One hundred and thirty male (6–8 weeks old) C57BL/6 mice (obtained from Laboratory Animal Center of Faculty of Medicine of University of São Paulo) were acclimatized for 1 week. All animal care and experimental procedures were approved by the University Ethics Committee (CEUA/CAPPesq).
Animals were anesthetized using 35 mg/kg of ketamine hydrochloride (Cristalia, Brazil) and 5 mg/kg of xylazine hydrochloride (Bayer, Brazil) prior to all procedures. The right chest was cleansed with an alcohol solution (Rioquimica, Sao Paulo, Brazil). The intrapleural injection was performed using a 23-gauge needle attached to a 1-mL syringe containing the solution of cells which was introduced into the chest cavity at 1 cm lateral to the right parasternal line. The plunger of the syringe was removed and the needle was slowly advanced until it reached the pleural space, where the sub-atmospheric intrapleural pressure allowed the fluid to enter the pleural cavity spontaneously. The mice were monitored after the procedure until they were completely recovered.
Three groups of 40 mice each received concentrations of LLC at 0.1, 0.5 or 1.5 × 105 cells intrapleurally. These animals were subdivided into two groups; the first (30 animals per concentration of cells) were euthanized after 7, 14 or 21 days and the second group (10 animals per concentration of cells) were evaluated for survival expectancy. A control group of 10 animals received saline solution intrapleurally.
Mice were killed according to the study calendar; the abdominal wall was opened and the viscera were retracted to visualize the diaphragm. Pleural fluid (PF), when present, was gently aspirated and the volume was measured and placed in tubes for evaluation.
Animals were anesthetized using 35 mg/kg of ketamine hydrochloride (Cristalia, Brazil) and 5 mg/kg of xylazine hydrochloride (Bayer, Brazil) prior to all procedures. The right chest was cleansed with an alcohol solution (Rioquimica, Sao Paulo, Brazil). The intrapleural injection was performed using a 23-gauge needle attached to a 1-mL syringe containing the solution of cells which was introduced into the chest cavity at 1 cm lateral to the right parasternal line. The plunger of the syringe was removed and the needle was slowly advanced until it reached the pleural space, where the sub-atmospheric intrapleural pressure allowed the fluid to enter the pleural cavity spontaneously. The mice were monitored after the procedure until they were completely recovered.
Three groups of 40 mice each received concentrations of LLC at 0.1, 0.5 or 1.5 × 105 cells intrapleurally. These animals were subdivided into two groups; the first (30 animals per concentration of cells) were euthanized after 7, 14 or 21 days and the second group (10 animals per concentration of cells) were evaluated for survival expectancy. A control group of 10 animals received saline solution intrapleurally.
Mice were killed according to the study calendar; the abdominal wall was opened and the viscera were retracted to visualize the diaphragm. Pleural fluid (PF), when present, was gently aspirated and the volume was measured and placed in tubes for evaluation.
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