Ultrastructural changes in colonic epithelial cells in a rat model of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a global, chronic intractable disease. The functions of drugs and food components have been evaluated in models of IBD induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Here, we used transmission (TEM) and osmium-maceration scanning (SEM) electron microscopy to evaluate the ultrastructure of colonic epithelial cells in rat models of IBD induced by TNBS. Histological evaluation revealed that the intestinal crypts in the most regions of the IBD-model colons were deformed and we classified them as having high cell migration rates (HMIG). The remaining regions in the intestinal crypts retained a relatively normal structure and we classified them as having low cell migration rates (LMIG). Osmium-maceration SEM revealed the mucosal fluid flowing in spaces without secretory granules in crypt goblet cells of both HMIG and LMIG regions, indicating the depletion of goblet cell mucin that is found in patients with IBD. The Golgi apparatus in absorptive cells was stacked and curled in both regions. Osmium-maceration SEM showed membrane network structures resembling endoplasmic reticulum that were large and expanded in absorptive cells with HMIG rather than with LMIG regions in IBD-model colons. These findings indicated that endoplasmic reticulum stress is associated with susceptibility to IBD and that the effects of various agents can be evaluated according to endoplasmic reticulum stress revealed by using electron microscopy in models of IBD induced by TNBS.

Polysplenia syndrome with duodenal and pancreatic dysplasia in a Holstein calf: a case report.

BACKGROUND: Laterality disorders of the abdominal organs include situs inversus totalis that mirrors the arrangements of all internal organs and heterotaxy syndrome (situs ambiguus) in which the thoracic or abdominal organs are abnormally arranged. Heterotaxy is often accompanied by multiple congenital malformations, and it generally comprises asplenia and polysplenia syndromes. To our knowledge, polysplenia syndrome has been reported in only three cattle, and computerized tomographic (CT) images of these animals were not obtained. CASE PRESENTATION: A six-month-old Holstein heifer had ruminal tympani and right abdominal distension. CT imaging showed that the rumen occupied the right side of the abdominal cavity, the omasum and abomasum occupied the left ventral side and the liver was positioned on the left. The colon and cecum were located at the left dorsum of the cavity, and the left kidney was located more cranially than the right. Postmortem findings revealed two spleens attached to the rumen. Significantly, the duodenum was too short to be divided into segments, except the cranial and descending parts, or flexures, except the cranial flexure, and the pancreas, which lacked a left lobe, was covered with mesojejunum. The liver comprised a relatively large right lobe and a small left lobe without quadrate and caudate lobes. The caudal vena cava that connected to the left azygous vein passed irregularly through the aortic hiatus of the diaphragm, and the common hepatic vein without the caudal vena cava passed through the caval foramen. Although the lungs and heart were morphologically normal, the right atrium received three major systemic veins. Polysplenia syndrome was diagnosed based on the CT and postmortem findings. CONCLUSION: We defined the positions of the abdominal organs and morphological abnormalities in various organs of a calf with polysplenia syndrome based on CT and postmortem findings. These findings will improve understanding of the malpositioning and malformations that can occur in the organs of cattle with polysplenia syndrome.

Structure and functions of the placenta in common minke (Balaenoptera acutorostrata), Bryde's (B. brydei) and sei (B. borealis) whales.

The structure and functions of placentas were examined in 3 species of rorqual whales, common minke (Balaenoptera acutorostrata), Bryde's (B. brydei) and sei (B. borealis) whales, with the aim of confirming the structural characteristics of the chorion, including the presence of the areolar part, and clarifying steroidogenic activities and fetomaternal interactions in the placentas of these whales. Placentas were collected from the second phase of the Japanese Whale Research Program under Special Permit in the North Pacific (JARPN II). Histological and ultrastructural examinations revealed that these whale placentas were epitheliochorial placentas with the interdigitation of chorionic villi lined by monolayer uninucleate cells (trophoblast cells) and endometrial crypts as well as folded placentation by fold-like chorionic villi. Moreover, well-developed pouch-like areolae were observed in the placentas, and active absorption was suggested in the chorionic epithelial cells of the areolar part (areolar trophoblast cells). Berlin blue staining showed the presence of ferric ions (Fe(3+)) in the uterine glandular epithelial cells and within the stroma of chorionic villi in the areolar part. An immunohistochemical examination revealed tartrate-resistant acid phosphatase (TRAP; known as uteroferrin in uteri) in the cytoplasm of glandular cells and areolar trophoblast cells. This result suggested that, in cetaceans, uteroferrin is used to supply iron to the fetus. Furthermore, immunoreactivity for P450scc and P450arom was detected in trophoblast cells, but not in areolar trophoblast cells, suggesting that trophoblast cells synthesize estrogen in whale placentas. Therefore, we herein immunohistochemically revealed the localization of aromatase and uteroferrin in cetacean placentas during pregnancy for the first time.

Macrophages are the determinant of resistance to and outcome of nonlethal Babesia microti infection in mice.

In the present study, we examined the contributions of macrophages to the outcome of infection with Babesia microti, the etiological agent of human and rodent babesiosis, in BALB/c mice. Mice were treated with clodronate liposome at different times during the course of B. microti infection in order to deplete the macrophages. Notably, a depletion of host macrophages at the early and acute phases of infection caused a significant elevation of parasitemia associated with remarkable mortality in the mice. The depletion of macrophages at the resolving and latent phases of infection resulted in an immediate and temporal exacerbation of parasitemia coupled with mortality in mice. Reconstituting clodronate liposome-treated mice at the acute phase of infection with macrophages from naive mice resulted in a slight reduction in parasitemia with improved survival compared to that of mice that received the drug alone. These results indicate that macrophages play a crucial role in the control of and resistance to B. microti infection in mice. Moreover, analyses of host immune responses revealed that macrophage-depleted mice diminished their production of Th1 cell cytokines, including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Furthermore, depletion of macrophages at different times exaggerated the pathogenesis of the infection in deficient IFN-γ(-/-) and severe combined immunodeficiency (SCID) mice. Collectively, our data provide important clues about the role of macrophages in the resistance and control of B. microti and imply that the severity of the infection in immunocompromised patients might be due to impairment of macrophage function.

Effectiveness of electroacupuncture at Zusanli (ST36) on the immunohistochemical density of enteroendocrine cells related to gastrointestinal function.

The purpose of this study was to examine the effects of electroacupuncture at Zusanli on the immunohistochemical density of enteroendocrine cells related to gastrointestinal function. The authors investigated the histochemical changes of mucous substances and immunohistochemical density of gastrin, serotonin, calcitonin gene-related peptide (CGRP), insulin, and pancreatic polypeptide (PP) secreting cells in rats. Staining density of mucous substances and the enteroendocrine cells of the gastrointestinal tract was observed with histochemical and immunohistochemical methods. Stainless steel needles with a diameter of 0.25 mm were inserted into Zusanli (St36, 5mm below the head of the fibula under the knee joint, and 2mm lateral to the anterior tubercle of the tibia) and connected to an electrical stimulator. The electroacupuncture (EA) stimulation was delivered for 30 minutes at 10 mA, 2 Hz in EA stimulation (2EA group) or 4 Hz in EA stimulation (4EA group) in each experimental group. In 4EA stimulation at the Zusanli, staining density of Alcian blue-periodic acid-Schiff on mucous substances of the stomach body was stronger than those of the 2EA and control groups. Periodic acid-Schiff staining density of pyloric mucosa in the 4EA group was stronger than that of the 2EA and control groups. The immunohistochemical staining density of gastrin, serotonin, and CGRP-secreting cells of pylorus in the 2EA and 4EA groups was stronger than that of the control group. Immunohistochemical staining density of insulin and PP secreting cells of islets of the pancreas in the 2EA and 4EA groups was stronger than that of the control group. These results suggest that EA stimulus at St36 has the potential to influence gastric mucous substances and enteroendocrine cells (gastrin, serotonin, CGRP, insulin, and PP) that subsequently modulate digestive functions.

Immunohistochemical study on the ontogenetic development of the regional distribution of peptide YY, pancreatic polypeptide, and glucagon-like peptide 1 endocrine cells in bovine gastrointestinal tract.

The regional distribution and relative frequency of peptide YY (PYY)-, pancreatic polypeptide (PP)-, and glucagon-like peptide 1 (GLP-1)-immunoreactive (IR) cells were determined immunohistochemically in the gastrointestinal tract at seven ontogenetic stages in pre- and postnatal cattle. Different frequencies of PYY-, PP-, and GLP-1-IR cells were found in the intestines at all stages; they were not found in the esophagus and stomach. The frequencies varied depending on the intestinal segment and the developmental stage. The frequencies of PYY- and PP-IR cells were lower in the small intestine and increased from ileum to rectum, whereas GLP-1-IR cells were more numerous in duodenum and jejunum, decreased in ileum and cecum, and increased again in colon and rectum. The frequencies also varied according to pre- and postnatal stages. All three cell types were most numerous in fetus, and decreased in calf and adult groups, indicating that the frequencies of these three types of endocrine cells decrease with postnatal development. The results suggest that these changes vary depending on feeding habits and adaptation of growth, secretion, and motility of intestine at different ontogenetic stages of cattle.

Immunohistochemical features of substance P-immunoreactive chromaffin cells and nerve fibers in the rat adrenal gland.

The distribution of substance P (SP) immunoreactivity and the colocalization of SP with other bioactive substances in chromaffin cells and nerve fibers were investigated in the rat adrenal gland at the light microscopic level. In the capsule and cortex, SP immunoreactivity was seen in some nerve fibers around blood vessels and in thick nerve bundles passing through the cortex directly into the medulla. In the medulla, the SP immunoreactivity was observed in a small number of chromaffin cells; these SP-immunoreactive chromaffin cells were either phenylethanolamine N-methyltransferase (PNMT) immunoreactive or immunonegative, indicating that they were either adrenaline cells or noradrenaline (NA) cells. SP-immunoreactive varicose nerve fibers were also found in the medulla and were in contact with a cluster of the NA cells showing catecholamine fluorescence, which suggests that SP from medullary nerve fibers may regulate the secretory activity of the NA cells. Because no SP-immunoreactive ganglion cell was present in the rat adrenal gland, the intra-adrenal nerve fibers were considered to be extrinsic in origin. The double-immunostaining method further revealed that the SP-immunoreactive chromaffin cells also exhibit immunoreactivities for calcitonin gene-related peptide (CGRP), and neuropeptide tyrosine (NPY), suggesting that these peptides can also be released from the chromaffin cells by certain stimuli. The intra-adrenal nerve fibers in the medulla were composed of SP-single immunoreactive, and SP/CGRP-, SP/choline acetyltransferase (ChAT)-, SP/nitric oxide synthase (NOS)-, SP/pituitary adenylate cyclase activating polypeptide (PACAP)-, ChAT/NOS-, and ChAT/PACAP-immunoreactive nerve fibers, which may affect the secretory activity of the NA cells. In the adrenal capsule, the nerve fibers were present around blood vessels and showed immunoreactivities for SP/ CGRP, SP/NPY, SP/NOS, and SP/vasoactive intestinal polypeptide, suggesting that the origin of nerve fibers in the capsule may differ from those in the medulla.

The mesencephalic trigeminal sensory nucleus is involved in acquisition of active exploratory behavior induced by changing from a diet of exclusively milk formula to food pellets in mice.

Post-weaning mice fed exclusively milk display low-frequency exploratory behavior [Ishii, T., Itou, T., and Nishimura, M. (2005) Life Sci. 78, 174-179] compared to mice fed a food pellet diet. This low-frequency exploratory behavior switched to high-frequency exploration after a switch from exclusively milk formula to a food pellet diet. Acquisition of the high-frequency exploratory behavior was irreversible. Recently, we demonstrated that the mesencephalic trigeminal nucleus (Me5) is involved in the control of feeding and exploratory behavior in mice without modulating the emotional state [Ishii, T., Furuoka, H., Itou, T., Kitamura, N., and Nishimura, M. (2005) Brain Res. 1048, 80-86]. We therefore investigated whether the Me5 is involved in acquisition of high-frequency exploratory behavior induced by the switch in diet from an exclusively milk formula to food pellets. Mouse feeding and exploratory behaviors were analyzed using a food search compulsion apparatus, which was designed to distinguish between the two behaviors under standard living conditions. Immunohistochemical analysis of immediate early genes indicated that the Me5, which receives signals from oral proprioceptors, is transiently activated after the diet change. The change from low-frequency to high-frequency exploratory behavior was prevented in milk-fed mice by bilateral lesion of the Me5. These results suggest that the Me5 is activated by signals associated with mastication-induced proprioception and contributes to the acquisition of active exploratory behavior.

One-stage unifocalization and palliative right ventricular outflow tract reconstruction.

We report a successful case of one-stage unifocalization concomitant with palliative right ventricular outflow tract reconstruction for pulmonary atresia and ventricular septal defect with major aortopulmonary collateral arteries and central pulmonary arterial absence. Through a median sternotomy, one-stage unifocalization was accomplished using autologous pericardial conduit and no prosthetic material. To achieve an adequate pulmonary blood flow, the right ventricular outflow tract was reconstructed so that it would be 70% of the normal pulmonary annular diameter, while the ventricular septal defect was left open. One-stage unifocalization concomitant with palliative right ventricular outflow tract reconstruction is considered to be a good surgical option for selected patients with these diseases.

Strategies for a schizophrenic patient who required aortic valve replacement.

An aortic valve was replaced in a schizophrenic patient with violent behaviour. The usual protocol was altered and strategies to prevent accidents before and after surgery were devised. Minimally invasive surgery, administering sedation during the critical recovery period, and therapeutic use of communication as a psychological intervention were effective strategies. The patient recovered well and was discharged on day 14 after surgery.

Left ventricular outflow tract obstruction due to anomalous insertion of papillary muscle.

A 56-year-old man who complained of quadrantic hemianopsia was admitted to determine its etiology. Cerebral angiography revealed no organic stenosis. Echocardiography showed clear direct continuity between a hypertrophied anterolateral papillary muscle and the anterior mitral leaflet, and the left ventricular (LV) outflow tract (LVOT) was narrowed by the presence of an accessory papillary muscle. The LVOT obstruction caused an intra-LV pressure overload that resulted in LV concentric hypertrophy. Arrhythmia, such as paroxysmal atrial fibrillation (PAF), was thought to have caused a cerebral embolism. Mitral valve replacement (MVR), septal myectomy, and myectomy of the abnormal papillary muscle were performed, and complete release of the LVOT obstruction was accomplished. Anomalous insertion of papillary muscle is a rare cause of LVOT obstruction. Echocardiography was useful in identifying the papillary muscle malformation, and surgery was completely curative.

Partial translocation for repair of left ventricular rupture after mitral valve replacement.

A new partial translocation technique to repair left ventricular rupture after mitral valve replacement and reimplant a mitral prosthesis is described. We repaired a tear from the interior by using mattress stitches buttressed with strips of Dacron felt, constructed a new annulus with a crescent-shaped piece of bovine pericardium on the left atrial wall above the repaired mitral annulus, and implanted a new prosthesis by using the newly constructed mitral annulus. We consider avoidance of mechanical stress by the prosthesis on the repaired site crucial to a successful outcome, and this new method is useful.

Thoracic surgery residency program and health care system in the United States.

The Japanese Board of General Surgery as well as the Japanese Board of Thoracic Surgery voted to change the educational and training requirements for certification eligibility. The health care system has been modified due to a reduction of the budget for national health care, such as introduction of Diagnosis Related Groups (DRG)/Prospective Payment System (PPS). Several problems regarding the system have already been discussed in the United States, ranging from 30-year experience of residency program to 20-year experience of health care system. In a residency requirement, training period became longer in Japan than in the United States, although number of cases required is ten times less in Japan. The General Surgery Board now became mandatory in Japan, whereas optional in the United States. Interprogram transfer is possible in the United States, but not in Japan. Quality control has been organized for Thoracic Surgery in the United States, but the Japanese counterpart needs more work. In health care system, health insurance is mandatory and operated publicly in Japan while the United States counterpart is mostly optional and managed privately. Malpractice suits are 7-8 times more frequent in the United States. DRG/PPS health care system was temporarily useful in reducing the national budget for health care in the United States. However, an incentive to treat patients with uncomplicated diseases and short hospital stay increased the expenditure. In order to control this, Peer Review Organization has been established and restricted the physician's activities. Because health insurance is optional in the United States, people opt for cheaper health insurance with greater benefits. So the health insurance companies with huge number of customers have had strong opinions over physicians and hospitals. This also has created restrictions on practice. These potential problems may occur shortly in the Japanese residency program and health care system as well. Thus, preventive strategies may be needed.

Induction of cell cycle arrest and p21(CIP1/WAF1) expression in human lung cancer cells by isoliquiritigenin.

Isoliquiritigenin is a natural flavonoid isolated from licorice, shallot and bean sprouts. The effect of isoliquiritigenin on cell proliferation and cell cycle progression was examined in the A549 human lung cancer cell line. Isoliquiritigenin significantly inhibited the proliferation of lung cancer cells in a dose- and time-dependent manner. Flow cytometric analysis demonstrated that isoliquiritigenin restrained the cell cycle progression at G2/M phase. Further examinations using cDNA arrays and real-time quantitative RT-PCR revealed that isoliquiritigenin enhanced the expression of p21(CIP1/WAF1), a universal inhibitor of cyclin-dependent kinases. These results suggest that isoliquiritigenin will be a promising agent for use in chemopreventive or therapeutics against lung cancer.

Protective effects of preischemic treatment with pioglitazone, a peroxisome proliferator-activated receptor-gamma ligand, on lung ischemia-reperfusion injury in rats.

OBJECTIVES: Lung injury induced by ischemia-reperfusion is the main cause of early graft failure after lung transplantation, which may result from oxygen-free radicals, inflammatory cytokine production, and polymorphonuclear leukocyte accumulation into the interstitium, resulting in severe lung edema. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) belongs to the nuclear receptor superfamily and has an anti-inflammatory effect by preventing the activation of transcription factors such as nuclear factor-kappaB (NF-kappaB). NF-kappaB regulates the expression of many genes of early response products in the development of acute inflammation. We examined the effects of pioglitazone, a synthetic ligand of PPAR-gamma, against lung ischemia-reperfusion injury in rats. METHODS: The left lungs of male Wistar rats were rendered ischemic for 90 min and then reperfused for 2 h. Treated animals received pioglitazone (10 mg/kg) 2 h before induction of ischemia. Lung injury was quantified in terms of lung microvascular permeability (Evans blue dye extravasation), tissue lipid peroxidation (thiobarbituric acid reactive substances), and tissue polymorphonuclear leukocyte accumulation (myeloperoxidase activity). The tissue concentrations of tumor necrosis factor-alpha (TNF-alpha) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) were also measured. Statistical analyses were performed by one-way analysis of variance, followed by Sheffe's multiple comparison test. RESULTS: The lung vascular permeability in pioglitazone-treated animals was reduced by 55% of the increase of Evans blue dye extravasation relative to control animals (P=0.003). The protective effects of pioglitazone treatment were correlated with the reduction by 79% of the increase of thiobarbituric acid reactive substances (P=0.045) and the reduction by 58% of myeloperoxidase activity increase (P<0.001). The production of TNF-alpha was reduced by 63% of the increase (P<0.001) and the reduction of CINC-1 was 45% (P<0.001). Pioglitazone did not affect the lung in the sham animals. CONCLUSIONS: Pioglitazone treatment before ischemia attenuated lung ischemia-reperfusion injury in rats. The mechanism of these protective effects involves inhibition of the production of proinflammatory cytokines, polymorphonuclear leukocyte accumulation, and tissue lipid peroxidation, resulting in reduced lung edema.

Localization and expression of gastrin-releasing peptide (GRP) in the bovine cervix.

Gastrin-releasing peptide (GRP) has been suggested as a novel regulatory peptide in the female reproductive tract but the presence of GRP and GRP mRNA in the non-neurogenic tissue of the cervix has not yet been clarified. In the present study, immunohistochemistry and in situ hybridization were used to reveal the distribution of GRP immunoreactivity and expression of GRP mRNA in the bovine cervix. The cervixes from 21 non-pregnant and 20 pregnant cows, and 6 fetuses were used in the study. In the fetus, adult non-pregnant and pregnant specimens, GRP and GRP mRNA were predominantly detected in the luminal epithelial cells of basal areas of peripheral regions of the cervix. Positive staining of GRP in the epithelial cells of the cervix was first detected in the CRL 37 cm of the fetus. During the estrous cycles, the staining intensity of GRP in the epithelial cells was stronger in the follicular phase than in the luteal phase. During the early gestational period, GRP immunoreactivity was detected at relatively similar intensity to the follicular phase. In situ hybridization results ascertained the expression of GRP mRNA in the superficial epithelial cells of the cervix of non-pregnant and pregnant cows. The results suggest that GRP may be important both in the development of the fetal cervix and secretory activity of the epithelial cells of the cervix.

A novel technique for removal of permanent pacemaker leads.

We removed from 4 patients pacemaker leads that had migrated or become infected. Case 1: A 62-year-old man developed uncontrollable infection of the pacing leads. Case 2: A 78-year-old man, whose infected pacemaker was removed, had a second one implanted in the contralateral side; the pacing lead infection from the first procedure, however, was uncontrollable. Case 3: A 56-year-old woman presented with dyspnea and hepatomegaly subsequent to the second implantation of a pacemaker; the pacing leads from the first procedure caused severe stenosis in both the superior and inferior vena cavae. Case 4: A 60-year-old woman had a ruptured and migrated pacing lead in the right ventricle. We operated using a cardiopulmonary bypass and a specially designed plastic tube for removal of the leads. Although Case 2 required reconstruction of the vena cavae, all patients recovered. When removal of pacing leads is necessary, it should be done as soon as possible with cardiopulmonary bypass.

Extrapericardial pulmonary vein clamp technique for pulmonary arteriovenous fistula: report of a case.

The major concern when operating on a patient with a neurologically symptomatic pulmonary arteriovenous fistula (PAVF) is how to prevent a thromboembolic event during surgery. We describe a new technique whereby the extrapericardial pulmonary vein is clamped before transecting the afferent and efferent vessels of the fistula. The potentially pooled clots that can form while manipulating lung are stopped by the clamp. Before the extrapericardial pulmonary vein is declamped, one of the drainage veins is incised halfway and the pooled blood containing the potential clots is completely washed out. We successfully performed segmentectomy using this technique in a 66-year-old man with chronic left hemianopia and a large PAVF in the left anteromedial and lateral basal segments, and no thromboembolic events occurred.

Antithrombin reduces the ischemia/reperfusion-induced spinal cord injury in rats by attenuating inflammatory responses.

Antithrombin (AT) reveals its antiinflammatory activity by promoting endothelial release of prostacyclin (PGI(2)) in vivo. Since neuroinflammation is critically involved in the development of ischemia/reperfusion (I/R)-induced spinal cord injury (SCI), it is possible that AT reduces the I/R-induced SCI by attenuating the inflammatory responses. We examined this possibility using rat model of I/R-induced SCI in the present study. AT significantly reduced the mortality and motor disturbances by inhibiting reduction of the number of motor neurons in animals subjected to SCI. Microinfarctions of the spinal cord seen after reperfusion were markedly reduced by AT. AT significantly enhanced the I/R-induced increases in spinal cord tissue levels of 6-keto-PGFIalpha, a stable metabolite of PGI2. AT significantly inhibited the I/R-induced increases in spinal cord tissue levels of TNF-alpha, rat interleukin-8 and myeloperoxidase. In contrast,Trp(49) -modified AT did not show any protective effects. Pretreatment with indomethacin significantly reversed the protective effects of AT. An inactive derivative of factor Xa, which selectively inhibits thrombin generation, has been shown to fail to reduce SCI. Taken together, these observations strongly suggested that AT might reduce I/R-induced SCI mainly by the antiinflammatory effect through promotion of endothelial production of PGI(2). These findings also suggested that AT might be a potential neuroprotective agent.