The changes of ultra-microstructure of transplanted VX-2 tumor caused by the injection of ozone into the tumor: an experimental study in rabbits

Objective: To investigate the changes of ultra-microstructure of transplanted VX-2 tumor in rabbits caused by injection of different concentrations of ozone (O₃) into the tumor in order to determine the proper concentration of O₃ which can be safely and effectively used in experimental study as well as in clinical practice, and based on the duration of the changes of ultra-microstructure to estimate the reasonable interval time for repeated injections of O₃. Methods: A total of 20 rabbits bearing VX-2 tumor on hind legs were randomly divided into four groups: control group (n = 2), group T20 (n = 6, using 20% O₃), group T40 (n = 6, using 40% O₃) and group T60 (n = 6, using 60% O₃). Under direct vision the mixed gas of O₂ and O₃ (20%, 40% or 60% O₃ concentration) with twice the amount of tumor volume was injected into each study rabbit. Only imitation puncturing was performed in the two rabbits of control group. Every two rabbits from each group were sacrificed immediately after the procedure, and the tumors were removed for fixation. And every two rabbits from the remaining rabbits in each study group were sacrificed at 4 and 8 days after the procedure respectively. The specimens were collected, fixed, sectioned, and then sent for transmission electron microscopic examination. Results: In Group T20, mild injury of tumor cells was observed immediately after O₃ injection, which was manifested as slight ridge lysis of mitochondria and sporadic swelling of mitochondria, dilation of endoplasmic reticulum, slight widening of peri-nuclear gap in some cells, but the intercellular space remained normal. In group T40 and group T60, obvious damage of tumor cells was observed immediately after O₃ injection, which was characterized by pronounced swelling of mitochondria, dilation of endoplasmic reticulum, swelling of nucleus, widening of peri-nuclear gap, a burst of cellular membrane and collapse of tissue structure in some tumor cells. On the 4th day, massive necrosis appeared with infiltration of inflammatory cells. Other changes included incomplete cellular membrane, mitochondria-lysosome formation (presenting as black crescent particle of high electron-density) or lysis of mitochondria, dilation of endoplasmic reticulum, partial lysis of cytoplasm, irregular nucleus and widening of peri-nuclear gap, etc., which were more significant and serious in group T60. Vascular endothelial cells appeared to be injured too. All these changes could be observed even on the 8th day after the treatment. The above mentioned changes could be gradually repaired. Conclusion After injection of 20%-60% O₃ into VX-2 tumor in rabbits, different degrees of damage or even death of the tumor cells and the vascular endothelial cells can be caused. The damage is slight when 20% O₃ is used, while the damage is much remarkable when 40% or 60% O₃ is employed. Taking both the safety and therapeutic effect into account the use of 40% O₃ might be more suitable for both experimental and clinical use. The reasonable interval time for repeated injection of O₃ should be 4-8 days as certain and definite cellular damage can still be observed at 8 days after the injection. (authors)