Influence of Bcg Administration Time on the in-Vivo Migration of Leukocytes

The temporal variation in the migration of polymorphonuclear leukocytes (PMN) induced by live BCG was studied in the mouse. Ten μl of a 5 ± 10⁶ live BCG/ml suspension or sterile saline were placed on a cell trap immediately before its subcutaneous implantation at different clock times: 0100, 0500, 0900, 1300, 1700 and 2100 in animals synchronized with L(0700-1900): D(1900-0700). Eight hours later, the cell trap was removed, prepared for histological identification and counted. PMN counts in the cell trap were maximal 480 min after implantation. Nonspecific migration was thought to occur and the peak value of leukocytes of 22.8 ± 6.1 cells/10,000 μm² was obtained when the saline cell-trap was implanted at 0500. In the BCG-treated mice, a circadian rhythm was observed in the migration of leukocytes. The acrophase was at 1700. The results support the hypothesis that the circadian stage of antigen encounter influences the magnitude of the immune response.     

Influence of Bcg Administration Time on the in-Vivo Migration of Leukocytes

The temporal variation in the migration of polymorphonuclear leukocytes (PMN) induced by live BCG was studied in the mouse. Ten μl of a 5 ± 10⁶ live BCG/ml suspension or sterile saline were placed on a cell trap immediately before its subcutaneous implantation at different clock times: 0100, 0500, 0900, 1300, 1700 and 2100 in animals synchronized with L(0700-1900): D(1900-0700). Eight hours later, the cell trap was removed, prepared for histological identification and counted. PMN counts in the cell trap were maximal 480 min after implantation. Nonspecific migration was thought to occur and the peak value of leukocytes of 22.8 ± 6.1 cells/10,000 μm² was obtained when the saline cell-trap was implanted at 0500. In the BCG-treated mice, a circadian rhythm was observed in the migration of leukocytes. The acrophase was at 1700. The results support the hypothesis that the circadian stage of antigen encounter influences the magnitude of the immune response.     

The role of fractions from Paracoccidioides brasiliensis in the genesis of inflammatory response

The influx of inflammatory cells towards the peritoneal cavity in rats inoculated intraperitoneally with subcellular preparations of the fungus Paracoccidioides brasiliensis was studied. In addition to the dead fungus, also fractions F1 of the cell wall, which mainly consisted of polysaccharides and the lipid extract, induced intense cell migration 4 hr after inoculation, with a greatly increased number of polymorphonuclear leucocytes (PMN). Study of the kinetics of cell influx showed that both fraction F1 and the lipid extract initially induced intense PMN migration between the 4th and 24th hr after inoculation of these agents, followed by migration of mononuclear cells (MN) around the 48th hr. We also observed that migration of these cells increased gradually after inoculation of growing doses of fraction F1. The present data suggest that polysaccharides and lipids isolated from P. brasiliensis may participate in the initial phase of the inflammatory response in paracoccidioidomycosis.     

Chronopharmacokinetic study of gentamicin in dogs

The purpose of this experiment was to determine whether the time of day of single intravenous doses of gentamicin affects the drug's pharmacokinetics in dogs maintained under a 12 h light (08:00 to 20:00 h), 12 h dark (20:00 to 08:00 h) cycle. Using a crossover design, 6 mixed-breed male dogs received a single dose of 2 mg/kg of gentamicin at 8:00 or 20:00 h. Serial blood samples were collected and pharmacokinetic parameters were calculated following each timed dose. The concentration of the antibiotic was lower following the 08:00 h compared to the 20:00 h administration. When gentamicin was administered at 20:00 h, the initial concentration, mean residence time, and area under the disposition curve were significantly higher (p < 0.05) and the apparent volume of distribution of the central compartment, apparent volume of distribution, apparent volume of distribution at steady-state, and total body clearance (1.73+/-0.55 at 20:00 h versus 3.31+/-0.67 L/min/kg at 08:00 h) were significantly lower than for the 08:00 h administration (p < 0.05). Our results show that the pharmacokinetics of gentamicin exhibits significant temporal variation when administered to dogs at different times of day.     

Action of prostaglandin F2 alpha on pregnancy in mice. Prevention of its abortive property by progesterone]

Prostaglandin F2alpha determines a high proportion of abortions in the mouse when administered before implantation at a dose level of 2 mg/kg. After implantation between days 6-8 or 7-9, doses 20 times higher are necessary to produce the effect. Daily progesterone administration, 5 mg per animal, from day 1 to day 17 allow the evolution of pregnancy in 60% of the mice even when 120 mg/kg prostaglandin is given. This dose determines usually 100% abortions. No teratogenic effect has been observed.     

Chronopharmacokinetic Study of Gentamicin in Dogs

The purpose of this experiment was to determine whether the time of day of single intravenous doses of gentamicin affects the drug's pharmacokinetics in dogs maintained under a 12 h light (08:00 to 20:00 h), 12 h dark (20:00 to 08:00 h) cycle. Using a crossover design, 6 mixed‐breed male dogs received a single dose of 2 mg/kg of gentamicin at 8:00 or 20:00 h. Serial blood samples were collected and pharmacokinetic parameters were calculated following each timed dose. The concentration of the antibiotic was lower following the 08:00 h compared to the 20:00 h administration. When gentamicin was administered at 20:00 h, the initial concentration, mean residence time, and area under the disposition curve were significantly higher (p<0.05) and the apparent volume of distribution of the central compartment, apparent volume of distribution, apparent volume of distribution at steady‐state, and total body clearance (1.73±0.55 at 20:00 h versus 3.31±0.67 L/min/kg at 08:00 h) were significantly lower than for the 08:00 h administration (p<0.05). Our results show that the pharmacokinetics of gentamicin exhibits significant temporal variation when administered to dogs at different times of day.     

Orcadian Variation in Amikacin Clearance and Its Effects on Efficacy and Toxicity in Mice with and Without Immunosuppression

A once-daily dosage regimen has been recently recommended in the use of aminoglycoside antibiotics since they induce a postantibiotic effect. In choosing this regimen, one must determine the most appropriate time of day for administration of the drug. We investigated the effects of the timing of amikacin (AMK) administration on the kinetics, the efficacy against intraperitoneal infection with Pseudomonas aeruginosa, and the toxicity of AMK in mice with and without immijnosuppression. We found circadian variations in the kinetics, efficacy, and toxicity of the drug in mice. Male and female ICR mice, which were housed under a light-dark (12:12 h) cycle with free food and water intake, were injected subcutaneously with AMK sulfate 50 mg/kg body wt. There was a circadian variation in AMK clearance for both sexes with the maximum value in the dark phase and the minimum in the light phase after a single administration. When AMK 500 mg/kg/day was repeatedly administered once daily for 30 days, higher toxicity was demonstrated in mice injected with the drug at the time of day with lower AMK clearance, although no difference was demonstrated in the toxicity between the two time points with different AMK clearance when AMK 1,500 mg/kg was administered in a single dose. The ED₅₀ of AMK to cure the infected mice in the midlight phase (13:00 h) with lower clearance was significantly lower than that in the middark phase (01:00 h) with higher clearance. In contrast, the ED₅₀ in the early light phase (09:00 h) was significantly lower than that in the early dark phase (21:00 h), although AMK clearance was not different between these two different time points. In mice premedicated with cyclophosphamide to suppress immune functions, the difference in the ED₅₀ of AMK was still demonstrated between 13:00 and 01:00 h, but not between 09:00 and 21:00 h. The present study shows not only that there were circadian variations in both AMK clearance and toxicity after repeated administration, but also that there was a circadian variation in the efficacy of AMK in mice infected with P. aeruginosa. These results suggest that the timing of drug administration should be considered in pharmacotherapy with AMK and that the most appropriate time of administration in mice and nocturnal animals may be in the midlight (resting) phase. They also suggest that the ED₅₀ of AMK. against P. aeniginosa infection may be influenced not only by the circadian variation in pharmacokinetics but also by the variations in immune systems suppressed by cyclophosphamide.     

Effects of nicotine on oviducal blood flow and embryo development in the rat

Nicotine (5.0 mg/kg) was injected (s.c.) twice daily on Day 1 or Days 1-4 or 1-5 of pregnancy. Cumulative doses of nicotine retarded embryo cell cleavage and substantially reduced embryo cell number (saline vs nicotine: 42.5 +/- 1.7 vs 22.1 +/- 1.9 nuclei/embryo, at 12:00 h on Day 5; P less than 0.05). However, treatment for even 1 day (Day 1) significantly reduced cell number (saline vs nicotine: 42.5 +/- 1.7 vs 30.5 +/- 0.9, at 12:00 h day on Day 5; P less than 0.01). Nicotine injection also resulted in a marked and prolonged reduction in oviduct blood flow (pretreatment vs 90 min after nicotine: 0.61 +/- 0.06 vs 0.37 +/- 0.10 ml/min . g-1; P less than 0.005). The results indicate that, in the rat, even a brief exposure to nicotine, the chief alkaloid of tobacco, reduces oviducal blood flow and the rate of embryo cell proliferation. The embryo is therefore susceptible to the effects of nicotine before implantation.     

Orcadian Variation in Amikacin Clearance and Its Effects on Efficacy and Toxicity in Mice with and Without Immunosuppression

A once-daily dosage regimen has been recently recommended in the use of aminoglycoside antibiotics since they induce a postantibiotic effect. In choosing this regimen, one must determine the most appropriate time of day for administration of the drug. We investigated the effects of the timing of amikacin (AMK) administration on the kinetics, the efficacy against intraperitoneal infection with Pseudomonas aeruginosa, and the toxicity of AMK in mice with and without immijnosuppression. We found circadian variations in the kinetics, efficacy, and toxicity of the drug in mice. Male and female ICR mice, which were housed under a light-dark (12:12 h) cycle with free food and water intake, were injected subcutaneously with AMK sulfate 50 mg/kg body wt. There was a circadian variation in AMK clearance for both sexes with the maximum value in the dark phase and the minimum in the light phase after a single administration. When AMK 500 mg/kg/day was repeatedly administered once daily for 30 days, higher toxicity was demonstrated in mice injected with the drug at the time of day with lower AMK clearance, although no difference was demonstrated in the toxicity between the two time points with different AMK clearance when AMK 1,500 mg/kg was administered in a single dose. The ED₅₀ of AMK to cure the infected mice in the midlight phase (13:00 h) with lower clearance was significantly lower than that in the middark phase (01:00 h) with higher clearance. In contrast, the ED₅₀ in the early light phase (09:00 h) was significantly lower than that in the early dark phase (21:00 h), although AMK clearance was not different between these two different time points. In mice premedicated with cyclophosphamide to suppress immune functions, the difference in the ED₅₀ of AMK was still demonstrated between 13:00 and 01:00 h, but not between 09:00 and 21:00 h. The present study shows not only that there were circadian variations in both AMK clearance and toxicity after repeated administration, but also that there was a circadian variation in the efficacy of AMK in mice infected with P. aeruginosa. These results suggest that the timing of drug administration should be considered in pharmacotherapy with AMK and that the most appropriate time of administration in mice and nocturnal animals may be in the midlight (resting) phase. They also suggest that the ED₅₀ of AMK. against P. aeniginosa infection may be influenced not only by the circadian variation in pharmacokinetics but also by the variations in immune systems suppressed by cyclophosphamide.     

Differential role of CD18 integrins in mediating lung neutrophil sequestration and increased microvascular permeability induced by Escherichia coli in mice

The in vivo contributions of CD18 integrin-dependent and -independent mechanisms in mediating the increases in lung neutrophil (polymorphonuclear leukocyte; PMN) sequestration and microvascular permeability are not well understood. We determined the time course of these responses to Gram-negative sepsis in the mouse lung and addressed the specific contributions of CD18 integrins and ICAM-1. PMN sequestration in the lung was assessed by morphometric analysis, and transalveolar PMN migration was assessed by bronchoalveolar lavage. Lung tissue PMN number increased by 6-fold within 1 h after i.p. Escherichia coli challenge; this value peaked at 3 h (7-fold above control) and decreased at 12 h (3.5-fold above control). PMN migration into the airspace was delayed; the value peaked at 6 h and remained elevated up to 12 h. Saturating concentrations of anti-CD18 and anti-ICAM-1 mAbs reduced lung tissue PMN sequestration and migration; however, peak responses at 3 and 6 h were inhibited by 40%, indicating that only a small component of PMN sequestration and migration was CD18 dependent at these times. In contrast to the time-dependent decreased role of CD18 integrins in mediating PMN sequestration and migration, CD18 and ICAM-1 blockade prevented the increase in lung microvascular permeability and edema formation at all times after E. coli challenge. Thus, Gram-negative sepsis engages CD18/ICAM-1-independent mechanisms capable of the time-dependent amplification of lung PMN sequestration and migration. The increased pulmonary microvascular permeability induced by E. coli is solely the result of engagement of CD18 integrins even when PMN accumulation and migration responses are significantly CD18 independent.     

Effect of unilateral ovariectomy and subsequent ovum implantation in mice.

Unilateral ovariectomy (ULO) was done on any stage of the cycle and the animals were mated within day 1 to day 21 to observe the acute and long term effect of ULO on ovum implantation. Implantation reduced in proportion to single ovary if the animals were mated within 24 hr of ULO. Increase in ovarian weight along with an increase in implantation number continued in mated mice and reached at peak on day 19-21 of ULO (sacrificed after 6 days i.e., 25-27 days of ULO). After ULO the remaining ovary compensated within day 5-6 of ULO even during pregnancy. Ovarian histology showed stimulation of small antral follicles in mice mated on day 3 of ULO (sacrificed after 6 days i.e., day 9 of ULO) along with a decrease of large antral follicles and pre-antral follicles. Preantral follicles were at peak on day 12-14. Large antral follicles attained a peak on day 4 which slowly decreased. The occurrence of implantation in such ULO conditions are discussed.     

Proliferation of erythroid and granulocyte progenitors in the spleen as a function of stem cell dose.

A study of the kinetics of cellular proliferation, in the morphologically unrecongizable haemopoietic progenitor cell compartment, as a function of injected CFU-S dose has been carried out in the spleens of lethally X-irradiated mice using 3H-TdR labelling. Amplification in this proliferating cell compartment was observed to decline as CFU-S dose increased. The number of divisions in the differentiated line arising from CFU-S up to the first appearance of recognizable erythroid precursors were calculated to be 9-2, 12-5, 15 and 17 for the 2, 0-35, 0-05 and 0-007 femur equivalent doses respectively. The growth of cell populations arising from CFU-S was biphasic, with a rapid initial phase having a doubling time of about 6-3 hr, and a slow phase of doubling time around 1 day. Analysis of the rapid phase by the FLM method gave a cycle time of 5-6 hr, Recognizable labelled erythroid precursors were detected at the same time as, or just after, the change in slope of the growth curve. Significant numbers of proliferating (labelled) granulocytes only appeared in the spleens of animals receiving the higher marrow doses (2 and 0-35 femur). The erythroid to granulocyte ratio was also a decreasing function of marrow dose.     

Delta5-3beta-hydroxysteroid dehydrogenase and estradiol-17beta-hydroxysteroid dehydrogenase activity in preimplantation hamster embryos

Preimplantation golden hamster (Mesocricetus auratus) embryos were recovered on days 1 (= day of finding spermatozoa in the vagina) through 4 of pregnancy. Postimplantation embryos were studied in sectioned gestation sacs excised on days 5 and 6. Δ⁵-3β-Hydroxysteroid dehydrogenase (3β-HSD) activity in embryos was determined histochemically. There was no enzyme activity on days 1 and 2. Weak activity was first observed at 08:00–09:00 hr on day 3, the activity then increased, peaked at 01:00–03:00 hr on day 4, considerably declined by 08:00–09:00 hr (day 4), and was absent on days 5 and 6. These results suggest that the preimplantation embryos synthesize steroid hormones. It was previously hypothesized (Dickmann and Dey, 1973, Dickmann and Dey, 1974) that, hormones synthesized by the preimplantation rat embryo participate in the regulation of morula to blastocyst transformation and implantation of the blastocyst. This hypothesis is applicable to the hamster.In addition to 3βHSD, estradiol-17β-hydroxysteroid dehydrogenase activity was observed in day 3 embryos, suggesting that the embryo synthesizes estrogen.     

Optimum dose of LH-RH analogue Fertirelin Acetate for the induction of superovulation in mice.

The optimum dose for establishing superovulation in mice of Fertirelin Acetate (FA), an LH-RH analogue, was examined. Mice were subcutaneously injected with 5 IU of hCG at 17:00 (Day 0), and with various doses of FA (0.001 to 1.0 microg) five times at 4 h intervals on and after 22:00 on Day 0. To induce ovulation, 5 IU of hCG was again injected subcutaneously at 17:00 on Day 2. In the groups administered with doses ranging from 0.01 to 0.5 microg of FA, the number of ovulated eggs was significantly (p<0.05) larger than in the control group (12.9 +/- 5.9). The greatest number of ovulated eggs (22.6 +/- 7.3) was obtained in the group administered with 0.025 microg of FA. The results indicate that the effective dose of LH-RH analogue, FA, is between 0.1 and 0.5 microg for superovulation induction in mice.     

Inhibiting effect of plasma from normal and tumour bearing mice on the mitotic rate of regenerating liver

Plasma from normal mice and from mice bearing the ES2 transplantable malignant tumour was injected intraperitoneally at a dose of 0.01 ml/g body weight in partially hepatectomized mice. Control animals were injected with a solution of sodium citrate in saline. The recipients were killed at the first (14:00 hours/48 h). These times are the time of day and the number of h after partial hepatectomy and second (14:00 hours/72 h) peak times after partial hepatectomy. The number of colchicine metaphases per 1000 nuclei was determined for hepatocytes and litoral cells. A different effect was obtained with plasma from tumour-bearing compared with normal mice. Plasma from both sources when injected 26 h after partial hepatectomy (16:00 hours/26 h) inhibited the mitotic activity of hepatocytes at the next peak of regenerative activity (14:00 hours/48 h). The plasma from tumour-bearing mice also inhibited the peak on the following day (14:00 hours/72 h), whereas plasma from normal mice had no inhibitory effect and, indeed, a compensatory wave was observed at this time. Furthermore, plasma from tumour-bearing mice also showed an inhibitory effect at the first peak (14:00 hours/48 h) when injected at the time of partial hepatectomy (14:00 hours/00 h) or at 22 h before partial hepatectomy (16:00 hours/-22 h) whereas the injection of plasma from normal mice at these times had no inhibitory effect. In the litoral cells the injection of plasma from tumour-bearing mice made 22 h before hepatectomy (16:00 hours/-22 h) led to a stimulation of mitotic activity which was controlled at 14:00 hours/48 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vaginal smear cycle in 4-day cyclic Chinese hamsters, Cricetulus griseus]

The contents of vaginal smear of 4-day cyclic Chinese hamster (Cricetulus griseus) was investigated every 3 hours for 5 days. A light-dark cycle of 14--10 hr was used with the lights turned on at 6 : 00 a.m. Estrous cycle of the Chinese hamster determined by vaginal smears can be divided into 6 periods. The proestrous phase started at about 0 : 00 of day 1, the day of the proestrous phase was designated as day 1 of the estrous cycle. In the afternoon of the same day 1, nucleated epithelial cells gradually increased in number (proestrus : I), and the vaginal contents became to consist solely of nucleated epithelial cells at about 18 : 00 to 21 : 00 (estrus : II). At about 0 : 00 of day 2, however, nucleated epithelial cells were superseded suddenly by cornified epithelial cells, and this phase lasted for 9 to 12 hr (metestrus I : III). Towards the end of the cornified stage, nucleated cells appeared in short duration (metestrus II : IV). And then, in the evening of day 2, leucocytes gradually increased in number with degeneration of nucleated cells (diestrus I : V-1). On day 3, vaginal smear contained a large amount of mucus as well as degenerated nucleated cells and leucocytes (diestrus II : V-2). At about 21 : 000 of day 4, some cornified epithelial cells were seen and then proestrous stage was returned. The females were mated with 3 to 5 males in the evening of day 1, copulation was confirmed in 83.7% females in the next morning,thus the copulation in the Chinese hamster may be thought to occur during the vaginal smear stage of nucleated epithelial cells (estrous phase), i.e. about 18 : 00 to 24 : 00 of day 1.     

IL-2-based immunotherapy alters circulating neutrophil Fc receptor expression and chemotaxis

We performed functional assays on polymorphonuclear (PMN) leukocytes from 21 patients with advanced cancers, before, during, and after IL-2 administration. Of these, 19 were treated with high dose bolus IL-2 infusions (10(5) U/kg every 8 h) and 2 patients received low dose continuous infusions of IL-2 (250 U/kg/h). Five of six patients studied after IL-2 therapy had a decrease in their PMN chemotactic response to FMLP after bolus IL-2 (mean 8 doses) or, after the 4th day of continuous infusion IL-2 (pre-IL-2 values of 82% +/- 17% to 45% +/- 1% post-IL-2, p2 less than 0.004) compared with normal control values. In 8 of 10 patients studied, PMN capacity to oxidize intracellular dichlorofluorescein dye, an indirect measurement of O2- production in response to PMA stimulation, decreased after IL-2 administration (pre-IL-2 mean dichlorofluorescein oxidation (by channel number) 243 +/- 128 vs 3-day post-IL-2 87 +/- 86, p2 less than 0.02). Furthermore, a marked decrease in Fc gamma R III (Leu-11, CD16) expression was observed in 12/13 patients' PMN studied after IL-2 therapy (mean percent of PMN population with positive FcR expression was 81.1 +/- 15.4% pre-IL-2 which decreased to 56.0 +/- 30.5% post-IL-2, p2 less than 0.001). Other PMN surface markers (My4, My7, ICAM-1, LFA1, LFA3, Mac1) did not change significantly. PMN-mediated antibody-dependent cellular cytotoxicity did not change after IL-2 therapy (only 4/15 patients demonstrated more than 50% reduction in antibody-dependent cellular cytotoxicity). PMN phagocytosis of Staphylococcus aureus was also not significantly altered by IL-2 administration in six patients studied (pre-IL-2, 99 +/- 17% vs 111 +/- 28% post-IL-2, p2 greater than 0.2). We conclude that the systemic administration of IL-2 by intermittent or continuous administration is associated with marked changes in PMN function and cell surface receptor expression. These alterations may contribute to the apparent increased susceptibility to bacterial infection observed in these patients.     

LH discharge induced by medial preoptic implantation of estrone or dopamine in the ovariectomized estradiol primed rat.

Effect of implantation of estrogens or catecholamines into the medial preoptic area through chronically implanted double cannula on the release of LH was examined in the ovariectomized estradiol-primed rat. Implantation of estrone at 12:00 on the second or third day after subcutaneous injection of estradiol benzoate (20 mug) increased the serum concentration of LH at 18:00 compared with that in the non-implanted controls, whereas implantation of estradiol benzoate on the third day did not affect it. Dopamine implantation at 12:00 on the third day also induced a significant increase of LH concentration at 18:00, and, in contrast, norepinephrine implantation decreased the concentration at 18:00. It may be said that the medial preoptic area is responsive to estrone and can induce LH release, whereas it is not to estradiol. Furthermore, dopamine was effective for the activation of the medial preoptic area in relation to the inducement of LH release.     

Effects of skin pressure by clothing on digestion and orocecal transit time of food

In order to reveal the influence of clothing skin pressure on digestion of food through the gastrointestinal tract, we examined the absorption of dietary carbohydrate and orocecal transit time of a test meal by means of a breath hydrogen test on 7 healthy young women. In this experiment, we collected breath samples from the participants wearing loose-fitting experimental garment on the second day of the experiment and from the same participants but wearing an additional tight-fitting girdle on the following day for 16 hours and 9 hours, respectively. Skin pressure applied by a girdle on participant's waist, abdomen and hip region was 15.5 +/- 0.4 mmHg (mean +/- SE), 11.0 +/- 0.2 mmHg, and 13.6 +/- 0.6 mmHg, respectively, and the values were 2-3 times larger than those of the experimental garment. The hydrogen concentration vs. time curve showed that breath hydrogen levels at its peaks (15:00, 15:30, 16:00, 16:30, and 17:00 hr) on the third day of the experiment were significantly higher than those of the corresponding time on the second day (p < 0.05 at 17:00 and 15:00, p < 0.01 at 15:00, 16:00 and 16:30). Consequently, significantly pronounced breath hydrogen excretion was observed under the "pressure" clothing condition (p < 0.01). On the other hand, the transit time of the test meal for the subjects wearing a girdle did not differ significantly from that for the subjects wearing the garment of less pressure (270 +/- 18 minutes and 263 +/- 21 minutes, respectively). These results indicate that the clothing skin pressure has an inhibitory effect on the absorption of dietary carbohydrate in the small intestine, but no effect on the orocecal transit time of a meal.     

Model for mediastinal lymph node metastasis produced by orthotopic intrapulmonary implantation of lung cancer cells in mice

This study is designed to establish a pulmonary tumor model to investigate the biology and therapy of lung cancer in mice. Current methods for forming a solitary intrapulmonary nodule and subsequent metastasis to mediastinal lymph nodes are not well defined. Lewis lung carcinoma cell (LLC) suspensions were orthotopically introduced into the lung parenchyma of C57/BL6 mice via a limited skin incision without thoracotomy followed by direct puncture through the intercostal space. The implantation process was performed within approximately 50 sec per mouse, and the operative mortality was less than 5%. Single pulmonary nodules developed at the implanted site in 93% of animals and subsequent mediastinal lymph nodes metastasis were observed in all mice that were succeeded to form a lung nodule after intrapulmonary implantation. The size of tumor nodule and the weight of mediastinal lymph node increased in a time-dependent manner. The mean survival time of mice implanted successfully with LLC cells was 21 +/- 2 days (range; 19-24 days). Histopathological analysis revealed that no metastatic tumor was detectable in the mediastinal lymph nodes on day 11, but metastatic foci at mediastinal lymph nodes were clearly observed on days 17 and 21 after implantation. Other metastases in distant organs or lymph nodes were not observed at 21 days after the implantation. Comparative studies with intrapleural and intravenous injections of LLC cells suggest that the mediastinal lymph node metastasis by intrapulmonary implantation is due to the release of tumor cells from the primary nodule, and not due to extrapulmonary leakage of cells. An intravenous administration of CDDP on day 1 after tumor implantation tended to suppress the primary tumor nodule and significantly inhibited the lymph node metastasis. Thus, a solitary pulmonary tumor nodule model with lymph node metastasis approximates clinical lung cancer, and may provide a useful basis for lung cancer research.