Taeko Hata

Examination of in vitro chemosensitivity test using collagen gel droplet culture method with colorimetric endpoint quantification.

To develop a simpler method of performing the collagen gel droplet‐embedded culture drug sensitivity test (CD‐DST), we examined the introduction of colorimetric quantitative determination of images for evaluation of anticancer effect against cancer cells alone in the presence of fibroblasts, based on differences in proliferative morphology and stainability with neutral red of cells within collagen gel drops determined using a video‐microscope and NIH Image software. In examinations using a human cancer cell line and a fibroblast cell line, a high degree of linearity between number of cancer cells and image‐optical density was found within the range of 102–106 cells/droplet (r2=0.933). Using NIH Image, fibroblast cells could be eliminated at a cut‐off value of 128, and an immunocytochemical method demonstrated that the cells eliminated from the image were indeed fibroblasts, and those remaining were cancer cells. CD‐DST was carried out with mixtures of cancer cells with fibroblasts at various ratios, and the feasibility of evaluating anticancer activity in cancer cells alone with no effect of fibroblasts at any mixing ratio was confirmed. In addition, for CD‐DST of primary cell cultures of human lung cancers collected at the time of surgery, a high correlation between results obtained with the volume supplementation method, a current cell quantification method, and those with the imaging colorimetric quantification method was obtained (r=0.933). These results indicate that introduction of imaging colorimetric quantification utilizing NIH Image makes CD‐DST a quick and simple method that should be highly useful for clinical chemosensitivity testing using primary cell cultures of human cancers.

Behavioral characteristics of SART-Stressed mice in the forced swim test and drug action

SART-stressed (repeated cold-stressed) mice exhibited shortened immobility time in forced swimming tests, and a time-dependent increase in the duration of immobility time of stressed mice was less compared to unstressed mice. These changes were blocked by diazepam and alprazolam without influence on the immobility time of unstressed mice. The shortening of immobility time caused by SART stress was inhibited by repeated pretreatment with imipramine and mianserin, but not by a single dose. In contrast, neither single nor repeated administrations of lithium carbonate had effect on the immobility time of SART-stressed mice. The SART stress technique may be a potential model to investigate the relationship between stress and depression with complex symptoms like excessive emotion- and anxiety-related depression.

Decrease in muscarinic acetylcholine receptors in the small intestine of mice subjected to repeated cold stress.

Abstract The maximal contraction of the small intestine by acetylcholine greatly decreased during repeated cold stress. This change was mainly due to decrease in muscarinic receptors in small intestine, whose amounts were measured by the binding of 3-quinuclidinyl benzilate. Injection of norepinephrine or a tricyclic antidepressant, carpipramine during the exposure to the stress prevented this decrease in muscarinic receptors. The physiological significance of this phenomenon is discussed in relation to vagal hyperactivity under the stress.

Attenuation by prolonged nitric oxide synthase inhibition of the enhancement of fibrinolysis caused by environmental stress in the rat.

1 Nitric oxide (NO) suppresses platelet aggregation and plasminogen activator inhibitor (PAI) release from platelets, playing physiological and/or pathological roles in the haemostatic system. We investigated the effect of NG‐nitro‐L‐arginine methyl ester (L‐NAME), an NO synthase inhibitor, on the disseminated intravascular coagulation (DIC)‐like phenomena in rats under environmental stress, induced by prolonged fluctuation in air temperature, known as SART (specific alternation of rhythm in temperature) stress. 2 Exposure of rats to SART stress for 7 days caused mild DIC‐like symptoms such as thrombocytopenia, hypofibrinogenemia, decreased factor VIII: coagulant activity and shortened euglobulin clot lysis time (ECLT). The enhanced fibrinolysis was accompanied by a marked decrease in the activity of plasma PAI. 3 L‐NAME, but not its D‐enantiomer, when administered orally at 0.3–10 mg kg−1, twice a day for 7‐day exposure to stress, inhibited the stress‐induced decrease in fibrinogen levels in a dose‐dependent manner, whereas it failed to alter platelet count, factor VIII:coagulant activity and plasma protein levels in stressed rats. All these parameters in unstressed rats were resistant to L‐NAME at 10 mg kg−1. 4 Repeated treatment with 10 mg kg−1 of L‐NAME blocked the shortening of ECLT and the decrease in PAI activity following stress exposure, although it was without effect in unstressed rats. 5 The inhibitory effects of L‐NAME at 10 mg kg−1 on the stress‐induced alterations in fibrinogen levels and in ECLT were significantly reduced by coadministered L‐arginine at 1000 mg kg−1. 6 These findings demonstrate that repeated administration of L‐NAME attenuates the enhanced fibrinolysis, without aggravating thrombocytopenia, in SART‐stressed rats. Endogenous NO appears to contribute to the stress‐induced development of fibrinolysis by suppressing plasma PAI activity, most probably as a result of inhibition of the PAI release from platelets.

Characterization of platelet hypofunctions in rats under sart stress (repeated cold stress)

Platelet hypoaggregability has been reported in rats exposed to a chronic form of environmental stress induced by long-lasting fluctuation in air temperature, known as SART (specific alternation of rhythm in temperature) stress. This study examines functional characteristics of platelets from stressed rats in more detail. Exposure to stress reduced aggregation and ATP release in platelets stimulated with collagen, as determined using platelet-rich plasma (PRP). The resting levels of ATP but not ADP in platelets from stressed rats were lower than those from unstressed ones. Collagen-induced release and resting level of serotonin also decreased in platelets from stressed rats. In contrast, stress failed to cause hypoaggregability of washed platelets. Circulating platelet aggregates were detected in stressed rats. From these data, SART stress appears to cause intravascular activation of platelets in spite of in vitro hypofunctions. Alteration in plasma milieu may be associated with stress-induced platelet hypofunctions in PRP.

Platelet hypoaggregability in rats exposed to SART stress (repeated cold stress)

A prolongation of bleeding time accompanied by thrombocytopenia and abnormalities in coagulation-fibrinolysis systems has been observed in laboratory animals exposed to a chronic form of environmental stress induced by severe fluctuations of air temperature, known as SART (specific alternation of rhythm in temperature) stress. In order to clarify the hemostatic profile under SART stress in more detail, the present study examined platelet aggregability in vitro as well as in vivo in stressed rats. During exposure to stress, thrombocytopenia developed from day 5, and remained up to at least day 14. In vitro aggregation of platelets stimulated by ADP or collagen was markedly decreased in stressed rats, compared with unstressed rats. Furthermore, stressed rats exhibited in vivo hypoaggregability of platelets, as estimated by the magnitude of the drop in circulating platelet counts following intravenous injection of ADP and collagen. Protein and cholesterol content in platelets remained constant after stress exposure. These results indicate that SART-stressed rats exhibit platelet dysfunctions in addition to thrombocytopenia. Considering the previous findings, the hemostatic system under SART stress appears to show a general tendency toward hemorrhage.

The kappa opioid receptor agonist SA14867 has antinociceptive and weak sedative effects in models of acute and chronic pain.

We examined the analgesic effect of the selective kappa opioid receptor agonist SA14867 and the balance of its antinociceptive and sedative effects. The ED(50) values of SA14867 after oral administration for acetic acid-induced writhing, first and second phases of the formalin test, and rotarod test in mice were 6.1, 9.3, 2.7, and 19.5mg/kg, respectively. These values were smaller than those of the conventional kappa receptor agonists asimadoline and U-50488H. However, the balance of the antinociceptive and sedative effects of SA14867 was better than those of the other two drugs. Orally administered SA14867 (0.1-1mg/kg) significantly improved the decreased pain threshold in a specific alternation of rhythm in an environmental temperature (SART)-stressed model by prophylactic and therapeutic treatment. Improvement in the decreased pain threshold of SA14867-treated animals was attenuated by the opioid receptor antagonist naloxone. Furthermore, orally administered asimadoline (10-100mg/kg) improved the decreased pain threshold in a SART-stressed model, but the doses were close to those known to induce sedative effects. In addition, SA14867 (0.1-1mg/kg) significantly inhibited the arthritis-induced decrease in the pain threshold. Subcutaneously administered morphine (0.1-1mg/kg) improved the decreased pain threshold in a SART-stressed model; on the contrary, morphine did not inhibit the arthritis-induced decrease in the pain threshold. Moreover, orally administered SA14867 (0.1-1mg/kg) strongly attenuated mechanical allodynia and thermal hyperalgesia in a sciatic nerve ligation model. These results suggest that SA14867 has analgesic effects on chronic pain and may serve as a new therapeutic agent for pain treatment.

Possible involvement of oxygen-derived free radicals in abnormal hemostasis induced by SART stress (repeated cold stress) in laboratory animals

Abnormal hemostatic profiles indicating hemorrhagic tendency have been reported in rodents exposed to prolonged fluctuation in ambient temperature, known as SART (specific alternation of rhythm in temperature)-stressed animals. In this study, investigation was made of possible involvement of oxygen-derived free radicals in the development of stress-induced hemostatic alteration. SART-stressed rats and mice exhibited marked decrease in platelet count, fibrinogen level and factor VIII:C activity. Superoxide dismutase, when administered s.c. twice a day to mice for 7 days of stress exposure, inhibited the above alterations. Catalase given in the same manner, had essentially the same effect, though to a lesser extent. Allopurinol administered orally once daily during stress reduced stress-induced thrombocytopenia, but caused considerable increase in fibrinogen and factor VIII:C activity in stressed and unstressed mice. Lipid peroxide significantly increased in the heart but not in the plasma following stress exposure in rats and mice. Active oxygens would thus appear to be, at least partially, involved in the development of abnormal hemostasis induced by SART stress.

Nasal Mucosal Hypersensitivity in Guinea Pigs Intermittingly Exposed to Cold

To develop an animal model for experimental nasal hypersensitivity and hyperreactivity, guinea pigs were subjected to intermittent exposure to cold temperature (intermittent cold stress, SART stress) for 5 consecutive days. In SART-stressed guinea pigs, nasal mucosal hypersensitivity to histamine evoking sneeze response and nasal hypersecretion in response to methacholine were observed. The hypersensitivity remained for further 7 days after being released from SART stress. On the other hand, such nasal mucosal hypersensitivity was not caused by a continuous cold stress alone, suggesting that intermittent exposure to cold may be of importance for the appearance of nasal mucosal hypersensitivity. In passively sensitized SART-stressed guinea pigs, the quantity of nasal secretion induced by an allergen was significantly increased compared with that of a group of normal animals. The expression of muscarinic acetylcholine receptor (m-ACh.R) became higher in SART-stressed guinea pigs. Thus, hypersensitivity and hyperreactivity in this system were found to be associated with an increase in density of m-ACh.R. SART-stressed guinea pigs will serve as an animal model for hypersensitivity in nasal mucosa, which would be useful for the study of nasal allergy.

Cross tolerance to environmental stress and endotoxin

Mild DIC-like phenomena develop in rodents exposed to environmental stress, induced by prolonged fluctuation in ambient temperature, known as SART (specific alternation of rhythm in temperature) stress, the symptoms being essentially similar to those produced by endotoxin. The aim of the present study was to investigate whether common mechanisms are involved in the stress-induced DIC-like phenomena and endotoxaemia. The mortality in mice after intraperitoneal injection with endotoxin at 8.3, 16.7 and 33.3 mg/kg increased in a dose-dependent manner. In the mice exposed to stress for 7 days, the death rate following endotoxin at 8.3 mg/kg was markedly lower than that in unstressed mice, although no difference was detected between the two groups following endotoxin at 16.7 and 33.3 mg/kg. Conversely, repeated administration of endotoxin at a low dose, 0.5 mg/kg, during exposure to stress for 7 days inhibited the stress-induced thrombocytopenia and hypofibrinogenemia. These findings indicate the development of cross tolerance to prolonged environmental stress and endotoxin, implying involvement of a common mechanism.