Intermittent blood flow in tumour microvasculature is believed to contribute to heterogeneity in tumour oxygen delivery; transient vessel nonperfusion is thought to result in acutely hypoxic cells resistant to conventional radiotherapy. This thesis describes three main areas of work: (1) the development of a histologic method capable of detecting intermittent blood flow in experimental tumours at the single vessel level; (2) the quantification and characterization of tumour blood flow fluctuations in the murine SCCVII carcinoma; and (3) the modification of tumour blood flow and the reduction of flow heterogeneity using vasoactive drugs. A double staining technique involving the sequential intravenous injection of two fluorescent vascular markers was used to detect transient episodes of tumour vessel nonperfusion. The stains employed were Hoechst 33342 and the carbocyanine dye, DiOC₇(3), both of which have short (< 3 minutes) circulation half-lives and preferentially stain cells adjacent to perfused blood vessels. When injections of the vascular markers are separated by some interval, each stain defines only those tumour vessels which were perfused during the few minutes immediately post-injection; thus, two "pictures" of tumour microvascular flow are obtained and tumour vessels subject to periods of nonperfusion can be easily visualized in frozen sections since they are outlined by one stain but not the other. Using the double staining technique, in which Hoechst 33342 and then DiOC₇(3) are administered intravenously 20 minutes apart to unrestrained C3H/He mice, staining mismatch (indicative of transient vessel nonperfusion) is regularly observed in subcutaneous SCCVII carcinoma. Vessels stained with DiOC₇(3) only (reperfusion of previously nonperfused vessels) or with H33342 only (nonperfusion of previously perfused vessels) are observed in approximately equal numbers. The percentage of tumour vessels subject to intermittent flow is a function of SCCVII tumour size: tumours ≤100 mg do not exhibit statistically significant amounts of mismatch. At sizes > 100 mg, overall staining mismatch is significantly increased over background levels and maximum mismatch is observed at tumour sizes >400 mg (8.6 ±2.9%). In most tumours, transient vessel nonperfusion is more pronounced in central tumour regions. In addition to mismatch observed in individual vessels, large "patches" of unequal staining are also seen. Anaesthesia or restraint do not significantly influence intermittent blood flow. The above information suggests that transient episodes of tumour vessel nonperfusion occur as a consequence of flow reduction in a feeding vessel; vessels in central regions of large tumours may be susceptible to collapse as a result of elevated tumour interstitial pressure. In the SCCVII tumour, a small number of peripheral vessels possess vascular smooth muscle and thus may be capable of vasomotor activity. The importance of perfusion pressure in the control of tumour microcirculatory flow was examined using vasoactive drugs. Hydralazine, a vasodilator which lowers blood pressure, causes a profound reduction in tumour RBC flow to 8.7 + 6.4% of pretreatment values in unanaesthetized mice. The drug causes collapse of central tumour vessels: following a dose of 10mg/kg intravenously, 36±16% of vessels are completely nonperfused, as detected using the double staining technique. Conversely, elevation of blood pressure using the vasoconstrictor angiotensin II results in a 2-3x increase in tumour blood flow. In addition, angiotensin II infusion significantly reduces the number of tumour vessels subject to transient nonperfusion from 8.1 % to 2.0%. However, intermittent blood flow in the SCCVII carcinoma can also be influenced by nonvasoactive drugs: nicotinamide, the amide form of vitamin B3, reduces episodes of transient nonperfusion. In summary, intermittent blood flow has been characterized in a transplanted murine squamous cell carcinoma using a novel fluorescent double staining method which allows the detection of flow fluctuations in solid tumours at the microvascular level. If transient episodes of nonperfusion occur in human tumours and result in impaired oxygen or drug delivery, then such flow fluctuations may be an important factor limiting tumour cure or local control by radiotherapy or chemotherapy. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

A radiação ionizante prévia promove alterações actínicas em tecidos peritumorais,o que poderia influenciar a demarcação do linfonodo sentinela.O presente estudo desenvolveu modelo experimental para demarcação do linfonodo sentinela do reto do rato e para definição da dose de radiação (curva de calibração). O objetivo foi avaliar a influência da radiação ionizante pré-operatória sobre a marcação, com corante azul patente, do linfonodo sentinela do reto de ratos. A amostra foi constituída por 40 ratos machos Wistar e dividida em 2 grupos:Grupo 1 (controle não irradiado; n = 20) e Grupo 2 (irradiado com 1200cGy e demarcado 2 dias após; n = 20). Foi observado aumento linear do tempo de coloração do linfonodo no Grupo 2. Concluindo,a irradiação pré-operatória não influiu na demarcação do linfonodo sentinela do reto do rato / Previous ionizing radiation induces actinic alterations in peritumoral tissues and thus might influence the localization of the sentinel lymph node. The present study developed an experimental model for the localization of the sentinel lymph node of the rectum of the rat and for the definition of the dose of radiation (calibration curve). The objective was to evaluate the influence of preoperative ionizing radiation on the staining of a patent blue dye in the sentinel lymph node of the rectum in rats.The sample was composed of 40 male Wistar rats and was divided in two groups: Group 1 (non-irradiated control; n = 20 ) and Group 2(irradiated with 1200cGy and stained 2 days afterwards; n = 20). It was observed a linear increase in the time for the staining of the lymph in Group 2.In conclusion, preoperative irradiation did not influence the staining of the sentinel lymph node of the rectum in rats

A radiação ionizante prévia promove alterações actínicas em tecidos peritumorais,o que poderia influenciar a demarcação do linfonodo sentinela.O presente estudo desenvolveu modelo experimental para demarcação do linfonodo sentinela do reto do rato e para definição da dose de radiação (curva de calibração). O objetivo foi avaliar a influência da radiação ionizante pré-operatória sobre a marcação, com corante azul patente, do linfonodo sentinela do reto de ratos. A amostra foi constituída por 40 ratos machos Wistar e dividida em 2 grupos:Grupo 1 (controle não irradiado; n = 20) e Grupo 2 (irradiado com 1200cGy e demarcado 2 dias após; n = 20). Foi observado aumento linear do tempo de coloração do linfonodo no Grupo 2. Concluindo,a irradiação pré-operatória não influiu na demarcação do linfonodo sentinela do reto do rato / Previous ionizing radiation induces actinic alterations in peritumoral tissues and thus might influence the localization of the sentinel lymph node. The present study developed an experimental model for the localization of the sentinel lymph node of the rectum of the rat and for the definition of the dose of radiation (calibration curve). The objective was to evaluate the influence of preoperative ionizing radiation on the staining of a patent blue dye in the sentinel lymph node of the rectum in rats.The sample was composed of 40 male Wistar rats and was divided in two groups: Group 1 (non-irradiated control; n = 20 ) and Group 2(irradiated with 1200cGy and stained 2 days afterwards; n = 20). It was observed a linear increase in the time for the staining of the lymph in Group 2.In conclusion, preoperative irradiation did not influence the staining of the sentinel lymph node of the rectum in rats