The Domestic Dog Embryo: In Vitro Fertilization, Culture, and Transfer.

Advances in embryo technologies in the domestic dog have made significant strides in the past decade. This progress has been spurred by interests in taking advantage of the dog as a biomedical research model for human and companion animal medicine, developing assisted reproductive technologies to manage genetic diversity in endangered canids maintained ex situ, and improving breeding in rare or working breeds of dogs. Here, we focus on recent advancements and techniques for collection of in vivo-matured oocytes, in vitro fertilization (IVF), in vitro culture of early (≤8-cell) and advanced stage (≥16-cell) embryos, and embryo transfer.

Endocrine and paracrine controls of canine follicular development and function.

Canid reproduction is unique among other mammals in that females experience long and variable periods of ovarian inactivity. While the domestic dog exhibits a non-seasonal, largely sporadic monoestrus occurring once or twice a year, most wild canids, such as the gray wolf (Canis lupus) and red wolf (Canis rufus), are seasonal breeders with onset apparently dependent on species, latitudinal location and/or variety of environment factors. Neuroendocrine controls of ovarian functions have been mostly studied in the dog, but less so in their wild counterparts, due to difficulties in regular blood sampling. Yet, development of non-invasive hormone monitoring has advanced the understanding of reproductive cycle in wild canids. Recent advances in in vitro follicle culture technology also have begun to provide insights into paracrine controls of canid ovarian folliculogenesis. This review highlights current knowledge on canid reproduction with emphasis on endocrine and paracrine controls of follicular development. We also discuss future research priorities, including advancing the understanding of anoestrous termination and role of paracrine factors in canine folliculogenesis.

Anti-Müllerian Hormone in the Domestic Dog during the Anestrus to Oestrous Transition.

The reproductive cycle of the domestic dog features a long period of relative ovarian inactivity or anestrus. The mechanism of anestrous termination/oestrous resumption is not yet fully understood, which presents a challenge to the development of oestrous induction protocols. In this study, we assess the possibility that anti-Müllerian hormone (AMH) might play a role in this transition by characterizing its patterns of expression in the circulation during the transition from anestrus to oestrous and in all stages of ovarian follicular growth. Serum samples from five beagles (2.0-4.5 years) were collected three times per week at least 30 days prior to the onset of oestrous and assessed for AMH concentrations. Serum AMH concentration increased significantly during the transition from anestrus to proestrus and then declined back to the anestrous baseline beginning on day -4 before the luteinizing hormone surge, which was determined by changes in serum progesterone concentrations. Cortical sections of ovaries from females undergoing routine ovariohysterectomy (aged 8 months-5 years, n = 4) were evaluated for AMH by immunohistochemistry. Pre-antral and small antral follicles were most strongly immunoreactive for AMH. These data suggest that the increase in the number of antral follicles is associated with the rise in serum AMH as the anestrous period comes to an end. The rise in AMH might be useful in predicting the onset of oestrus and therefore assist with the optimization of oestrous induction protocols and possibly other assisted reproductive technologies.

The domestic dog and cat as models for understanding the regulation of ovarian follicle development in vitro.

The culture of ovarian follicles is an important tool for understanding the mechanisms controlling follicle development and differentiation of the oocyte. The benefit of recovering meiotically and developmentally competent oocytes from early stage follicles (primordial, primary, pre-antral and early antral) also would be significant, ranging from rescue of genomes from endangered species to preserving fertility in women facing cancer treatments. This research field is at an early stage of scientific discovery. To-date, live offspring from cultured primordial follicles that produced fertilizable oocytes has occurred only in the mouse. Progress in other more complex species has been limited because larger animals have longer durations of natural folliculogenesis, thereby requiring more culture time to generate fully grown follicles and oocytes. We believe the dog and cat are excellent models for understanding more about folliculogenesis in vitro. This review highlights what is known about this topic for these two species as well as future priorities. We have discovered that it is more challenging to maintain viability of primordial follicles within ovarian tissues in vitro in the dog than the cat. Nonetheless, it is possible to grow both isolated cat and dog pre-antral follicles in culture. Although the follicles of both species have the capacity to increase in size and produce steroids, only cat oocytes appear morphologically normal. The reason for this striking difference between these two species is an area of high research priority. While much more fundamental data are required, we envision advanced technology that will allow harvesting oocytes from the vast, unused follicle stores sequestered within carnivore ovaries. These gametes have utility for reproducing genetically valuable dogs and cats that are 'companions' or biomedical models for investigating human disorders as well as for salvaging the genomes of rare canid and felid species that die before contributing to genetic management programs.

Permanent, lowered HLA class I expression using lentivirus vectors with shRNA constructs: Averting cytotoxicity by alloreactive T lymphocytes.

Transplantation of many tissues requires histocompatibility matching of human leukocyte antigens (HLA) to prevent graft rejection, to reduce the level of immunosuppression needed to maintain graft survival, and to minimize the risk of graft-versus-host disease, particularly in the case of bone marrow transplantation. However, recent advances in fields of gene delivery and genetic regulation technologies have opened the possibility of engineering grafts that display reduced levels of HLA expression. Suppression of HLA expression could help to overcome the limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors, necessitate the maintenance of large donor registries, and complicate the logistics of procuring and delivering matched tissues and organs to the recipient. Accordingly, we investigated whether knockdown of HLA by RNA interference (RNAi), a ubiquitous regulatory system that can efficiently and selectively inhibit the expression of specific gene products, would enable allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors, which provide a delivery system that can achieve integration into genomic DNA, thereby permanently modifying transduced graft cells. Our results show that lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA can achieve efficient and dose-dependent reduction in surface expression of HLA in human cells, associated with enhanced resistance to alloreactive T lymphocyte-mediated cytotoxicity, while avoiding MHC-non-restricted killing. We hypothesize that RNAi-induced silencing of HLA expression has the potential to create histocompatibility-enhanced, and, eventually, perhaps "universally" compatible cellular grafts.

Left ventricular chamber size predicts the race time of Japanese participants in a 100 km ultramarathon.

OBJECTIVE: As a subanalysis of an echocardiographic study performed on 291 Japanese participants in a 100 km ultramarathon, to estimate predictors of race time. METHODS: A total of 247 male participants in a 100 km ultramarathon (age 20-73 years) were examined by echocardiography. Correlations between age, body surface area, monthly running distance, or echocardiographic variables and the race time were examined. RESULTS: According to simple regression analysis, age (r = 0.299, p < 0.0001), monthly running distance (r = -0.388, p < 0.0001), left ventricular end diastolic diameter (r = -0.300, p < 0.0001), and left ventricular end systolic diameter (r = -0.325, p < 0.0001) correlated significantly with the race time. When multiple regression analysis was performed, age (f = 2.364), monthly running distance (f = -0.113), and left ventricular end systolic diameter (f = -2.361) remained significant predictors of the race time. CONCLUSION: Left ventricular diameter predicts the race time for a 100 km ultramarathon, in addition to age and amount of training.

Reliability of MIBG myocardial scintigraphy in the diagnosis of Parkinson's disease.

AIM: To evaluate the reliability of [(123)I]meta-iodobenzylguanidine (MIBG) myocardial scintigraphy for diagnosing Parkinson's disease (PD). PATIENTS/METHODS: A series of 391 outpatients showing one or more parkinsonian-like symptoms was longitudinally followed up for accurate clinical diagnosis. MIBG scintigraphy was performed in the patients and 10 normal controls of similar age. The heart to mediastinum uptake ratio was calculated in each person, and the values were considered abnormal if they were greater than two standard deviations below the control mean. RESULTS: MIBG uptake was decreased in most patients with PD (87.7%), and was seen in all advanced cases with Hohen-Yahr stage III or more; the sensitivity and specificity of scintigraphy for detecting PD were 87.7% and 37.4%, respectively. Surprisingly, over half of the patients without PD (66.5%) also exhibited low uptake, resulting in considerable overlap in the ratios between PD and the other disorders. CONCLUSION: MIBG scintigraphy is a sensitive, but not specific, test for PD. Low MIBG uptake does not necessarily indicate PD, but is essential for diagnosing advanced PD.

Cerebral blood flow index image as a simple indicator for the fate of acute ischemic lesion.

BACKGROUND AND PURPOSE: To evaluate the feasibility of utilizing cerebral blood flow (CBF) index images, we attempted to investigate 1) whether CBF index images can reveal the resulting infracted area, 2) whether the CBF index can correlate other modality (SPECT). METHODS: DWI and DPI were obtained in 17 patients within 12 hours of stroke onset and follow up MRI. On three DPI delivered images, namely relative regional cerebral blood volume (rrCBV), uncorrected mean transit time (MTTu) and CBF index images, correlations between initial lesion volume of and follow up infarction volume of three images and rCBF images delivered with singular value decomposition (SVD) methods were assessed. Then 99mTc-ECD SPECT was taken immediately after MRI to correlate to MRI data. RESULTS: Among the three images, lesion volume of CBF index images against follow up infarct volume had the highest correlation (r = 0.995) to a linear fit and the slope was closest to 1.0 (0.91) and had identical accuracy to the regression coefficient of rCBF images. CBF index well correlated to SPECT delivered CBF. CONCLUSION: CBF index images can accurately predict final infarct volume. Evaluating CBF index images together with DWI can guide the initial assessment in the acute stage of cerebral ischemia.

Assessment of reflection pulse wave in patients with cardiomyopathy: evaluation of noninvasive measurement of wave intensity.

OBJECTIVE: We assessed the reflection pressure wave using noninvasive measurement of wave intensity (WI) in patients with cardiomyopathy. METHODS AND RESULTS: Subjects included 8 patients with hypertrophic cardiomyopathy (HCM group) and 10 patients with dilated cardiomyopathy (DCM group). Twelve healthy subjects were used as a control group. By using a combined Doppler and echo-tracking system, changes in vascular diameter (dD) and blood flow velocity (dV) were recorded simultaneously at the common carotid artery and dD x dV was measured as WI. In the components of WI, the positive component of early systolic phase (FE) and the negative component following FE (B) were significantly reduced in the DCM group. There was a significant positive correlation between FE and B in all 3 groups. The appearance time of B was significantly shorter in the HCM group and significantly longer in the DCM group compared with the control group. CONCLUSIONS: It was suggested that the value of the reflection pressure wave was influenced by the left ventricular contractility, and that the effect of the reflection pressure wave appeared earlier in patients with HCM and later in patients with DCM compared with the control subjects.

Treatments of non-parasitic giant hepatic cysts.

This retrospective study presents the results of surgical treatments for large cyst of the liver over 10 cm in diameter in 9 patients diagnosed and treated at Kurume University Hospital. There were 8 women and 1 man, with an average age of 71.6 years. Although the chief complaints were abdominal pain or fullness, 1 had obstructive jaundice due to biliary compression by a large cyst. Cyst size ranged from 10 cm to 27 cm. There are several treatment modalities for giant hepatic cyst, such as cyst resection, unroofing, and sclerotherapy after cyst drainage. Operative procedures in the patients reported here were surgical resection of the liver cyst in 3 patients, unroofing with ethanol sclerotherapy in 1 patient and laparoscopic treatment in 2 patients. Sclerotherapy after percutaneous transhepatic cyst drainage was performed in 3 patients because their general condition was poor. There were no postoperative complications among these patients. Costs varied among the patients and depended mainly on the method of treatment and period of hospital stay. We discuss operative procedures and costs of treatment of each patient and review the literature.

Xanthogranulomatous cholecystitis.

Seven cases of xanthogranulomatous cholecystitis are presented, and their clinicopathological appearance is described. Three men and 4 women with xanthogranulomatous cholecystitis, aged 53-72 years old, were reviewed. Five patients had had previous attacks of acute cholecystitis lasting from 3 weeks to 6 months. Abdominal ultrasonography was performed in all patients, and computed tomography in 5 patients. Cholelithiasis and sludge were present in all patients. The gallbladder wall was thickened in all patients. On computed tomography, one patient showed no abnormal finding, and 4 patients had abnormal findings such as increased wall thickness and irregularity, and pericholecystic abnormalities. A diagnosis of gallbladder carcinoma was made preoperatively in 1 patient. During laparotomy, the gallbladders in all patients showed signs of chronic cholecystitis, and cholecystectomies were performed. Histological findings showed xanthogranulomatous cholecystitis, and 4 patients had stones in the gallbladder wall. Despite the characteristic histologic appearance of xanthogranulomatous cholecystitis, radiologic findings are nonspecific, varying from signs observed in other forms of cholecystitis to the appearance of a gallbladder neoplasm. We report here 7 cases of xanthogranulomatous cholecystitis and review the literature.

A case of hepatocellular carcinoma rupturing after angiography.

We report a case of primary hepatocellular carcinoma (referred to as hepatocellular carcinoma below) apparently rupturing after angiography. The patient was a 62-year-old male who was admitted for the treatment of hepatocellular carcinoma. Ultrasonography (US) and computed tomography (CT) on admission showed a tumor occupying the entire left lobe of the liver and partly protruding outside the liver and a tumor embolus in the portal vein. We performed preoperative angiography, after which fever and abdominal discomfort appeared. Two days after the angiography, abdominal pain and a rapid increase in the size of the abdominal tumor were noted. US also revealed an unquestionable increase in the size of the tumor, leading to a diagnosis of intratumoral hemorrhage due to the rupture of hepatocellular carcinoma. Since child classification A, clinical stage I and ICG 11.7% indicated an adequate functional reserve of the liver, we performed an emergency operation. Laparotomy revealed that the tumor occupied almost the entire left lobe of the liver, partly protruded outside it, and was bleeding from part of its anterior surface. The volume of intra-abdominal hemorrhage was about 100 ml. A portal tumor embolus was present in the portal vein from the horizontal part to the trunk. We performed resection of the left and caudate lobes of the liver with removal of the portal tumor embolus. The resected specimens showed a hemorrhage in and around the tumor. We speculated that in a hepatocellular carcinoma that involves the surface of the liver and is complicated by tumor embolism of the portal vein, angiography could trigger the rupture of the carcinoma.

Spatial and temporal analysis of left ventricular filling flow propagation in hypertensive patients before and after regression of myocardial hypertrophy with alacepril therapy.

The purpose of this study was to assess the clinical significance of spatial and temporal analysis of left ventricular (LV) filling-flow propagation using color M-mode Doppler echocardiography before and after regression of LV hypertrophy in patients with hypertension. Seven patients with hypertensive LV hypertrophy were studied. Echocardiographic and Doppler examinations were performed both before and after 6 months administration of alacepril. LV mass index (LVMI), LV flow propagation velocity (FPV), and the maximal early transmitral flow velocity (E) were measured. LVMI, FPV, and FPV/E ratio were compared to before and after administration of alacepril. In addition, the correlation between LVMI and FPV/E ratio was evaluated. Results showed that LVMI was significantly decreased (P < 0.05) and the FPV/E ratio was significantly increased (P < 0.05) after treatment with alacepril. There was no significant change in FPV. In addition, there was a significant negative correlation between LVMI and the FPV/E ratio (r = -0.662, P < 0.001). The present study indicates that the FPV/E ratio could be a useful noninvasive parameter to assess the diastolic dysfunction associated with LV hypertrophy in patients with hypertension.

[A case of sepsis by Neisseria meningitidis beginning with pneumonia during a trip abroad].

A 62-year-male presented a high fever and a dry cough during a trip to Australia. He was admitted to a hospital as soon as be returned to Japan. The next day after returning to Japan, he was transferred to our hospital with septic shock and loss of consciousness. Neisseria meningitidis was cultured from his blood. N. meningitidis is rare in Japan. However its seems common, in some foreign countries. With these findings, it can be postulated that N. meningitidis might be one of the etiological agents of the imported infectious disease.

Evaluation of simultaneous excision of pancreatic cancer and the surrounding blood vessels.

Of the 139 patients who underwent excision for invasive cancer in the pancreatic duct at Kurume University Hospital between January 1965 and December 1998, the subjects were 38 patients in whom blood vessels around the cancer were simultaneously excised. The surgical methods were pancreatoduodenectomy (PD) in 31 patients, distal pancreatectomy (DP) in 5, and total pancreatectomy (TP) in 2. The excised blood vessels were the portal vein alone in 32 patients, the artery alone in 1, and both portal vein and artery in 5. Excision of the portal vein was performed by circumcision in 25 patients and by segmentectomy in 12. The range of circumcision was 1.0-7.0 cm (mean, 3.5 +/- 1.4 cm), and the blocking time of the portal vein was 8-36 min (mean, 19.5 +/- 8.8 min). Of the 25 patients who underwent circumcision, reconstruction was performed by end-to-end anastomosis in 23 and by transplantation of the autologous vein between the ends in 2. Of the 12 patients who underwent segmentectomy, direct suture was performed in 10, and transplantation of an autologous vein patch was performed in 2. Postoperative complications occurred in 14 of the 32 patients. In 5 of the 6 patients who underwent excision of the artery, reconstruction was performed by end-to-end anastomosis in 3 and by transplantation of the autologous vein between the ends in 2. Postoperative complications did not occur in the patient who had undergone excision of the artery alone, but 4 of the 5 patients who had undergone simultaneous excision of the portal vein and artery had postoperative complications, of whom 2 died during the period of hospitalization. Three patients with pv0, pv1 or pv2 survived for more than 3 years. Because some of the patients who had undergone excision of the portal vein alone survived for a long time and this method is relatively safe, this surgery can be generally applied, but simultaneous excision of the portal vein and artery should be carefully applied because the incidences of postoperative complications and death during the period of hospitalization are high. With the development of surgical techniques and postoperative control, simultaneous excision of pancreatic cancer and the surrounding blood vessels has become safe, but this method should only be applied to patients who have the potential to recover completely.

Effects of prolonged strenuous exercise on plasma levels of atrial natriuretic peptide and brain natriuretic peptide in healthy men.

BACKGROUND: Now that marathon racing is growing in popularity, many thousands of enthusiastic athletes are participating in various ultramarathons all over the world each year. However, it remains controversial whether such a sport contributes to the promotion of health. The occurrence of transient cardiac dysfunction and irreversible myocardial injury has been reported in association with such exercise in healthy individuals. Brain natriuretic peptide (BNP) is a cardiac hormone, as is atrial natriuretic peptide (ANP), and its measurement has been widely used for clinical evaluation of cardiac dysfunction. However, little is known about the response of plasma BNP to prolonged strenuous exercise. We hypothesized that confirmation of minimal cardiac dysfunction or myocardial injury may be made by measurements of plasma BNP. METHODS: Levels of plasma ANP, BNP, catecholamines, blood lactate, and serum cardiac troponin T (cTnT) were determined before and after a 100-km ultramarathon in 10 healthy men to examine the effects of the exercise on levels of ANP and BNP and correlations between the natriuretic peptides and cTnT as a marker for myocardial damage. RESULTS: Whereas all variables significantly increased after the race, increased levels of ANP and BNP were most strongly correlated with increases in cTnT levels. The cTnT level after the race was greater than the upper reference limit in 9 of 10 men. CONCLUSIONS: Such exercise significantly increased ANP and BNP levels in healthy men, and the increases could be partially attributed to myocardial damage during the race.

Congenital giant aneurysm of the left atrial appendage mimicking pericardial absence case report.

A 25-year-old man was found to have an abnormal cardiac contour on a chest radiograph, and was referred. Transesophageal echocardiography suggested herniation of the left atrial appendage (LAA) through a gap in the pericardium, and magnetic resonance imaging indicated congenital partial absence of the pericardium. Cardiac dysfunction was caused by compression from the enlarged left atrium and thrombi were thought to be present in the appendage, so surgery was performed. The intraoperative diagnosis was congenital LAA aneurysm. Although distinguishing between congenital LAA aneurysm and congenital absence of the pericardium is reported to be possible with magnetic resonance imaging, we were unable to so in this case.

Clinicopathological evaluation of surgical treatment for early gallbladder cancer.

We evaluated the therapeutic principles for early gallbladder cancer based on clinicopathological characteristics and outcomes in 27 patients encountered at the Kurume University Hospital between January, 1975 and December, 1999. Concerning the depth of wall penetration, 15 patients had mucosal cancers (m-cancers), and 12 patients muscularis propria cancers(mp-cancers). The gross patterns were lp (pedunculated) in 16 patients, ls (sessile) in 3 patients, IIa (flat elevated) in 4 patients, and IIb (flat) in 4 patients. The operative procedure used was cholecystectomy (C) in 12 patients, 4 of whom underwent lymph node dissection. Full-thickness cholecystectomy (FTC) was carried out in 3 patients, one of whom had lymph node dissection. Combination of C and gallbladder bed resection (GbBR) was performed in 7 patients, 6 of whom had lymph node dissection. Combination of C and bile duct resection (BDR), and lymph node dissection was performed in 1 patient. Combination of C and GbBR and BDR, and lymph node dissection was performed in 6 patients. All the patients who underwent lymph node dissection were negative for metastasis. Of the 27 patients, 2 underwent laparoscopic operation: one with m-cancer was 79 years old, and the other with mp-cancer 86 years old. In the m-cancers, no lymphatic, venous or perineural infiltration was observed. In contrast, in the mp-cancers, lymphatic and venous infiltration each were observed in 4 patients (33.3%), although no perineural infiltration was observed. A diagnosis of gallbladder cancer was made postoperatively in 6 patients, of whom 4 had the IIb pattern and all were complicated by gallstone, indicating the difficulty of diagnosing the IIb pattern. The 5-year survival rates for the m- and mp-cancers were as high as 90.9% and 80.8%, respectively. As a curative surgical technique for m- and mp-cancers, lymph node dissection should be performed in addition to FTC, GbBR, and BDR, in combination. When a postoperative histopathologic diagnosis of gallbladder cancer has been made, no second-look operation should be performed for m-cancers, but lymph node dissection of up to the second group should be performed for mp-cancers in a second-look operation.