Fluorescent visualisation of the hypothalamic oxytocin neurones activated by cholecystokinin-8 in rats expressing c-fos-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 fusion transgenes.

The up-regulation of c-fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c-fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c-fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c-fos gene regulatory sequences that drive the expression of a c-fos-eGFP fusion protein. In c-fos-eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)-8, which selectively activates oxytocin (OXT)-secreting neurones in the hypothalamus. In double transgenic rats that express c-fos-eGFP and an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1-positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK-8. It is possible that not only a plane image, but also three-dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.

PARG dysfunction enhances DNA double strand break formation in S-phase after alkylation DNA damage and augments different cell death pathways.

Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for the degradation of poly(ADP-ribose). PARG dysfunction sensitizes cells to alkylating agents and induces cell death; however, the details of this effect have not been fully elucidated. Here, we investigated the mechanism by which PARG deficiency leads to cell death in different cell types using methylmethanesulfonate (MMS), an alkylating agent, and Parg(-/-) mouse ES cells and human cancer cell lines. Parg(-/-) mouse ES cells showed increased levels of γ-H2AX, a marker of DNA double strand breaks (DSBs), accumulation of poly(ADP-ribose), p53 network activation, and S-phase arrest. Early apoptosis was enhanced in Parg(-/-) mouse ES cells. Parg(-/-) ES cells predominantly underwent caspase-dependent apoptosis. PARG was then knocked down in a p53-defective cell line, MIAPaCa2 cells, a human pancreatic cancer cell line. MIAPaCa2 cells were sensitized to MMS by PARG knockdown. Enhanced necrotic cell death was induced in MIAPaCa2 cells after augmenting γ-H2AX levels and S-phase arrest. Taken together, these data suggest that DSB repair defect causing S-phase arrest, but p53 status was not important for sensitization to alkylation DNA damage by PARG dysfunction, whereas the cell death pathway is dependent on the cell type. This study demonstrates that functional inhibition of PARG may be useful for sensitizing at least particular cancer cells to alkylating agents.

Cyclooxygenase-2 activity is important in craniofacial fracture repair.

The aim of this study was to examine the effect of cyclooxygenase (COX)-2 on bone repair after craniofacial fracture in mice. A 4-mm fracture was created in the parietal bone of 8-week-old male COX-2 wild-type (COX-2(+/+)) and knockout (COX-2(-/-)) mice. Ribonucleic acid was extracted from the fractured bone and analysed. For morphological and histological analysis, the mice were killed 8 and 12 weeks after treatment, and sections were prepared. Three-dimensional computed tomography was performed, and the sections were stained with hematoxylin-eosin for histological examination. Expression of COX-2 messenger ribonucleic acid was induced in COX-2(+/+) mice, but not in COX-2(-/-) mice. Ossification at the fracture site was almost complete 12 weeks after fracture in COX-2(+/+) mice. In COX-2(-/-) mice, incomplete union had occurred at the fracture site. In both types of mice, the fracture site contained no cartilaginous tissue, and the callus formed from the periosteal side. These results suggest that COX-2 plays an important role in craniofacial fracture repair and that COX-2-selective non-steroidal anti-inflammatory drugs might interfere with fracture repair of the membranous viscerocranium in the clinical setting.

Poly(ADP-ribose) Glycohydrolase deficiency sensitizes mouse ES cells to DNA damaging agents.

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme for degradation of poly(ADP-ribose) by splitting ribose-ribose bonds. Parg-deficient (Parg(+/-) and Parg(-/-)) mouse ES cell lines have been established by disrupting both alleles of Parg exon 1 through gene-targeting. A transcript encoding a full length isoform of Parg was eliminated and only low amounts of Parg isoforms were detected in Parg(-/-) embryonic stem (ES) cells. Poly(ADP-ribose) degradation activity was decreased to one-tenth of that in Parg(+/+) ES cells. Parg(-/-) ES cells exhibited the same growth rate as Parg(+/+) ES cells in culture. Sensitivity of Parg(-/-) ES cells to various DNA damaging agents, including an alkylating agent dimethyl sulfate, cisplatin, gemcitabine, 5-fluorouracil, camptothecin, and gamma-irradiation was examined by clonogenic survival assay. Parg(-/-) ES cells showed enhanced lethality after treatment with dimethyl sulfate, cisplatin and gamma-irradiation compared with wild-type (Parg(+/+)) ES cells (p<0.05, respectively). In contrast, a sensitization effect by Parg-deficiency was not observed with gemcitabine and camptothecin. These results suggest the possibility that functional inhibition of Parg leads to sensitization of tumor cells to some chemo- and radiation therapies.

A case of tumoural calcinosis in the temporomandibular joint associated with systemic sclerosis.

Systemic sclerosis (SSc) is a relatively rare condition characterized by the excessive production and deposition of collagen within tissue. This condition is thought to be immunologically mediated and, in addition to its notorious cutaneous manifestations, often involves multiple organs. A case is presented of systemic sclerosis associated with extensive tumoural calcinosis in the temporomandibular joint. There has been no evidence of recurrence or complications during approximately 2 years of follow up, but long-term follow up is essential.

Effects of the short chain sugar acid 2-buten-4-olide on the hypothalamo-pituitary-adrenal axis in normal and adjuvant-induced arthritic rats.

The effects of intraperitoneal (i.p.) administration of 2-buten-4-olide (2-B4O), an endogenous sugar acid, on the hypothalamo-adenohypophysial system were examined in Lewis rats that were normal and in adjuvant-induced arthritic (AA) rats. In comparison with vehicle-treated rats, the plasma corticosterone and c-fos mRNA levels in the paraventricular nucleus (PVN) of normal rats increased significantly after i.p. administration of 2-B4O. Dual immunostaining revealed that almost all corticotrophin-releasing factor (CRF)-immunopositive neurones in the parvocellular division of the PVN exhibited Fos-like immunoreactivity (LI) 120 min after i.p. administration of 2-B4O (100 mg/kg). In the AA rats, repeated i.p. administration of 2-B4O (100 mg/kg) after immunisation significantly suppressed the expression of clinical symptoms and significantly increased plasma concentrations of corticosterone. Further, repeated i.p. administration of 2-B4O significantly increased CRF mRNA levels in the PVN and pro-opiomelanocortin mRNA levels in the anterior pituitary; however, they did not change arginine vasopressin mRNA levels in the parvocellular division of the PVN. These results suggest that i.p. administration of 2-B4O activates the hypothalamo-pituitary-adrenal (HPA) axis via the activation of CRF neurones in the PVN, and the activation of the HPA axis by i.p. administration of 2-B4O may be associated with the inhibition of AA in rats.

Prolactin-releasing peptide is a potent mediator of stress responses in the brain through the hypothalamic paraventricular nucleus.

The effects of i.c.v. administration of prolactin-releasing peptide on neurons in the paraventricular nucleus of rats and plasma corticosterone levels were examined by measuring changes in Fos-like immunoreactivity, c-fos mRNA using in situ hybridization histochemistry, and plasma corticosterone using a specific radioimmunoassay. Approximately 80% of corticotropin-releasing hormone immunoreactive cells exhibited Fos-like immunoreactivity in the parvocellular division of the paraventricular nucleus 90 min after i.c.v. administration of prolactin-releasing peptide. The greatest induction of the c-fos mRNA expression in the paraventricular nucleus was observed 30 min after administration of prolactin-releasing peptide, and occurred in a dose-related manner. Plasma corticosterone levels were also significantly increased 30 min after administration of prolactin-releasing peptide. Next, the effects of restraint stress, nociceptive stimulus and acute inflammatory stress on the expression of the prolactin-releasing peptide mRNA in the dorsomedial hypothalamic nucleus, nucleus of the solitary tract and ventrolateral medulla were examined using in situ hybridization histochemistry for prolactin-releasing peptide mRNA. Restraint stress and acute inflammatory stress upregulated the prolactin-releasing peptide mRNA expression in the nucleus of the solitary tract and ventrolateral medulla. Nociceptive stimulus upregulated the prolactin-releasing peptide mRNA expression in the ventrolateral medulla. Finally, we observed that pretreatment (i.c.v. administration) with an anti-prolactin-releasing peptide antibody significantly attenuated nociceptive stimulus-induced c-fos mRNA expression in the paraventricular nucleus. These results suggest that prolactin-releasing peptide is a potent and important mediator of the stress response in the brain through the hypothalamic paraventricular nucleus.

Expression of immediate early genes and vasopressin heteronuclear RNA in the paraventricular and supraoptic nuclei of rats after acute osmotic stimulus.

Monitoring the expression of immediate early genes (IEGs) is useful for following stress-induced cellular responses in the neuroendocrine system. We have examined the transcriptional activities of four IEGs (c-fos, junB, NGFI-A and NGFI-B) and of the arginine vasopressin (AVP) gene in the hypothalamic paraventicular (PVN) and supraoptic nuclei (SON) of rats after acute osmotic stimuli, using in situ hybridization histochemistry. After intraperitoneal (i.p.) administration of hypertonic saline (2% body weight, 900 mOsm/kg), the expression levels of all IEG mRNAs were increased significantly both in the PVN and SON at as early as 10 min, peaked at 30 min and remained elevated until 60 min. The expression of AVP heteronuclear (hn)RNA also peaked at 30 min, and remained elevated until 180 min. Thirty min after i.p. administration of hypertonic saline (600 mOsm/kg), the expression levels of all IEG mRNAs in the PVN and SON were significantly increased in comparison with those after i.p. administration of isotonic saline (290 mOsm/kg). Regression analysis revealed that expression levels of the IEG mRNAs and AVP hnRNA were positively correlated with the plasma concentration of sodium, and the rates of increase of the expression levels of all IEG mRNAs were similar. The expression levels of all IEG mRNAs examined are useful markers for following the changes of the AVP gene transcription in the PVN and SON after acute osmotic stimuli in rats.

Involvement of presurgical pain in preemptive analgesia for orthopedic surgery: a randomized double blind study.

Preemptive analgesia (PA) is effective in animal models but its clinical effectiveness remains controversial. We examined the effect of preexisting pain on PA. Subjects were recruited from patients needing orthopedic surgery. Some had presurgical pain (fracture surgery and arthritic surgery), while others had no presurgical pain (removal surgery for a tumor, nail or plate). Epidural morphine or a saline control was given preemptively before surgery and maintained until skin closure. Following skin closure, naloxone or placebo was injected intravenously to erase the aftereffects of the morphine. After total recovery, the PCA pump was set to inject epidural morphine. Pain intensity after surgery was measured by a visual analogue scale (VAS), and the amount of morphine used within 48h after surgery. PA was significantly effective for removal surgery, but ineffective for fracture or arthritic surgery. For the fracture and arthritic surgery PA treatment groups, there was a significant correlation between pre- and postsurgical (6h) spontaneous pain, while the corresponding control groups showed no significant correlation. Postsurgical VAS values in the fracture and arthritic surgery control groups increased significantly compared with presurgical VAS values. PA was effective when presurgical pain was absent, but ineffective when presurgical pain was present. We propose that central sensitization is already established by presurgical pain, and preserved until the termination of surgery. The ineffectiveness of PA did not depend on whether the pain was acute (fracture surgery) or chronic (arthritic surgery).

Enhanced beta-receptor-mediated vasorelaxation in hypoxic porcine coronary artery.

To investigate the beta-adrenoceptor-mediated responses in hypoxic coronary arteries, we studied the effect of isoproterenol (Iso) on isolated porcine coronary arteries contracted with endothelin-1 in media aerated with 0, 5, 7.5, and 95% O(2). The concentration-response curve of Iso was significantly shifted to the left by hypoxia (0 and 5% O(2)). In oxygenated and hypoxic arteries, 3 x 10(-8), 10(-6), and 10(-5) M Iso significantly increased the contents of cAMP. However, there was no difference in the increases of cAMP content induced by 3 x 10(-8) M Iso between oxygenated and hypoxic arteries. The content of cAMP induced by high concentrations of Iso (10(-6) and 10(-5) M) was significantly larger in hypoxic than in oxygenated arteries. Furthermore, the potentiation by hypoxia of the Iso-induced vasorelaxation was inhibited by glibenclamide and depolarization by KCl, but not by removal of endothelium and indomethacin. The vasodilatory response to forskolin and dibutyryl cAMP was unaffected by hypoxia. We conclude that activation of the ATP-sensitive K(+) channel may account for the potentiation of the response to Iso in hypoxic coronary arteries.

Inhibition of RhoA translocation and calcium sensitization by in vivo ADP-ribosylation with the chimeric toxin DC3B.

Pretreatment of intact rabbit portal vein smooth muscle with the chimeric toxin DC3B (10(-6) M, 48 h; ; ) ADP-ribosylated endogenous RhoA, including cytosolic RhoA complexed with rhoGDI, and inhibited the tonic phase of phenylephrine-induced contraction and the Ca2+-sensitization of force by phenylephrine, endothelin and guanosine triphosphate (GTP)gammaS, but did not inhibit Ca2+-sensitization by phorbol dibutyrate. DC3B also inhibited GTPgammaS-induced translocation of cytosolic RhoA () to the membrane fraction. In DC3B-treated muscles the small fraction of membrane-associated RhoA could be immunoprecipitated, even after exposure to GTPgammaS, which prevents immunoprecipitation of non-ADP-ribosylated RhoA. Dissociation of cytosolic RhoA-rhoGDI complexes with SDS restored the immunoprecipitability and ADP ribosylatability of RhoA, indicating that both the ADP-ribosylation site (Asn 41) and RhoA insert loop (Wei et al., 1997) are masked by rhoGDI and that the long axes of the two proteins are in parallel in the heterodimer. We conclude that RhoA plays a significant role in G-protein-, but not protein kinase C-mediated, Ca2+ sensitization and that ADP ribosylation inhibits in vivo the Ca2+-sensitizing effect of RhoA by interfering with its binding to a membrane-associated effector.

Down-regulation of G-protein-mediated Ca2+ sensitization in smooth muscle.

Prolonged treatment with guanosine 5'-[gamma-thio]triphosphate (GTP gamma S; 5-16 h, 50 microM) of smooth muscle permeabilized with Staphylococcus aureus alpha-toxin down-regulated (abolished) the acute Ca2+ sensitization of force by GTP gamma S, AIF-4, phenylephrine, and endothelin, but not the response to phorbol dibutyrate or a phosphatase inhibitor, tautomycin. Down-regulation also abolished the GTP gamma S-induced increase in myosin light chain phosphorylation at constant [Ca2+] and was associated with extensive translocation of p21rhoA to the particulate fraction, prevented its immunoprecipitation, and inhibited its ADP ribosylation without affecting the immunodetectable content of G-proteins (p21rhoA, p21ras, G alpha q/11, G alpha i3, and G beta) or protein kinase C (types alpha, beta 1, beta 2, delta, epsilon, eta, theta, and zeta). We conclude that the loss of GTP gamma S- and agonist-induced Ca2+ sensitization through prolonged treatment with GTP gamma S is not due to a decrease in the total content of either trimeric (G alpha q/11, G alpha i3, and G beta) or monomeric (p21rhoA and p21ras) G-protein or protein kinase C but may be related to a structural change of p21rhoA and/or to down-regulation of its (yet to be identified) effector.

Somatosensory evoked potentials recorded from the posterior pharynx to stimulation of the median nerve and cauda equina.

Somatosensory evoked potentials (ppSEPs) in response to stimulation of the median nerve at the wrist and the cauda equina at the epidural space (the L4 level) were recorded from the posterior wall of the pharynx in 15 patients who underwent spinal surgery under general anesthesia, using disc electrodes attached to the endotracheal tube, and compared with segmental spinal cord potentials (seg-SCPs) that were recorded simultaneously from the posterior epidural space (PES). ppSEPs consisted of the initially positive spike (P9) followed by slow positive (P13) and negative (N22) waves. The P13 and N22 of ppSEPs had phase reversal relationship with the P2 and N2 recorded from the PES, respectively. The peak latencies of P9 (9.40 +/- 0.7 ms) (mean +/- SD), P13 (13.1 +/- 0.9 ms), and N22 (22.0 +/- 2.1 ms) of ppSEPs coincided with those of P1, N1 and P2 of seg-SCPs, respectively, ppSEPs were recorded more clearly with a reference electrode on the dorsal surface of the neck than with the reference electrode at the earlobe or back of the hand. The threshold and maximal stimulus intensities were also similar between the ppSEPs and seg-SCPs. Thus, the P9, P13, and N22 components of ppSEPs were thought to have the same origin as the P1, N1 and P2 of seg-SCPs, respectively. Therefore, the P9, P13 and N22 of ppSEPs may reflect incoming volleys through the root, synchronized activities of the interneurons and primary afferent depolarizations (PAD), respectively. ppSEPs in response to cauda equina stimulation showed that the latencies of the two initial components (4.6 +/- 0.4 and 6.4 +/- 0.6 ms) corresponded to those of the SCPs recorded from the PES (4.6 +/- 0.3 and 6.3 +/- 0.5 ms), suggesting that these potentials reflect impulses conducting through the spinal cord, similar to epidurally recorded SCPs.

Relationship between tissue ischemia and venous endothelin-1 during abdominal aortic aneurysm surgery.

OBJECTIVES: Several substances may be released from ischemic tissues with the declamping shock that occurs during abdominal aortic aneurysm surgery. To clarify the relationship between tissue ischemia and venous endothelin-1 (ET-1) level in humans, plasma ET-1 and oxygen content in the iliac vein were measured before anesthesia, after the induction of anesthesia, after the release of the proximal and first distal clamps, after the release of the second distal clamp, and 1 hour after the second clamping. DESIGN: Prospective study. SETTING: A University hospital. PARTICIPANTS: Seven patients who underwent abdominal aneurysmectomy and replacement with bifurcated graft. INTERVENTIONS: A 20G catheter was inserted into the radial artery for the direct measurement of blood pressure and for collecting arterial blood. An 18G, 20-cm catheter was inserted into the femoral vein for collecting venous blood from the lower extremities. MEASUREMENTS AND MAIN RESULTS: The arterial and venous ET-1 levels did not change after the induction of anesthesia. Immediately after the initial release of the proximal and distal clamps, venous oxygen content dramatically decreased from 11.3 to 3.6 mL/dL (vol%) with significant increases in venous ET-1 concentration from 2.3 to 4.9 pg/mL. Concomitant with the decrease in venous oxygen content, venous plasma pH and base excess decreased with increase in PCO2, suggesting that ischemic changes in tissues distal to the cross-clamp may occur during aortic clamping. Venous ET-1 levels were significantly correlated with venous oxygen content, pH, PO2, oxygen saturation, base excess, blood sodium concentration, and potassium concentration. One hour after the second declamping, the venous ET-1 level remained high in comparison with the preanesthetic level, whereas the venous oxygen content returned to the preanesthetic level. There was no correlation between venous plasma ET-1 and venous plasma norepinephrine or epinephrine concentration. CONCLUSIONS: Tissue ischemia may increase venous ET-1 levels in humans. Factor(s) other than tissue ischemia may provoke the increase in venous ET-1 that occurs after the release of the second distal clamp.

Hypoxia modifies the vasodilatory effects of nitroglycerin, prostaglandin E1, and hydralazine on isolated porcine coronary arteries.

To evaluate the potency of vasodilatory drugs in hypoxia, we studied the effects of nitroglycerin (NTG), prostaglandin E1 (PGE1), and hydralazine on porcine coronary artery constricted with endothelin-1 (ET-1) in both oxygenated and hypoxic conditions. Removal of endothelium potentiated NTG-induced relaxation in oxygenated conditions. Hypoxia potentiated relaxation of endothelium-intact arteries induced by NTG, but not relaxation of endothelium-denuded arteries. These findings suggest that hypoxia may modify endothelial function in NTG-induced relaxation. The relaxation of endothelium-intact and -denuded arteries induced by PGE1 in hypoxia was significantly greater than that in the oxygenated condition. PGE1 significantly increased the content of cyclic AMP in the hypoxic condition; it was much greater than that in the oxygenated condition, suggesting that hypoxia may enhance PGE1-induced relaxation by increasing cyclic AMP levels. Hypoxia attenuated hydralazine-induced relaxation in both endothelium-intact and denuded arteries. Indomethacin and aspirin attenuated hydralazine-induced relaxation in the oxygenated condition, suggesting that cyclooxygenase-related eicosanoid(s) may be involved in hydralazine-induced relaxation. However, indomethacin did not alter relaxation of hypoxic arteries induced by hydralazine. These findings suggest that hypoxia may inactivate cyclooxygenase in hydralazine-induced relaxation. Hypoxia may greatly modify the action of vasodilators on porcine coronary smooth muscle.

SPECT demonstration of splenosis.

Splenosis is defined as the heterotopic autotransplantation of splenic tissue. The main cause of splenosis is splenic rupture following abdominal trauma, in which fragments of splenic tissue are seeded throughout the peritoneal cavity. Demonstration of splenosis by scintigraphy or CT imaging has been reported, but there is no previous report of simultaneous demonstration by SPECT and CT imaging. Autotransplantation of splenic tissue in the abdominal cavity forms a solid tumor-like image on CT and is difficult to differentiate from other abdominal masses. In such cases, SPECT demonstration of splenosis is very important for identification of its exact site. A case report of splenosis is presented, in which simultaneous demonstration by SPECT and CT was performed. This was confirmed at laparotomy for gastrectomy.