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 10 2 -10 6 cells/droplet (r 2 = = = =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.

ABSTRACT-To clarify the relationship between SART (specific alternation of rhythm in temperature) stress (repeated cold stress) and anxiety, the effects of various types of stress on the behavior of mice were studied in elevated plus-maze tests and then the effects of anxiolytics were evaluated. The percentage of time spent in the open arms of the plus-maze apparatus decreased in mice subjected to SART stress without change in the total number of arm entries. No change was noted in mice subjected to other stresses, such as 1-h, 2-day and 5-day cold stress and 1-h, 15-h and 5´15-h restraint stress. The reduction in the percentage of time spent in the open arms caused by SART stress was inhibited by single and repeated administrations of diazepam and alprazolam and by a single administration of buspirone, which have no influence on the percentage of time spent in the open arms in nonstressed mice, but not by flumazenil, WAY-100635 and chronic treatment with buspirone. The effects of diazepam and buspirone were antagonized by flumazenil and WAY-100635, respectively. The behavior of SART-stressed mice in the plus-maze would thus appear to arise from anxiety, to which benzodiazepine and serotonin receptors are related, but the diazepam binding inhibitor, an endogenous anxiogenic protein, is not. Thus SART-stressed animals may be useful for investigating the psychopharmacological and neuropharmacological basis of anxiety.Keywords: Elevated plus-maze, Stress, SART stress, Anxiety, Repeated cold stressThe relationship between anxiety and stress is a point of much interest. Chronic stress induces mood disorder-like behavior in mammals including humans, and it may be a main factor in the development of anxiety (1, 2). Exposure to various types of stress results in anxiogenic behavior in tests for anxiety in animals. Social stress (3, 4), inescapable electric foot-shocks (5), immersion in water (6) and exposure to unpleasant smells such as cat odor (7) reduce the exploration of open spaces in an elevated plus-maze and cause reduction in social interactions in mice (7, 8).The behavioral characteristics of animals exposed to SART (specific alternation of rhythm in temperature) stress (9 -13) were studied. SART stress (14) is stimulation by repeated and sudden changes in environmental temperature from room temperature to cold temperature, an event that may be encountered by humans in daily life such as in early spring or autumn or when leaving an air-conditioned room in summer or a heated room in winter. Animals exposed to SART stress are a model of autonomic imbalance (15) and show adverse biological events (16 -19) and physiological abnormalities (14, 20 -23). Abnormal behavior is shown in open-field (9), step-down (11) and forced swimming tests (12, 13). This abnormal behavior is normalized by antianxiety agents (9, 12, 13). Changes in neurotransmitters related to abnormal behavior have been noted (24 -26). Certain aspects of these abnormalities are thought to be related to anxiety.To clarify the relationship between SART ...

Exposing mice to 24 and 4°C in alternate 1 hr periods in the day time and maintaining 4°C at night for several days decreases the tail clamp pressure re quired to evoke pain behavior. This model is referred to as SART (specific alterna tion rhythm of temperature) stress. An extract from inflamed skin of rabbits inocu lated with vaccinia virus (neurotropin) clearly normalized the hyperalgesia in this SART stress model. To clarify the mechanism of the hyperalgesia in SART mice and the mode of the antinociceptive action of neurotropin in this model, the influence of systemically administered neurotransmitter related drugs was studied. 1) Neurotropin, 5-hydroxytryptophan and L-dihydroxyphenylalanine significantly normalized the de crease in nociceptive threshold, and muscimol tended to inhibit it in nociceptive threshold in SART stressed mice. 2) Haloperidol, phenoxybenzamine, reserpine, bicu culline, scopolamine, physostigmine and naloxone alone did not influence the nocicep tive threshold in SART stressed mice.3) The antinociceptive effect of neurotropin was significantly attenuated by p-chlorophenylalanine, haloperidol and phenoxybenza mine; and it was completely inhibited by reserpine. 4) Naloxone, bicuculline, scopola mine and physostigmine had no influence on the antinociceptive effect of neurotropin. These results suggest that hypofunction mainly of the monoaminergic systems contrib utes to hyperalgesia in SART stressed mice and that neurotropin produces the anti nociceptive effect by restoring these neural functions.

Abstract-In order to explore the peripheral microcirculation and to obtain an outline of autonomic innervation in SARI (specific alternation of rhythm in temper ature)-stressed (repeated cold-stressed) animals, which are regarded as model animals for clinical vagotonic-type dysautonomia, peripheral tissue blood flow was determined in mice, using the hydrogen clearance method.SART-stressed mice showed a decrease in gastric blood flow, no change in hepatic blood flow and an increase in dermal blood flow. In the mice exposed to the restraint and water immersion stress (RWIS), a type of acute stress, in contrast with SARI stress which is a subacute type, remarkable decreases were observed in gastric, hepatic and dermal blood flows.Changes of both gastric and dermal blood flow in SART stressed mice were dose-dependently prevented and maintained within normal limits by the treatment with Neurotropin, a sedative analgesic which is an extract isolated from vaccinia virus-inoculated and inflamed skin of rabbits. In RWIS loaded mice, Neurotropin exhibited a great preventive effect on changes of blood flow in the stomach, a slight effect in the liver, and no effect in the cutis. When mice were loaded with SARI stress after left-cervical vagotomy, SARI stress failed to elicit any decrease in gastric blood flow. In SARI-stressed mice treated with 6 hydroxydopamine, gastric and dermal blood flows tended to show a further decrease and increase, respectively, over and above the changes caused by SART stress. From these results, it is suggested that SART-stressed mice may have decreased gastric parasympathetic tone, a decrease in sympathetic tone and also other anomalies such as increased tension of the sympathetic cholinergic vasodilator nerves in the cutis.There have been numerous reports that SART (specific alternation of rhythm in temperature) -stressed (repeated cold stressed) animals (1, 2), vagotonic-type model animals (3) for clinical autonomic imbalance, show various physiological changes (3-7). With respect to the circu latory system of SART-stressed animals, several abnormalities (5,6,8,9) such as a continuous lowering of blood pressure (5), sympathicotonic-type ECG (electrocardio gram) (6), increases in blood flow in the abdominal aorta and superior mesenteric artery, and decreased blood flow in the common carotid artery (5), have been previously reported.

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