Longitudinal optical monitoring of blood flow in breast tumors during neoadjuvant chemotherapy.

We measure tissue blood flow markers in breast tumors during neoadjuvant chemotherapy and investigate their correlation to pathologic complete response in a pilot longitudinal patient study (n = 4). Tumor blood flow is quantified optically by diffuse correlation spectroscopy (DCS), and tissue optical properties, blood oxygen saturation, and total hemoglobin concentration are derived from concurrent diffuse optical spectroscopic imaging (DOSI). The study represents the first longitudinal DCS measurement of neoadjuvant chemotherapy in humans over the entire course of treatment; it therefore offers a first correlation between DCS flow indices and pathologic complete response. The use of absolute optical properties measured by DOSI facilitates significant improvement of DCS blood flow calculation, which typically assumes optical properties based on literature values. Additionally, the combination of the DCS blood flow index and the tissue oxygen saturation from DOSI permits investigation of tissue oxygen metabolism. Pilot results from four patients suggest that lower blood flow in the lesion-bearing breast is correlated with pathologic complete response. Both absolute lesion blood flow and lesion flow relative to the contralateral breast exhibit potential for characterization of pathological response. This initial demonstration of the combined optical approach for chemotherapy monitoring provides incentive for more comprehensive studies in the future and can help power those investigations.

Heterodyne frequency-domain multispectral diffuse optical tomography of breast cancer in the parallel-plane transmission geometry.

PURPOSE: The authors introduce a state-of-the-art all-optical clinical diffuse optical tomography (DOT) imaging instrument which collects spatially dense, multispectral, frequency-domain breast data in the parallel-plate geometry. METHODS: The instrument utilizes a CCD-based heterodyne detection scheme that permits massively parallel detection of diffuse photon density wave amplitude and phase for a large number of source-detector pairs (10(6)). The stand-alone clinical DOT instrument thus offers high spatial resolution with reduced crosstalk between absorption and scattering. Other novel features include a fringe profilometry system for breast boundary segmentation, real-time data normalization, and a patient bed design which permits both axial and sagittal breast measurements. RESULTS: The authors validated the instrument using tissue simulating phantoms with two different chromophore-containing targets and one scattering target. The authors also demonstrated the instrument in a case study breast cancer patient; the reconstructed 3D image of endogenous chromophores and scattering gave tumor localization in agreement with MRI. CONCLUSIONS: Imaging with a novel parallel-plate DOT breast imager that employs highly parallel, high-resolution CCD detection in the frequency-domain was demonstrated.

Correlation between inhibition of myelin basic protein (arginine) methyltransferase by sinefungin and lack of compact myelin formation in cultures of cerebral cells from embryonic mice.

Sinefungin, a known inhibitor of protein methylation, inhibited the myelin basic protein (arginine) methyltransferase activity in homogenates of cultured cerebral cells from embryonic mice. Fifty percent inhibition was achieved with 25 microM sinefungin. Electron microscopic examination of the myelin fraction, isolated by gradient density centrifugation and obtained from untreated cells, revealed numerous ringlike multilamellar membranous substructures that had a major dense line periodicity, compactness, and the general appearance expected of myelin obtained by the same technique from whole brain. Cells treated with 30 microM sinefungin, which inhibits myelin basic protein methyltransferase in broken cell preparations about 60%, produced ringlike structures that were devoid of multilamellar periodicity and compactness reminiscent of the vacuolated myelin observed in subacute combined degeneration and in nitrous-oxide- or cycloleucine-treated animals in which methyltransferase activity is also inhibited. The sinefungin-induced change in multilamellar periodicity cannot be attributed to a lack of myelin basic protein, since the ratio of myelin basic protein to total protein did not decrease in sinefungin-treated cells. This primary culture system should be useful for further evaluating the hypothesis that the methylation of myelin basic protein is related to the formation of compact myelin.

Solubility properties of neuronal tubulin: evidence for labile and stable microtubules.

The solubility properties of tubulin and microtubules in pure cultures of sympathetic neurons were examined by electron microscopic and biochemical techniques. For morphological analyses, neurons were extracted with Triton X-100 in the presence or absence of 1 mM CaCl2, and the resulting detergent-extracted residues were examined for microtubules. In parallel experiments, the solubility of tubulin was determined under various solution conditions. Detergent-extracted residues of neurons prepared without Ca2+ contained many microtubules. Neurite residues prepared in the presence of Ca2+ also contained microtubules, but at substantially lower numbers than in residues prepared without Ca2+. The biochemical data parallel the morphological observations. Following detergent-extraction under microtubule stabilizing conditions, 30% of the tubulin was detergent-soluble (i.e. unpolymerized), while 70% was detergent-insoluble (i.e. polymerized). A more detailed examination of the solubility properties of tubulin indicated that 62% was detergent-insoluble but soluble in buffers containing mM CaCl2, while 5-8% was detergent and Ca2+-insoluble. A variety of control experiments indicated that non-specific adsorption of tubulin onto detergent-insoluble components of the cultures, assembly of tubulin onto pre-existing microtubules, and incomplete extraction of tubulin from cells contributed minimally to the levels of Ca2+-soluble and insoluble tubulin obtained with the extraction conditions used. These results indicate that (a) the majority of neuronal tubulin is assembled into microtubules which disassemble upon treatment with Ca2+ and (b) a portion of the neuronal tubulin is assembled into microtubules which show the unusual property of Ca2+-stability.

Factors affecting schwann cell basal lamina formation in cultures of dorsal root ganglia from mice with muscular dystrophy.

Pure populations of sensory neurons (N), Schwann cells (S) and fibroblasts (Fb) were established in culture from normal and dystrophic (dy) mice in order to investigate the cellular origin(s) of the peripheral nervous system abnormalities present in murine muscular dystrophy. These cell types were placed together in various combinations and their subsequent interactions were monitored with the light and electron microscope. The formation of the basal lamina (BL) which in normal tissue, completely surrounds the external aspect of the Schwann cell (when in contact with axons) was documented by morphometric analysis of electron micrographs. Defects in Schwann cell BL formation, observed throughout the PNS of the dy mouse in vivo, were used as a marker for the expression of the dystrophic abnormality in culture. Initially mature cultures of dy tissues containing only S and N (SN) without Fb were examined and found to contain an incomplete BL that surrounded only 82.8 +/- 12.2% of the externally directed plasmalemma of axon-related Schwann cells. The following recombination cultures were established: (1) normal S were placed on dystrophic N; (2) dystrophic S were placed on dystrophic N; (3) dystrophic S were placed on normal N; and (4) normal Fb were added to a dystrophic SN culture. After a 5-week period, the BL formed by normal S in direct contact with dystrophic N was thick and continuous (97.7 +/- 2.2 coverage). On the other hand, in culture situations (without Fb) containing dystrophic S in contact with either dystrophic or normal neurites, the BL coverage was considerably less (58.5 +/- 14.8% and 55.4 +/- 13.2%, respectively). The addition of normal Fb obtained from sciatic nerve explants to dystrophic SN cultures in time resulted in the formation of a morphologically complete BL (98.9 +/- 1.4% coverage). We conclude that neuronal signal(s) are adequate to induce complete BL formation by Schwann cells in the dystrophic tissue but that dystrophic Schwann cells are incapable of forming a complete BL. Furthermore, this deficiency of dy Schwann cells is apparently corrected by the presence of normal Fb by an unknown mechanism.