and a contrast agent technique, with the probe on the brain surface during surgery. Fusion images were obtained with the probe on the head before craniotomy in some patients. Animated US images with SMI could differentiate hematoma containing no vessels from brain tissue, and flow images using SMI and contrast agent techniques clarified the borderlines. Animated fusion images of intraoperative US and preoperative CT provided information on the extent of hematoma and residual hematoma during emergency surgery. We made various fusion CT images showing intracranial hematoma with US probes and decided on the skin incision line before beginning surgery, as if we were using a neuronavigation system. US with SMI, contrast agent, and fusion techniques provide information on the extent of intracranial hematoma and residual hematoma with no vessels and no flow. Monitoring by US and fusion CT images is useful for ICH surgery as a next-generation neuronavigator.

Novel system for degeneration of blood vessels by UV irradiation and subsequent regeneration using chick bone marrow cells.

Recently, many results have been reported regarding the pluripotency of bone marrow cells (BMCs) with the aim of benefiting regenerative medicine for humans. Particularly, vessel formation by hematopoietic stem cells or vascular endothelial stem cells which were derived from bone marrow has received considerable interest, since the mechanism of vessel formation has been found to be involved in neoangiogenesis of serious diseases such as cancer. Most work on neoangiogenesis and regeneration has involved mammalian experimental systems, however the avian model is useful since the process of neoangiogenesis and regeneration of vessels can be observed with the whole embryo culture system. We have established a novel system using early chick embryos, where a portion of blood vessels are degenerated by UV irradiation, and vessel regeneration is then studied. Incubated embryos were partially covered with aluminum foil, from the embryonic body to the dorsal marginal vein, and irradiated with UV for 1 min. Donor BMCs were obtained from the femurs and tibias of chicks aged 10 days, fluorescently labeled with PKH26 and injected into the anterior vitelline vein of the recipients. In BMC-treated embryos the donor BMCs were observed around the UV-degenerated vessels, and regeneration of blood vessels occurred, in contrast to the untreated embryos. These results indicate that avian BMCs have the ability to participate in vessel regeneration, and the avian model used here may be a useful tool for studies of vessel neoangiogenesis and repair.