Laura Perissin

Tonic pain response in mice: effects of sex, season and time of day

Seasonal and diurnal variations in tonic pain reactions were examined in female and male CBA/J mice maintained in a 12/12 dark/light cycle, at controlled temperature and humidity conditions. Animals were injected into the dorsum of one hindpaw with a dilute (20 microl, 1%) formalin solution. Pain-related behaviors were quantified as the time spent licking the injected paw and the number of flinching episodes. The experiments were performed during the first part of the light phase (Light: from 7 to 10 a.m.) or during the first part of the dark phase of the diurnal cycle (Dark: from 7 to 10 p.m.), in two different periods of the year: Spring (March-June) and Winter (November-January). Considering all data, females showed a slightly enhanced licking response, as well as an increase in the time spent in self-grooming, in comparison with males. In Spring, the licking and flinching responses were higher during the Dark phase than during the Light phase. This held for both sexes and for both phases of the behavioral response to formalin injection. By contrast, no significant diurnal variation in pain reactions was found in Winter. These seasonal and diurnal differences were not due to nonspecific changes in motor behavior, inasmuch as locomotor activity and self-grooming showed a different pattern: during the second phase after formalin, self-grooming was higher in the Light period in the experiments performed in Spring, whereas locomotor activity showed no significant seasonal changes. These results show that the behavioral reactions to prolonged noxious input, integrated both at spinal and supraspinal sites, undergo similar seasonal and diurnal variations in both sexes, strengthening the importance of chronobiological factors in the modulation of nociception.

Diurnal variations in tonic pain reactions in mice

Diurnal changes in the behavioural reactions to subcutaneous formalin injection (20 microl, 1%) into the dorsum of an hindpaw were examined in female CBA/J mice aged 70-75 days, maintained in a 12/12 dark/ light cycle (light on at 07.00 h; light off at 19.00 h). Mice showed higher pain scores, as expressed by the amount of time spent licking the injected paw and by the number of flinching episodes, when tested under red light at the beginning of the dark phase (19.00-22.00: Dark group) than when tested either under white or red light at the beginning of the light phase of the diurnal cycle (7.00-10.00). The increases in pain reactions at night were found both during the first (0-10 min) and the second (11-55 min) phase of the behavioural response to formalin injection. They were not due to aspecific increases in motor behaviour, since self-grooming actually decreased in the Dark group during the second phase of the response, and the amount of locomotor activity after the injection was similar to, or lower than, that found in mice tested in the morning under white or red light, respectively. In another group of female CBA/J mice tested in the hotplate apparatus (at a temperature of 52 degrees), paw-lick latencies were significantly higher in mice tested at dark during the night, whereas jump (escape) latencies were higher in the morning. These results demonstrate different diurnal variations in the reactions to brief or prolonged noxious stimulation in mice, with greater responses to tonic pain at the onset of the dark phase.

RESTRAINT STRESS REDUCES THE ANTITUMOR EFFICACY OF CYCLOPHOSPHAMIDE IN TUMOR-BEARING MICE

Treatment with the cytotoxic antitumor drug cyclophosphamide is highly effective in mice bearing Lewis lung carcinoma, causing the absence of macroscopically detectable tumors at necroscopy after sacrifice. When the effects of the treatment on survival are determined, a significant increase in survival time and in the proportion of long-term survivors is observed. When restraint stress is further applied, tumors develop in all of the mice treated with cyclophosphamide, and survival time and the fraction of long-term survivors are significantly reduced. Flow cytometry of splenic T-lymphocyte subsets in normal mice indicates a significant decrease in the number of CD3+, CD4+, and CD8+ subsets after treatment with cyclophosphamide and after application of restraint stress; the interaction of the two treatments is significant for CD3+ and marginally significant for CD4+ subsets. The attenuation by restraint stress which was observed for the effects of cyclophosphamide on the presence of tumors at necroscopy and for the survival of the treated mice might thus be interpreted as follows: restraint stress attenuates the immune functions of the host directed toward the weakly immunogenic tumor, an effect which, in the absence of restraint stress, interacts effectively with the cytotoxic action of cyclophosphamide toward tumor cells. The results obtained using this animal model thus indicate that experimental stress reduces the therapeutic efficacy of a cytotoxic antitumor drug; experimental and clinical implications are discussed.

Melatonin decreases bone marrow and lymphatic toxicity of adriamycin in mice bearing TLX5 lymphoma

When CBA male mice bearing TLX5 lymphoma were treated in the evening with a single i.v. dose of adriamycin (20-40 mg/Kg), the administration of a single pharmacological dose of melatonin (10 mg/kg s.c.) 1 hr earlier reduced the acute mortality from 10/24 to 2/24. The increase in survival time caused by adriamycin over drug untreated controls was not reduced by melatonin. The administration of melatonin alone did not cause any antitumor or evident toxic effect. Melatonin also attenuated the reduction caused by adriamycin in the number of bone marrow GM-CFU, and of CD3+, CD4+ and CD8+ splenic T-lymphocyte subsets. Reduced and total glutathione levels were decreased in the bone marrow and in the liver cells of the animals treated with adriamycin, and were significantly restored by melatonin. Moreover, lipid peroxidation by adriamycin was reduced by melatonin, as indicated by malondialdehyde measurement in the liver of the treated animals. These data indicate that the protective effects of melatonin against the host toxicity of the prooxidant antitumor drug, adriamycin, might be attributed at least partially to its antioxidant properties. These findings appear of interest in relation to the physiological rhythmic levels of endogenous melatonin and to the chronotoxicology of anthracyclines.

Increase in therapeutic index of doxorubicin and vinblastine by aptameric oligonucleotide in human T lymphoblastic drug-sensitive and multidrug-resistant cells.

Aptameric GT oligomers are a new class of potential anticancer molecules that inhibit the growth of human cancer cell lines by binding to specific nuclear proteins. We demonstrated that an aptameric GT oligonucleotide increased the therapeutic index of doxorubicin and vinblastine in T lymphoblastic drug-sensitive and multidrug-resistant (MDR) cells. The doxorubicin ID50 decreased 6.5-fold by coadministration of 1 microM GT to CCRF-CEM cells and by 24-fold by coadministration of 0.75 microM GT to CEM-VLB300 cells. In CEM-VLB300 cells, the vinblastine ID50 decreased 11-fold by coadministration of 0.5 microM GT. Control CT sequence did not potentiate the drugs in either CCRF-CEM or CEM-VLB300 cells. The ability of GT to bind to specific nuclear proteins in cancer cells related to the increase in the therapeutic index of doxorubicin and vinblastine. No cooperation was detected by the administration of GT oligomer together with doxorubicin to rat differentiated thyroid FRTL-5 cells and to normal human lymphocytes. These cells did not show binding of GT to the specific nuclear proteins, and they were not sensitive to the cytotoxic action of the GT sequence. Drug potentiation by GT not involving normal human lymphocytes might be exploited to develop a more selective treatment of drug-sensitive and MDR tumors.

Seasonal effects of rotational stress on Lewis lung carcinoma metastasis and T-lymphocyte subsets in mice.

Rotational stress specifically increases the formation of spontaneous lung metastasis in mice bearing Lewis lung carcinoma, without significantly modifying the growth of primary tumor. The increase in metastasis number and volume caused by rotational stress varies in magnitude with a highly significant circannual rhythm; the acrophase approximately coincides with summer solstice. Rotational stress causes a significant reduction in the number of CD3+ and CD4+ T-lymphocyte subsets in summer, whereas in winter the number of CD3+ subset is significantly increased; the CD4+/CD8+ ratio and the number of NK 1.1 antigen positive cells are not significantly modified by rotational stress in both periods considered. The increase in metastasis formation by rotational stress thus appears to negatively correlate with the number of splenic CD3+ and CD4+ T-lymphocyte subsets. This seasonal behavior occurs in spite of the control of light cycle, temperature and humidity in the animal housing, suggesting the existence in the host of an endogenous oscillator with a circannual period. These data indicate the opportunity to consider endogenous rhythms within the host, as well as seasonal factors, in studies on stress and neuroimmunomodulation in experimental oncology.

Posterior Juxtascleral Infusion of Modified Triamcinolone Acetonide Formulation for Refractory Diabetic Macular Edema : One-Year Follow-Up

PURPOSE To evaluate prospectively the efficacy and safety of posterior juxtascleral infusion of a new formulation of triamcinolone acetonide for refractory diffuse diabetic macular edema. METHODS This was an interventional case series. Twenty-two consecutive eyes of 18 patients with refractory diffuse diabetic macular edema were included in the study. Each patient underwent a complete ophthalmic examination, including optical coherence tomography (OCT) and digital fluorescein angiography (FA). All patients received a suspension of 40 mg triamcinolone acetonide, 20 mg sodium chondroitin sulfate, and 15 mg sodium hyaluronate (1.5 mL), delivered posteriorly through a conjunctival and Tenons incision. All patients completed the 1-year follow-up. RESULTS On average, studied eyes received 1.5 treatments. Mean preoperative foveal thickness (+/-SD) and visual acuity (+/-SD) were 474.2 +/- 136.6 microm and 0.6 +/- 0.37 logarithm of the minimum angle of resolution (logMAR), respectively. The central foveal thickness was significantly reduced from baseline at every follow-up visit (P < 0.001). Mean (+/-SD) reductions in macular thickness were 136 +/- 108 microm at 1 week and 128 +/- 122 microm after 1 year of follow-up. Mean (+/-SD) improvement in visual acuity at 12 months was 0.15 +/- 0.21 logMAR (P = 0.008). Visual acuity improvement of one or more lines and three or more lines were observed in 14 (63.6%) and 6 (27.3%) eyes, respectively. Seven eyes (31.8%) required topical treatment due to a significant intraocular pressure increase. CONCLUSIONS Posterior juxtascleral infusion of a new formulation of triamcinolone acetonide is an effective treatment for diffuse diabetic macular edema unresponsive to conventional grid laser photocoagulation. A randomized, larger study is warranted.

Stress and Chemotherapy: Combined Effects on Tumor Progression and Immunity in Animal Models

Abstract: In mice bearing Lewis Lung Carcinoma, rotational and restraint stress specifically increases the formation of lung metastasis, and restraint stress markedly attenuates the antitumor effects of cyclophosphamide. The aim of this investigation was therefore to examine the effects of restraint stress on tumor metastasis in mice bearing MCa mammary carcinoma, and on the effectiveness of CNU and DTIC. Restraint stress increases MCa mammary carcinoma metastasis, caused a marked reduction in cyclophosphamide activity, and a minor attenuation of the effects of CCNU and DTIC. The possible occurence of F seasonal factors, observed for the increase by rotational stress of Lewis lung carcinoma metastasis, was also determined for cyclophosphamide effectiveness. The survival time of control mice is longer in February than in June, and is not appreciably modified by rotational stress. The effects of cyclophosphamide are similar in both seasonal periods, and are similarly attenuated by rotational stress. The seasonal effects of rotational stress, and the reduction of the effects of cyclophosphamide caused by rotational stress, are accompanied by corresponding variations in the number of CD3+ and CD4+ splenic T‐lymphocyte subsets and in the CD4+/CD8+ ratio, respectively. The reported effects of stress on tumor progression and on the effectiveness of cyclophosphamide thus appear to occur via modulation of immune responses of the host directed against the tumor. These data appear of interest for their experimental implications, and suggest the opportunity to consider the role that the stress during treatment may play in determining the effectiveness of clinical antitumor chemotherapy.

Seasonal Dependency of the Effects of Rotational Stress and Cyclophosphamide in Mice Bearing Lewis Lung Carcinoma

The antitumor effects of cyclophosphamide were previously shown to be markedly reduced by the application of restraint stress in mice bearing Lewis lung carcinoma. The aim of this work was to determine the effects of rotational stress on the antitumor action of cyclophosphamide in the same animal-tumor system. Since the effects of rotational stress on metastasis were found to display a circannual rhythm, with a maximum in summer and a minimum in winter, the experiments were performed in June and February. Groups of 10 young female mice were kept under low stress housing conditions, with a 12-12 h light/dark cycle, starting 2 weeks before and during each experiment. Rotational stress caused an increase of metastasis volume to 361% of nonstressed controls in June and a decrease to 32.4% in February. In both seasons, the treatment with cyclophosphamide (240 mg/kg/day for 6 days) caused the absence of detectable metastasis at sacrifice in all mice; its combination with rotational stress caused the presence of metastases in similar proportions (6/10 and 10/10 for June vs February, respectively). The survival time of control mice was approximately twice as long in February as in June and was not appreciably modified by rotational stress; cyclophosphamide was similarly active in both seasons (4/10 and 6/10 long-term survivors for June vs February, respectively), and the number of long-term survivors was reduced to 0/10 in both seasons by rotational stress. The survival of the different experimental groups inversely correlated with the number of metastases as determined at sacrifice at the end of treatment and also with the number of CD3(+) and CD4(+) splenic T-lymphocyte subsets. These results do not appear to depend on the disruption of the circadian organization of the mice by rotational stress or by seasonal differences in cyclophosphamide activity. On the other hand, they can be interpreted assuming that cyclophosphamide reduces tumor metastasis and that T-lymphocyte-mediated immune responses of the host, amenable to modulation by stress and displaying seasonal differences uncoupled from circadian rhythms, further contribute to the tumor inhibitory effects of the drug. The observed differences in tumor metastasis caused by rotational stress and survival time in two different seasons, and the marked attenuation of cyclophosphamide antitumor action by rotational stress, appear of interest for their experimental and clinical implications.

The effect of triamcinolone acetonide, sodium hyaluronate, and chondroitin sulfate on human endothelial cells: an in vitro study

Purpose. TO report the in vitro effect of triamcinolone acetonide (TA), sodium hyaluronate (SH), chondroitin sulfate (CS), and their combination on human endothelial cells. Methods. The antiangiogenic proprieties of 2 formulations of TA (TA-1 and TA-2), 2 formulations of glycosaminoglycan-containing viscoelastic agents (V-1 and V-2), and the association of TA-1 and V-1 were investigated. Their effect on angiogenesis was tested using human endothelial cells cocultured with human fibroblasts and myoblasts. After fixation and staining for CD31, a colorimetric output was obtained. A BCIP/NBT-buffered substrate allowed image analysis of tubule formation. Results. Both formulations of TA significantly reduced tubule formation as compared with controls (p<0.01). Moreover, the antiangiogenic effect of TA-1 was maintained when combined with V-1 (p<0.01). Conclusions. Triamcinolone acetonide alone or in combination with hyaluronate and chondroitin sulfate is able to significantly reduce human endothelial cell proliferation in an in vitro model.