RESUMEN
PURPOSE: To evaluate the ability of a quantified power Doppler ultrasonography (US) system to help quantitate differences in tumor vascularity after radiation therapy and administration of tumor necrosis factor (TNF). MATERIALS AND METHODS: Murine glioblastoma tumors were grown in the thighs of two sets of 25 mice each. Each mouse was assigned to one of four treatment groups: control (no treatment), radiation therapy, TNF therapy, or combination therapy (both radiation and TNF therapies). Mice were then evaluated with quantified power Doppler US, and a vascularity index (color area) was calculated for different tumor regions in each group. The tumors were then excised, and histologic evaluation was performed by using an immunofluorescence-tagged monoclonal antibody against blood vessel endothelium. The number of stained blood vessels per high-power field was correlated with the sonographically determined vascularity index. RESULTS: The color area of the total tumor decreased to 37% of that in the control group in mice treated with radiation therapy alone (P: =.02), 26% of that in the control group in mice treated with TNF alone (P: =.05), and 8% of that in the control group in those treated with both TNF and radiation (P: =.006). These results correlated well with the quantified results from immunofluorescent staining (r = 0.98). CONCLUSION: Quantified power Doppler US is a noninvasive method for the evaluation of tumor vascularity and blood flow.
Asunto(s)
Glioblastoma/irrigación sanguínea , Neoplasias Experimentales/irrigación sanguínea , Ultrasonografía Doppler en Color , Animales , Fraccionamiento de la Dosis de Radiación , Glioblastoma/diagnóstico por imagen , Glioblastoma/patología , Glioblastoma/radioterapia , Ratones , Ratones Endogámicos C57BL , Microcirculación , Microscopía Fluorescente , Trasplante de Neoplasias , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/patología , Flujo Sanguíneo Regional/fisiología , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
This study was designed to evaluate the accuracy of a system to quantitate tumor vascularity with amplitude (power) color Doppler sonography two- and three-dimensionally. The vascularity of 20 transplanted murine tumors was determined with quantitated amplitude color Doppler sonography both two- and three-dimensionally and compared to tumor vascularity estimated by histologic examination. Serial examinations were performed 15, 30, 45, and 60 min after the injection of the exotoxin CM-101 and saline solution to assess changes in tumor vascularity. Three-dimensional amplitude color Doppler sonography best depicted the overall vascularity of tumor when compared to histologic estimation of vessel density. However, neither two- nor three-dimensional amplitude color power angiography correlated well to the microvessel count, probably a reflection of the difference in the method for vessel quantification using sonographic versus histologic techniques. Three-dimensional amplitude Doppler sonography correlated better with counts of large vessels (> 100 microm) as opposed to small vessels (> 15 microm). Time-activity curves showed no difference in tumor flow at the times measured in the experimental group injected with CM-101 or when compared to saline solutions in either the peripheral or central portions of the tumor. This three-dimensional amplitude color Doppler sonographic system affords global quantification of tumor vascularity and flow that may, in turn, be useful in determining the probability of malignancy (by determination of branching patterns and vessel regularity) or tumor response or both to treatment.
Asunto(s)
Adenocarcinoma/irrigación sanguínea , Neoplasias Experimentales/irrigación sanguínea , Neovascularización Patológica/diagnóstico por imagen , Ultrasonografía Doppler en Color , Adenocarcinoma/patología , Adenocarcinoma/fisiopatología , Animales , Toxinas Bacterianas/farmacología , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/patología , Neoplasias Experimentales/fisiopatología , Neovascularización Patológica/patología , Neovascularización Patológica/fisiopatología , Polisacáridos Bacterianos/farmacología , Reproducibilidad de los Resultados , Streptococcus agalactiaeRESUMEN
A new spin-labeled maleimide derivative of the anion exchange inhibitor 4-4'-diaminodihydrostilbene-2,2'-disulfonate (H2DADS) has been synthesized as a site-specific molecular probe of the stilbenedisulfonate binding site of the anion exchange protein 1 (AE-1; band 3) in human erythrocytes. This probe, SL-H2DADS-maleimide, specifically and covalently labels the Mr 17 kDa integral membrane segment of band 3 with a 1:1 stoichiometry and inhibits essentially 100% of the band 3-mediated anion exchange. The linear V1 EPR spectrum of spin-labeled intact erythrocytes is indicative of a spatially isolated probe which is effectively immobilized on the submicrosecond time scale. Several independent lines of experimental evidence have shown that the nitroxide moiety of SL-H2DADS-maleimide-labeled band 3 is sequestered in a highly protected protein environment. These results are consistent with the observation that the spin-label is rigidly linked to band 3 in a fixed orientation with respect to the membrane normal axis [Hustedt, E. J., & Beth, A. H., (1996) Biochemistry 35, 6944-6954]. The nitroxide moieties of the SL-H2DADS-maleimide-labeled band 3 dimer are greater than 20 A from each other and are also more than 20 A from a monomer-monomer contact surface defined by cross-linking with the spin-labeled reagent BSSDA [bis(sulfo-N-succinimidyl)doxyl-2-spiro-5'-azelate]. These properties make SL-H2DADS-maleimide an extremely useful molecular probe for characterization of the physical properties of the band 3 stilbenedisulfonate binding site, determination of distances between the stilbenedisulfonate site and other segments of band 3, and investigation of the global rotational dynamics of human erythrocyte band 3.
Asunto(s)
Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Marcadores de Afinidad/síntesis química , Proteína 1 de Intercambio de Anión de Eritrocito/química , Membrana Eritrocítica/metabolismo , Marcadores de Spin/síntesis química , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/síntesis química , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/química , Proteína 1 de Intercambio de Anión de Eritrocito/aislamiento & purificación , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Transporte Biológico , Espectroscopía de Resonancia por Spin del Electrón/métodos , Membrana Eritrocítica/ultraestructura , Humanos , Indicadores y Reactivos , Sulfatos/sangreRESUMEN
Two new spin-label derivatives of 4,4'-diaminodihydrostilbene-2,2'-disulfonate (H2-DADS) have been chemically synthesized and employed in electron paramagnetic resonance (EPR) studies of binding to the anion exchange protein (band 3) in intact human erythrocytes. Equilibrium binding studies with the 4-monoacyl-spin-label derivative (mono-SL-H2-DADS) indicated an effective dissociation constant of 11 microM and substantial negative cooperativity in isotonic citrate buffer, pH 7.4, at 20 degrees C. The 4,4'-diacyl-spin-label derivative (di-SL-H2-DADS) bound with an effective dissociation constant of 54 microM and no detectable cooperativity under the same binding conditions. The findings of substantial negative cooperativity in binding of the less bulky mono-SL-H2-DADS and no cooperativity for di-SL-H2-DADS suggest the presence of an allosteric coupling between the stilbenedisulfonate sites on adjacent band 3 monomers rather than steric interactions. There were approximately 1 x 10(6) binding sites per erythrocyte for both the mono- and di-SL-H2-DADS derivatives, and the binding of each was blocked by pretreatment of intact cells with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), a highly specific covalent inhibitor of anion exchange. EPR spectra collected over a wide range of concentrations of mono-SL-H2-DADS indicated that binding resulted in immobilization of the probe and that, even upon near saturation of available binding sites, there were no detectable dipole-dipole interactions between bound probes. EPR spectra collected using di-SL-H2-DADS revealed the presence of intramolecular dipole-dipole interactions between spin-label moieties on opposite ends of this biradical probe, but no intermolecular dipole-dipole interactions between separate bound probes. These data indicate that di-SL-H2-DADS binds to the stilbenedisulfonate binding site on band 3 in a bent conformation and further suggest that the termini of these binding sites on adjacent monomers are greater than 20 A apart.
Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Eritrocitos/metabolismo , Estilbenos/metabolismo , Sitios de Unión , Células Cultivadas , Espectroscopía de Resonancia por Spin del Electrón , Electrones , Humanos , Modelos Moleculares , Marcadores de SpinRESUMEN
The location of the medullary chemoreceptors is not conclusively established. The original experiments, which were believed to suggest a shallow surface location in the ventrolateral medulla, have been questioned because substances, particularly CO2, applied on the surface of the medulla could diffuse into small arterioles. Because the whole tissue blood flow is supplied by surface arterioles, they could transport substances from the surface into the tissue to the respiratory centers. We studied simple transport equations describing movement of CO2 in arterioles bathed by rapidly flowing cerebrospinal fluid (CSF) and arterioles in tissue perfused by capillaries. Substantial exchange of CO2 could occur across the arteriole wall for all expected sizes of vessels when the partial pressure of CO2 at the outside wall was determined by CSF. When an arteriole is surrounded by tissue, only vessels with inside diameters (ID) less than or equal to 50 micron will exchange substantial amounts of CO2 but the smallest arterioles may be nearly in equilibrium with the tissue. The CO2 gradient in tissue around the arteriole will extend approximately 1 mm. Our simple theoretical description of CO2 transport in arterioles predicts substantial exchange in precapillary vessels. CO2 picked up by the smallest surface arterioles when the medulla is perfused at a high rate with CSF will not stay in the blood past the putative depth of the chemoreceptors. In arterioles greater than 30 micron, however, the CO2 could be carried to the respiratory centers.