Protrudin-deficient mice manifest depression-like behavior with abnormalities in activity, attention, and cued fear-conditioning.

Protrudin is a protein that resides in the membrane of the endoplasmic reticulum and is highly expressed in the nervous system. Although mutations in the human protrudin gene (ZFYVE27, also known as SPG33) give rise to hereditary spastic paraplegia (HSP), the physiological role of the encoded protein has been largely unclear. We therefore generated mice deficient in protrudin and subjected them to a battery of behavioral tests designed to examine their intermediate phenotypes. The protrudin-deficient mice were found to have a reduced body size and to manifest pleiotropic behavioral abnormalities, including hyperactivity, depression-like behavior, and deficits in attention and fear-conditioning memory. They exhibited no signs of HSP, however, consistent with the notion that HSP-associated mutations of protrudin may elicit neural degeneration, not as a result of a loss of function, but rather as a result of a gain of toxic function. Overall, our results suggest that protrudin might play an indispensable role in normal neuronal development and behavior.

A resected perivascular epithelioid cell tumor (PEComa) of the pancreas diagnosed using endoscopic ultrasound-guided fine-needle aspiration.

Primary perivascular epithelioid cell tumors (PEComas) of the pancreas are extremely rare. We herein report our experience with a patient who had a primary PEComa of the pancreas that was diagnosed by the preoperative histopathological examination of a biopsy specimen obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). The patient was a 43-year-old woman whose chief complaint was abdominal pain. Imaging studies revealed a pancreatic tumor. Gastrointestinal stromal tumor (GIST), solid pseudopapillary tumor and neuroendocrine tumor were considered in the differential diagnosis. A histopathological examination of a specimen of the tumor obtained using EUS-FNA showed spindle-shaped tumor cells with enlarged nuclei and eosinophilic cytoplasm. The tumor cells proliferated in a sheet-like fashion and stained positive for the melanoma-associated antigen HMB-45. A PEComa was thus diagnosed. If an adequate tumor specimen can be obtained using EUS-FNA, immunostaining may facilitate the diagnosis of extremely rare diseases and therefore assist in deciding the treatment policy.

Perineural invasion and preoperative serum CA19-9 as predictors of survival in biliary tract cancer.

BACKGROUND: Billiary tract cancer requires invasive surgical procedures for cure, and the risk factors related to patient prognosis remain controversial. PATIENTS AND METHODS: Out of the 111 patients who underwent resection of extrahepatic biliary tract tumors from 1986 to 2010, the records for 88 with both ampullary and extrahepatic bile duct cancer, which included all information for evaluation of the clinicopathological factors, were employed in a multivariate analysis. RESULTS: On univariate analysis, significant prognostic factors of poor survival unrelated to TNM factors were preoperative biliary drainage, high preoperative CA19-9 value, high preoperative CEA value, lymphatic invasion, perineural invasion, macroscopic growth pattern, histology, operative procedures (surgery), tumor persistence, high postoperative CA19-9 value, and postoperative chemotherapy. On multivariate analysis, perineural invasion (p=0.025) was the only prognostic factor independent of stage, for survival of patients with biliary tract cancer including ampullary cancer. When ampullary cancer was excluded, both perineural invasion and preoperative CA19-9 were the remaining prognostic factors independent of stage. The combination of both factors can very accurately identify long-term and short-term survivors of biliary tract cancer. CONCLUSION: The present study, to our knowledge, for the first time shows that both perineural invasion and preoperative CA19-9 are important prognostic factors in biliary tract cancer, and this would be beneficial for clinical clarification of the optimal strategies for this type of cancer.

Dynamic development of the first synapse impinging on adult-born neurons in the olfactory bulb circuit.

The olfactory bulb (OB) receives and integrates newborn interneurons throughout life. This process is important for the proper functioning of the OB circuit and consequently, for the sense of smell. Although we know how these new interneurons are produced, the way in which they integrate into the pre-existing ongoing circuits remains poorly documented. Bearing in mind that glutamatergic inputs onto local OB interneurons are crucial for adjusting the level of bulbar inhibition, it is important to characterize when and how these inputs from excitatory synapses develop on newborn OB interneurons. We studied early synaptic events that lead to the formation and maturation of the first glutamatergic synapses on adult-born granule cells (GCs), the most abundant subtype of OB interneuron. Patch-clamp recordings and electron microscopy (EM) analysis were performed on adult-born interneurons shortly after their arrival in the adult OB circuits. We found that both the ratio of N-methyl-D-aspartate receptor (NMDAR) to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), and the number of functional release sites at proximal inputs reached a maximum during the critical period for the sensory-dependent survival of newborn cells, well before the completion of dendritic arborization. EM analysis showed an accompanying change in postsynaptic density shape during the same period of time. Interestingly, the latter morphological changes disappeared in more mature newly-formed neurons, when the NMDAR to AMPAR ratio had decreased and functional presynaptic terminals expressed only single release sites. Together, these findings show that the first glutamatergic inputs to adult-generated OB interneurons undergo a unique sequence of maturation stages.

Adult neurogenesis promotes synaptic plasticity in the olfactory bulb.

To explore the functional consequences of adult neurogenesis in the mouse olfactory bulb, we investigated plasticity at glutamatergic synapses onto GABAergic interneurons. We found that one subset of excitatory synapses onto adult-born granule cells showed long-term potentiation shortly after their arrival in the bulb. This property faded as the newborn neurons matured. These results indicate that recently generated adult-born olfactory interneurons undergo different experience-dependent synaptic modifications compared with their pre-existing mature neighbors and provide a possible substrate for adult neurogenesis-dependent olfactory learning.

Preoperative serum CA19-9 and dissected peripancreatic tissue margin as determiners of long-term survival in pancreatic cancer.

BACKGROUND: Pancreatic cancer, a particularly deadly form of malignancy, has increased in the last decade worldwide. The purpose of this study is to identify markers for determining and identifying possible long-term survivors in cases of advanced pancreatic cancer. PATIENTS AND METHODS: 117 patients with pancreatic ductal carcinoma, including 89 with invasive tubular adenocarcinoma of the pancreas, Japan Pancreas Society (JPS) stage III-IVb patients, who underwent tumor resection between 1986 and 2006. RESULTS: Univariate prognostic analyses of the 5-year disease-specific survival (DSS) revealed that JPS stage (P < 0.0001), preoperative serum carbohydrate antigen 19-9 (CA19-9) level (preCA19-9; P < 0.0001), dissected peripancreatic tissue margin (DPM; P < 0.0001), residual tumor (R factor; P = 0.0007), lymph node metastasis density over 10% (ND10; P = 0.006), volume of the stromal connective tissue (stroma factor; P = 0.008), growth pattern (P = 0.01), and histology (P = 0.03) were all significantly associated with poor outcome in advanced pancreatic cancer. Multivariate logistic analysis confirmed that preCA19-9 [P = 0.0006, relative risk (RR) = 2.16] and DPM (P = 0.04, RR = 1.62) were prognostic factors that remained, independent of JPS stage (P = 0.001). The higher preCA19-9 was, the worse the prognosis was. Astonishingly, among JPS stage III cases, 76.9% of the patients with preCA19-9 below 37 U/ml survived more than 5 years. This, combined with an analysis of DPM, allowed us to identify those with the potentiality for long-term survival. CONCLUSION: Our results reveal for the first time that it is possible with JPS stage III-IVb invasive tubular adenocarcinomas of the pancreas to differentiate prognostic groups and potential survival rates, like with other cancers.

TNF-alpha induces thromboxane receptor signaling-dependent microcirculatory dysfunction in mouse liver.

TNF-alpha is a critical mediator of hepatic microcirculatory dysfunction during endotoxemia. The present study was to investigate the role of thromboxane A2 (TXA2) and the biological significance of thromboxane prostanoid (TP) receptor signaling in TNF-alpha-mediated hepatic microcirculatory dysfunction in male C57Bl/6 mice. The number of leukocytes adhering to the endothelial cells of the hepatic microvessels (the portal venules, sinusoids, and central venules) and the percentage of nonperfused sinusoids were determined using in vivo fluorescence microscopy. FR167653, an inhibitor of TNF-alpha, was administered 0 and 2 h after LPS injection. A TXA2 synthase inhibitor, OKY-046, was administered 30 min before TNF-alpha injection. Thromboxane prostanoid receptor knockout mice were used to investigate whether TNF-alpha-induced hepatic microcirculatory dysfunction is mediated by endogenously produced TXA2. FR167653 reduced LPS-induced leukocyte adhesion (50%-80%) and the percentage of nonperfused sinusoids (55%). The leukocyte adhesion was increased in the portal venules (8-fold), sinusoids (51-fold), and central venules (73-fold) in TNF-alpha-treated mice, accompanied with an increase in sinusoidal perfusion deficits (8-fold). Alanine aminotransferase levels rose as the adhesion of leukocytes increased. OKY-046 administration before TNF-alpha administration reduced leukocyte adhesion (41%-49% decrease) and sinusoid perfusion deficits (34% decrease). In TP receptor knockout mice, the number of adhering leukocytes, the percentage of nonperfused sinusoids, and alanine aminotransferase levels were lower (by 43%-56%, 41%, and 29%, respectively) than in wild-type counterparts. The results suggest that TP receptor signaling may promote hepatic microcirculatory dysfunction elicited by TNF-alpha. Blockade of TNF-alpha generation and TP receptor signaling may be a good strategy for managing endotoxin-induced hepatic injury.

The advantage of Kakita's method with pancreaticojejunal anastomosis for pancreatic resection.

In 1996, we reported the technical aspects of our new method for end-to-side pancreatojejunostomy (Kakita's method) that we performed in combination with the Whipple procedure without any complications related to failure in the anastomosis. In this chapter, we will introduce our technique in end-to-end style pancreatojejunal anastomosis with fewer anastomotic complications. The purpose of this study was to review Kakita's method with pancreatoduodenectomy. From April 1990 to December 2005, 324 consecutive cases of pancreatoduodenectomy were performed in the Department of Surgery at Kitasato University. In our institute, reconstruction in pancreatoduodenectomy is basically performed according to a modified Child's procedure. Our method is simple and can be applied wherever an end-to-side pancreatojejunal anastomosis is required. It consists of three steps: First, a drainage tube is inserted into the pancreatic duct. The second step, which is the unique aspect of our method, is an attachment of the jejunal wall and the cut surface of the pancreas using a single-layer suture technique. This allows us not only to reduce the number of sutures but also to eliminate some of the complicated manipulations required by other methods. The jejunal wall fully covers the cut surface of the pancreas, leaving no uncovered area between the wall and the pancreas. Third, a direct anastomosis between the pancreatic duct and the mucosal layer of the jejunal loop is applied. In our series, pancreatojejunal anastomotic leakage occurred only in 4 out of 324 patients, which was 1.23%. All patients were successfully treated with conservative therapy using drainage for an extended period postoperatively. The newly devised pancreatojejunostomy in our department is a simple, safe, and reliable procedure with excellent results.

Urinary trypsin inhibitor improves viability of the liver in brain-dead rats.

BACKGROUND/AIMS: The present study examined the effect of urinary trypsin inhibitor (UTI) on liver injury in hypotensive brain-dead rats. METHODS: Brain death was induced by inflating a balloon catheter placed in the epidural space. UTI (100,000 units/kg/hour) was intravenously administered from 30 min until 6 hours after the induction of brain death. Systemic hemodynamics and hepatic tissue flow (HTF) were measured, and blood samples and hepatic tissue specimens for morphological examinations were obtained during the experiments. RESULTS: The induction of brain death caused a 30% decrease in both mean arterial pressure and HTF, and an increase in the serum transaminase level in comparison with sham-operated rats. Brain death also increased the serum concentration of cytokine-induced neutrophil chemoattractant (CINC) (4.4-fold), as well as the number of CINC-positive cells (4.4-fold) and sequestered neutrophils in the sinusoids (3.1-fold). Post-treatment of brain-dead rats with UTI restored the HTF and reduced serum transaminase level. UTI decreased plasma CINC level and the number of neutrophils and CINC-positive cells in the sinusoids. CONCLUSIONS: The results suggest that treatment with UTI after the establishment of brain death improved the viability of the liver in hypotensive brain-dead rats by inhibiting CINC production.

Leukotriene B4/leukotriene B4 receptor pathway is involved in hepatic microcirculatory dysfunction elicited by endotoxin.

Leukotrienes (LTs), metabolites of arachidonic acid through 5-lipoxygenase (5-LOX), have been known to play a role in leukocyte recruitment. However, the contribution of LTB4 to liver microcirculatory dysfunction during endotoxemia remains unknown. LTB4 receptor (BLT1) has been identified as a high-affinity receptor specific for LTB4. The present study was conducted to examine the roles of LTB4 and BLT1 in hepatic microcirculatory dysfunction elicited by LPS in mice. The number of leukocytes adhering to the endothelial cells of the hepatic microvessels and perfused sinusoids was determined 4 h after the administration of LPS (0.3 mg/kg, i.v.) to male C57Bl6 mice by in vivo microscopy. A 5-LOX synthase inhibitor, AA-861 (10 or 100 mg/kg, s.c.), was administered 30 min before LPS injection. BLT1 knockout mice were used to investigate whether LPS-induced hepatic microcirculatory dysfunction is mediated by BLT1 signaling. The expression of 5-LOX, intercellular adhesion molecule (ICAM) 1, and TNF-[alpha] in the liver was measured by real-time reverse-transcriptase-polymerase chain reaction. The administration of LPS caused significant accumulation of leukocyte adhesion to the hepatic microvessels and reduced sinusoidal perfusion when compared with saline-treated mice. The hepatic microcirculatory dysfunction elicited by LPS was minimized in mice pretreated with AA-861 or in BLT1 knockout mice. This was associated with the suppression of hepatic expression of 5-LOX, ICAM-1, and TNF-[alpha]. These findings suggest that the LTB4/BLT1 pathway mediates hepatic microcirculatory dysfunction by enhanced expression of ICAM-1 and TNF-[alpha] in a murine model of endotoxemia.

Optimization of somatic inhibition at critical period onset in mouse visual cortex.

Local GABAergic circuits trigger visual cortical plasticity in early postnatal life. How these diverse connections contribute to critical period onset was investigated by nonstationary fluctuation analysis following laser photo-uncaging of GABA onto discrete sites upon individual pyramidal cells in slices of mouse visual cortex. The GABA(A) receptor number decreased on the soma-proximal dendrite (SPD), but not at the axon initial segment, with age and sensory deprivation. Benzodiazepine sensitivity was also higher on the immature SPD. Too many or too few SPD receptors in immature or dark-reared mice, respectively, were adjusted to critical period levels by benzodiazepine treatment in vivo, which engages ocular dominance plasticity in these animal models. Combining GAD65 deletion with dark rearing from birth confirmed that an intermediate number of SPD receptors enable plasticity. Site-specific optimization of perisomatic GABA response may thus trigger experience-dependent development in visual cortex.

Roles of thromboxane A(2) and prostacyclin in the development of atherosclerosis in apoE-deficient mice.

Production of thromboxane (TX) A2 and PG I2/prostacyclin (PGI2) is increased in patients with atherosclerosis. However, their roles in atherogenesis have not been critically defined. To examine this issue, we cross-bred atherosclerosis-prone apoE-deficient mice with mice deficient in either the TXA receptor (TP) or the PGI receptor (IP). Although they showed levels of serum cholesterol and triglyceride similar to those of apoE-deficient mice, apoE-/-TP-/- mice exhibited a significant delay in atherogenesis, and apoE-/-IP-/- mice exhibited a significant acceleration in atherogenesis compared with mice deficient in apoE alone. The plaques in apoE-/-IP-/- mice showed partial endothelial disruption and exhibited enhanced expression of ICAM-1 and decreased expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) in the overlying endothelial cells compared with those of apoE-/-TP-/- mice. Platelet activation with thrombin ex vivo revealed higher and lower sensitivity for surface P-selectin expression in platelets of apoE-/-IP-/- and apoE-/-TP-/- mice, respectively, than in those of apoE-/- mice. Intravital microscopy of the common carotid artery revealed a significantly greater number of leukocytes rolling on the vessel walls in apoE-/-IP-/- mice than in either apoE-/-TP-/- or apoE-/- mice. We conclude that TXA2 promotes and PGI2 prevents the initiation and progression of atherogenesis through control of platelet activation and leukocyte-endothelial cell interaction.

Role of thromboxane derived from COX-1 and -2 in hepatic microcirculatory dysfunction during endotoxemia in mice.

Although thromboxanes (TXs), whose synthesis is regulated by cyclooxygenase (COX), have been suggested to promote inflammation in the liver, little is known about the role of TXA(2) in leukocyte endothelial interaction during endotoxemia. The present study was conducted to investigate the role of TXA(2) as well as that of COX in lipopolysaccharide (LPS)-induced hepatic microcirculatory dysfunction in male C57Bl/6 mice. We observed during in vivo fluorescence microscopic study that LPS caused significant accumulation of leukocytes adhering to the hepatic microvessels and non-perfused sinusoids. Levels of serum alanine transaminase (ALT) and tumor necrosis factor alpha (TNF alpha) also increased. LPS raised the TXB(2) level in the perfusate from isolated perfused liver. A TXA(2) synthase inhibitor, OKY-046, and a TXA(2) receptor antagonist, S-1452, reduced LPS-induced hepatic microcirculatory dysfunction by inhibiting TNF alpha production. OKY-046 suppressed the expression of an intercellular adhesion molecule (ICAM)-1 in an LPS-treated liver. In thromboxane prostanoid receptor-knockout mice, hepatic responses to LPS were minimized in comparison with those in their wild-type counterparts. In addition, a selective COX-1 inhibitor, SC-560, a selective COX-2 inhibitor, NS-398, and indomethacin significantly attenuated hepatic responses to LPS including microcirculatory dysfunction and release of ALT and TNF alpha. The effects of the COX inhibitors on hepatic responses to LPS exhibited results similar to those obtained with TXA(2) synthase inhibitor, and TXA(2) receptor antagonist. In conclusion, these results suggest that TXA(2) is involved in LPS-induced hepatic microcirculatory dysfunction partly through the release of TNF alpha, and that TXA(2) derived from COX-1 and COX-2 could be responsible for the microcirculatory dysfunction during endotoxemia.

Experience-dependent slow-wave sleep development.

Sleep enhances plasticity in neocortex, and thereby improves sensory learning. Here we show that sleep itself undergoes changes as a consequence of waking experience during a late critical period in cats and mice. Dark-rearing produced a robust and reversible decrement of slow-wave electrical activity during sleep that was restricted to visual cortex and impaired by gene-targeted reduction of NMDA receptor function.

Separable features of visual cortical plasticity revealed by N-methyl-D-aspartate receptor 2A signaling.

How individual receptive field properties are formed in the maturing sensory neocortex remains largely unknown. The shortening of N-methyl-d-aspartate (NMDA) receptor currents by 2A subunit (NR2A) insertion has been proposed to delimit the critical period for experience-dependent refinement of circuits in visual cortex. In mice engineered to maintain prolonged NMDA responses by targeted deletion of NR2A, the sensitivity to monocular deprivation was surprisingly weakened but restricted to the typical critical period and delayed normally by dark rearing from birth. Orientation preference instead failed to mature, occluding further effects of dark rearing. Interestingly, a full ocular dominance plasticity (but not orientation bias) was selectively restored by enhanced inhibition, reflecting an imbalanced excitation in the absence of NR2A. Many of the downstream pathways involved in NMDA signaling are coupled to the receptor through a variety of protein-protein interactions and adaptor molecules. To further investigate a mechanistic dissociation of receptive field properties in the developing visual system, mice carrying a targeted disruption of the NR2A-associated 95-kDa postsynaptic density (PSD95) scaffolding protein were analyzed. Although the development and plasticity of ocular dominance was unaffected, orientation preference again failed to mature in these mice. Taken together, our results demonstrate that the cellular basis generating individual sensory response properties is separable in the developing neocortex.

Neutrophil elastase inhibitor attenuates lipopolysaccharide-induced hepatic microvascular dysfunction in mice.

The present study was conducted to elucidate the role of neutrophil elastase in lipopolysaccharide (LPS)-induced hepatic microvascular injury by using in vivo microscopy. The intravenous (i.v.) injection of LPS (0.1 mg/kg) in male C3H/HeN mice caused significant hepatic microcirculatory dysfunction: leukocyte adhesion to the sinusoids as well as to the venule, and reduced sinusoidal perfusion, in comparison with vehicle-treated mice. Concomitantly, the serum alanine aminotransferase (ALT) activity at 4 h after LPS injection was significantly increased. The serum concentrations of tumor necrosis factor (TNFalpha) and interleukin-1beta (IL-1beta) at 1 h and at 4 h after LPS injection, respectively, were significantly elevated. Neutrophil elastase inhibitors, ONO-5046 (30 and 90 mg/kg, i.v., 0 and 2 h after LPS injection) or FK706 (30 and 100 mg/kg, i.v., 0 and 2 h after LPS injection) minimized the LPS-induced hepatic microcirculatory dysfunction in a dose-dependent manner. Treatment with ONO-5046 and FK706 significantly reduced the ALT level as well as the serum concentrations of TNFalpha and IL-1beta. In addition, ONO-5046 and FK706 attenuated both hepatic microcirculatory dysfunction and liver injury mediated by TNFalpha and IL-1beta (10 microg/kg i.v.). Furthermore, both ONO-5046 and FK706 improved human neutrophil elastase (10 microg/kg i.v.)-induced hepatic microcirculatory dysfunction, although neutrophil elastase did not increase the levels of TNFalpha and IL-1beta. These results suggest that neutrophil elastase aggravates the LPS-induced hepatic microvascular dysfunction. Neutrophil elastase inhibitors attenuate hepatic microvascular dysfunction in response to LPS by inhibiting TNFalpha and IL-1beta production. Neutrophil elastase inhibitors also reduce the microvascular dysfunction mediated by TNFalpha and IL-1beta as well as by neutrophil elastase.

Effect of FR167653, a novel inhibitor of tumor necrosis factor-alpha and interleukin-1beta synthesis on lipopolysaccharide-induced hepatic microvascular dysfunction in mice.

Tumor necrosis factor-alpha (TNFalpha) and interleukin-1 (IL-1) have been recognized as key proinflammatory mediators in the pathogenesis of lipopolysaccharide (LPS)-induced liver injury. In the present study we examined the effect of FR167653, a novel inhibitor of TNFalpha and IL-1 synthesis, on the hepatic microvascular response to LPS using in vivo microscopy. Significant hepatic microvascular responses comprising leukocyte adhesion to the sinusoidal wall and central venules and reduced sinusoidal perfusion appeared 2 and 4 h after LPS (0.1 mg/kg, i.v.) injection in male C3H/HeN mice (LPS sensitive) when compared with male C3H/HeJ mice (LPS resistant). The serum concentrations of TNFalpha at 1.5 h and IL-1beta at 4 h after injection of LPS, as determined by enzyme-linked immunosorbent assay, were significantly higher in C3H/HeN mice than in C3H/HeJ mice. Administration of murine TNFalpha or IL-1beta (10 microg/kg., i.v., respectively) in both C3H/HeN and C3H/HeJ mice elicited the hepatic microvascular responses that were similar to those produced by LPS injection in C3H/HeN mice. FR167653 (1 and 10 mg/kg, i.v., 0 and 2 h after LPS injection) significantly reduced leukocyte adhesion and restored sinusoidal perfusion in a dose-dependent manner in C3H/HeN mice 4 h after LPS injection. The levels of TNFalpha, IL-1beta, and alanine aminotransferase also were significantly lower in FR167653-treated endotoxemic C3H/HeN mice than those in vehicle-treated endotoxemic animals. The results suggest that the hepatic microvascular response to LPS is partly mediated by TNFalpha and IL-1beta, and that FR167653 prevents LPS-induced hepatic microcirculatory dysfunction by inhibiting the production of TNFalpha and IL-1beta.