Proteomic analysis of nuclei dissected from fixed rat brain tissue using expression microdissection.

Expression microdissection (xMD) is a high-throughput, operator-independent technology that enables the procurement of specific cell populations from tissue specimens. In this method, histological sections are first stained for cellular markers via either chemical or immuno-guided methods, placed in close contact with an ethylene vinyl acetate (EVA) film, and exposed to a light source. The focal, transient heating of the stained cells or subcellular structures melts the EVA film selectively to the targets for procurement. In this report, we introduce a custom-designed flashcube system that permits consistent and reproducible microdissection of nuclei across an FFPE rat brain tissue section in milliseconds. In addition, we present a method to efficiently recover and combine captured proteins from multiple xMD films. Both light and scanning electron microscopy demonstrated captured nuclear structures. Shotgun proteomic analysis of the samples showed a significant enrichment in nuclear localized proteins, with an average 25% of recovered proteins localized to the nucleus, versus 15% for whole tissue controls (p < 0.001). Targeted mass spectrometry using multiple reaction monitoring (MRM) showed more impressive data, with a 3-fold enrichment in histones, and a concurrent depletion of proteins localized to the cytoplasm, cytoskeleton, and mitochondria. These data demonstrate that the flashcube-xMD technology is applicable to the proteomic study of a broad range of targets in molecular pathology.

[Eye fundus autofluorescence technique in the early diagnosis of age-related macular degeneration].

Autofluorescence (AF) of the eye fundus is one of the most promising studies. AF provides specific molecular information on the retinal pigment epithelium and enables one to diagnose early phenotypic changes that are predictors for progression of age-related macular degeneration. Many investigations have demonstrated that AF is a valuable clinical technique that should be improved in order to have information accessible to a patient on the diagnosis and prediction of age-related macular degeneration.

General statistics of stochastic process of gene expression in eukaryotic cells.

Thousands of genes are expressed at such very low levels (< or =1 copy per cell) that global gene expression analysis of rarer transcripts remains problematic. Ambiguity in identification of rarer transcripts creates considerable uncertainty in fundamental questions such as the total number of genes expressed in an organism and the biological significance of rarer transcripts. Knowing the distribution of the true number of genes expressed at each level and the corresponding gene expression level probability function (GELPF) could help resolve these uncertainties. We found that all observed large-scale gene expression data sets in yeast, mouse, and human cells follow a Pareto-like distribution model skewed by many low-abundance transcripts. A novel stochastic model of the gene expression process predicts the universality of the GELPF both across different cell types within a multicellular organism and across different organisms. This model allows us to predict the frequency distribution of all gene expression levels within a single cell and to estimate the number of expressed genes in a single cell and in a population of cells. A random "basal" transcription mechanism for protein-coding genes in all or almost all eukaryotic cell types is predicted. This fundamental mechanism might enhance the expression of rarely expressed genes and, thus, provide a basic level of phenotypic diversity, adaptability, and random monoallelic expression in cell populations.

A comparison of the microvascular response in the healing wound in the spontaneously hypertensive and non-hypertensive rat.

BACKGROUND: In the Spontaneously Hypertensive Rat (SHR), there is a significantly greater blood flow at the paw but not at the back than in the non-hypertensive Wistar Kyoto (WKY) rat. We wanted to assess the effect of this higher blood flow on wound healing at the paw. MATERIALS AND METHODS: We characterized the microvascular composition of wounds at the back and paw of 9 SHR rats and 10 WKY rats using a quantitative imaging program. Blood flow was compared using laser Doppler technology. RESULTS: The blood flow response to wounding at the back was identical in the SHR and WKY rats. There was an immediate sharp increase in flow at the center of the wound. Blood flow reached a peak at 3 days and then decreased somewhat by day 7, but still remained five-fold higher than the prewound baseline values. There was also a two-fold increase at the back wound perimeter. There were no differences in microvascular composition at the back between the SHR and WKY rats. In contrast, there was an immediate enormous increase in blood flow at paw wound center in the SHR rats. Flow increased to 75 ml/min/100 gm by 24 h then fell back sharply. Blood flow at the paw in the WKY rats changed very little over the 7 days post wounding. At 3 days, the flow was about twice as high in the SHR than in the WKY wound, but, by day 7, flow was similar in the two rat strains. At the SHR wound perimeter, there was a small increase in flow which was sustained through day 7. Although the microvascular composition at the paw wound center was similar in the SHR and WKY rats, there was a notable difference at the paw perimeter. At baseline, there was a slightly greater capillary density in the SHR paw (32 +/- 1 per mm3) than the WKY paw (25 +/- 8 per mm3). At 7 days after wounding, there was a substantial increase in capillary number in the SHR rats (48 +/- 8 per mm3) as compared to baseline (p = 0.05). In contrast, there was no significant difference in capillary number in the WKY paw wound perimeter (20 +/- 3 per mm3) as compared to baseline. CONCLUSIONS: There is a substantial difference in wound blood flow response between the hypertensive and the non-hypertensive rat. At the back, the blood flow effects of wounding are similar, but, at the paw, the SHR rat shows a dramatic transient increase in flow in the early phases of wound healing. There is apparently no capability to upmodulate microvascular resistance in response to increased pressure at this early stage of wound healing. However, within several days, the granulation tissue microvasculature becomes capable of controlling the effects of raised pressure in the SHR rat. In the SHR paw wound perimeter, there are significantly more capillaries than in the WKY rat. It is possible that greater capillary proliferation in the SHR rat results from higher blood flow in the early phase of wounding. The contrast between the WKY rat and the SHR rat serves to further illustrate the complexity of blood flow regulation which occurs during wound healing.

Visible-light photon migration through myocardium in vivo.

Empirical data between 510 and 590 nm of diffuse reflected light from the pig heart in vivo have shown that myoglobin and cytochrome c absorption peaks with little apparent contribution of red blood cell (RBC) Hb. Monte Carlo simulations of photon migration in tissue were performed to compare the effects of myoglobin and cytochromes with those of blood Hb on photon pathlengths and diffuse reflectance of visible wavelengths (450-600 nm) from the pig heart in vivo. Wavelength dependence of the input parameters, including the transport-corrected scattering coefficients (1.1-1.2 mm(-1)) and the absorption coefficients of blood-free solubilized heart tissue (0.43-1.47 mm(-1)), as well as the absorption coefficients of Hb, were determined by an integrating sphere method and standard spectrophotometry, respectively. The Monte Carlo simulations indicate that in the 510- to 590-nm range the mean path length within the myocardium for diffusely reflected light varies from 1.4 to 1.2 mm, whereas their mean penetration depth within the epicardium is only 330-400 micrometer for blood-free heart tissue. Analysis shows that the blood Hb absorption extrema are only observable between 510 and 590 nm when RBC concentration in tissue is >0.5%. Blood within vessels much larger than capillaries does not contribute significantly to the spectral features, because virtually all light in this spectral range is absorbed during transit through large vessels (>100 micrometer). This analysis suggests that diffuse reflected light in the 510- to 590-nm region will show spectral features uniquely associated with myoglobin and cytochrome c oxygenation states within 400 micrometer of the surface of the heart in situ as long as the capillary RBC concentration remains <0.5%.

A comparison of the cutaneous microvascular properties of the spontaneously hypertensive rat and the Wistar-Kyoto rat.

The Spontaneously Hypertensive rat (SHR) and its non-hypertensive companion strain, the Wistar-Kyoto (WKY) rat, provide an excellent comparative model to permit study of the differential properties of cutaneous microvascular beds. We explored the possibility that chronically elevated vascular pressures in the SHR rat might affect the microvascular constitution of the skin. We measured skin blood flow at the back and at the paw of a group of 20-week-old WKY rats and a contrast group of SHR rats. We then performed skin biopsies at these two locations and used the NIH Image program to count and measure the size of capillaries, arterioles, and venules. We also determined microvascular density as percentage of total tissue area. At basal temperature, skin blood flow was similar in the two rat strains at both the back and paw. Heat induced vasodilatation resulted in a 50% increase in blood flow at the back, reaching the same level in the two rat groups. However, at the paw site, thermal stimulation resulted in significantly greater flow (39.3 +/- 3.1 ml/100 gm tissue per min) in the SHR rats than the WKY rats (28.6 +/- 1.9 ml/100 gm tissue per min, P < 0.05). The ratio of systemic arterial pressure to skin blood flow was computed as an index of vascular resistance to flow. At basal temperature, this index was 50% greater for the SHR rats at both skin sites. At 44 degrees C, the resistance index decreased at both sites in both rat groups but was still approximately 50% higher at the back of the SHR than the WKY rats. In contrast, the resistance index at 44 degrees C at the paw site fell to the same level in both the SHR and WKY rats. There were twice as many capillaries at the back of the WKY rats than at the back of the SHR rats (9.2 +/- 2.0 per mm2 vs. 4.7 +/- 1.2 per mm2, P < 0.05). Expressed as a percentage of total tissue area, the capillary density at the back in the WKY rats was 0.064 +/- 0.010% as compared to 0.034 +/- 0.008% in the SHR rats (P < 0.05). There were five times more arterioles at the paw compared to the back in both rat groups with no significant difference between the groups. We measured the diameter of the lumen and the thickness of the wall of each arteriole and computed their ratio as an index of possible media hypertrophy. There were minimal differences seen in these parameters between the two rat groups at the back and paw sites. The venular density was significantly higher at the paw than at the back in both rat groups with no significant difference between them. Reduced capillary density at the back of the SHR rats may be a developmental adaptation to high blood pressure. Such a reduction in the pathways of blood flow may help account for increased flow resistance at that site, independent of arteriolar vasoconstriction.

Laser capture microdissection of single cells from complex tissues.

Laser capture microdissection (LCM) is a new method used to select and procure cell clusters from tissue sections. Once captured, the DNA, RNA or protein can be easily extracted from the isolated cells and analyzed by conventional PCR, reverse transcription (RT)-PCR or polyacrylamide gel electrophoresis, including protein zymography for specific macromolecular changes. In LCM, a thermoplastic polymer coating [ethylene vinyl acetate (EVA)] attached to a rigid support is placed in contact with a tissue section. The EVA polymer over microscopically selected cell clusters is precisely activated by a near-infrared laser pulse and then bonds to the targeted area. Removal of the EVA and its support from the tissue section procures the selected cell aggregates for molecular analysis. This initial NIH LCM approach using a flat transfer EVA film has been recently commercialized and has proven to be an effective routine microdissection technique for subsequent macromolecular analysis in many laboratories around the world. However, reliable and precise capture of individual cells from tissue sections has been difficult to perform with the current LCM instruments. In this report, we describe the capture of individual cells with a new NIH LCM microscope, which epi-irradiates the EVA polymer overlying individual cells with 1-ms laser pulses focused to 6 microns. A computer-controlled arm precisely positions a 40-micron-wide strip of a cylindrical EVA surface onto a sample with a light contact force (ca. 0.1 g). The small contact force and contact area on the film on the sample diminishes nonspecific transfer to negligible levels. By slightly rotating the cylinder to provide a renewable transfer surface, concentration of a distinct cell type on a single cylinder is possible. Using this novel adaptation, we demonstrate the rapid and practical capture of single cells from different types of tissue sections, including immunostained cells.

Optical simulations of a noninvasive technique for the diagnosis of diseased salivary glands in situ.

A simulation experiment for three-dimensional (3D) imaging of exogenous fluorescinated antibodies that specifically bind to infiltrating lymphocytes in minor salivary glands was carried out. Small (approximately 1 mm3 volume) rhodamine targets, which mimic diseased minor salivary glands labeled with fluorescent antibodies to infiltrating lymphocytes in Sjøgren's syndrome, were embedded in a highly scattering tissue phantom consisting of a thick Delrin disk covered by index matched Delrin slabs of various thickness. In this way the variation of fluorescence profiles on the surface of tissue could be examined corresponding to the range of depths of the salivary glands in vivo. Surface images were obtained for different target depths and radial distances from laser excitation to target fluorophore. These images were analyzed and compared to calculations based on random walk theory in turbid media, using previously determined scattering and absorption coefficients of the Delrin. Excellent agreement between the surface profiles experimentally measured and those predicted by our random walk theory was obtained. Derivation of these theoretical expressions is a necessary step toward devising an inverse algorithm which may have the potential expressions to perform 3D reconstruction of the concentration distribution of fluorescent labels within tissue.

Laser-capture microdissection: opening the microscopic frontier to molecular analysis.

As the list of expressed human genes expands, a major scientific challenge is to understand the molecular events that drive normal tissue morphogenesis and the evolution of pathological lesions in actual tissue. Laser capture microdissection (LCM) has been developed to provide a reliable method to procure pure populations of cells from specific microscopic regions of tissue sections, in one step, under direct visualization. The cells of interest are transferred to a polymer film that is activated by laser pulses. The exact morphology of the procured cells (with intact DNA, RNA and proteins) is retained and held on the transfer film. With the advent of LCM, cDNA libraries can be developed from pure cells obtained directly from stained tissue, and microhybridization arrays of thousands of genes can now be used to examine gene expression in microdissected human tissue biopsies. The fluctuation of expressed genes or alterations in the cellular DNA that correlate with a particular disease stage can ultimately be compared within or between individual patients. Such a fingerprint of gene-expression patterns can provide crucial clues for etiology and might, ultimately, contribute to diagnostic decisions and therapies tailored to the individual patient. Molecules found to be associated with a defined pathological lesion might serve as imaging ot therapeutic targets.

The relationship of laser-Doppler skin blood flow measurements to the cutaneous microvascular anatomy.

The hairless plantar paw surface of the rat shows high skin blood flow with a substantial response to thermal stimulation. This contrasts with hair-covered areas such as the back, where there is much lower basal flow and thermal response. These properties are similar to the differences seen in humans between skin sites which have a high density of arterioles and venules (AV areas) and sites with predominantly nutritive (NUTR) capillary perfusion. However, there has been no previous study of the microvascular anatomy of rodent skin. We used NIH Image, a quantitative imaging program, to count the capillaries, arterioles, and venules in the skin of the plantar paw surface and the back of 14 Wistar-Kyoto rats. We also used laser-Doppler techniques to determine skin blood flow at these sites. We found significantly more vessels per unit area at the paw. There were twice as many capillaries in the paw (19.6 +/- 2.4 per mm2) compared to the back (9 +/- 1.5 per mm2) (P < 0.001). Similarly, there were three times as many venules (11.8 +/- 1.2 per mm2 vs 3. 48 +/- 0.45 per mm2; P < 0.001). The largest difference was in the number of arterioles (7.76 +/- 0.74 per mm2 vs 0.79 +/- 0.13 per mm2 at the back; P < 0.001). The greater microvascular density at the paw was reflected in a threefold higher basal blood flow (6.6 +/- 0. 44 ml/min/100 g) compared to that in the back (1.99 +/- 0.07 ml/min/100 g) (P < 0.001). Microvascular volume at the back was 0.14 +/- 0.01 x 10(6) RBC/ml in the basal state compared to 0.31 +/- 0.01 x 10(6) RBC/ml at the paw. Thus, the increased number of vessels at the paw resulted in a twofold increase in microvascular volume. The plantar paw surface has considerably more vessels than the back. As might be expected, there is a higher proportion of arterioles and venules compared to capillaries at the paw than at the back. Thus, the plantar paw surface is an AV site compared to the back, which is a NUTR site. Although our prior studies have largely assumed that we could use the paw and back as contrast sites comparable to AV and NUTR sites in humans, we have now for the first time conclusively established this fact. The increased microvascular density at the paw results in higher skin blood flow at this site.

The microvascular composition of the healing wound compared at skin sites with nutritive versus arteriovenous perfusion.

Background. In the rat, there is a significantly greater blood flow response to wounding at the back, a site perfused mainly by small capillaries, than at the paw, which has a much higher density of arterioles and venules. Materials and methods. We characterized the microvascular composition of wounds at the two skin sites in 11 Wistar Kyoto rats using a quantitative imaging program. Blood flow was compared using laser Doppler technology. Results. Prior to wounding, skin blood flow was much greater at the paw (7.1 +/- 0.5 ml 100 g tissue-1 min-1) than at the back (2.1 +/- 0.1 ml 100 g tissue-1 min-1, P < 0.01) at baseline. Seven days after wounding, blood flow both at the center (8.3 +/- 1.4 ml 100 g tissue-1 min-1) and at the perimeter of the back wound (4.1 +/- 0.5 ml 100 g tissue-1 min-1) had increased substantially. In contrast, skin blood flow at the perimeter of the paw wound had increased moderately (12. 7 +/- 2.0 ml 100 g tissue-1 min-1), but there was no change at the center of the wound (6.9 +/- 0.9 ml 100 g tissue-1 min-1). There were three times more microvessels per mm2 at the paw site (39.3 +/- 3.6) than at the back (13.1 +/- 1.5) prior to wounding. The wound granulation tissue was very vascular; the numerical density of vessels was identical at back (166 +/- 9) and at paw (154 +/- 6). Despite the marked increase in blood flow at the perimeter of the back wound, there was no difference in the microvascular density (15. 2 +/- 1.4) compared to baseline, nor was there a difference at the paw perimeter (39.4 +/- 3.6) compared to baseline. Conclusions. This study demonstrates that the microvascular constitutions of granulation tissues at the paw and back are identical. Thus, the rise in flow at the back wound and reduction in flow at the paw wound are entirely consistent with similar microvascular compositions of these two sites. Yet, there is increased flow at the back wound perimeter where there is no significant change in microvascular constitution compared to unwounded skin. Therefore, a microvascular structure no different from that prior to wounding functions very differently after wounding. Clearly vasoregulatory factors impact on the wound to modify flow through the microvascular network.

Thermal modeling of laser capture microdissection.

A first-order thermal analysis is applied to Laser Capture Microdissection (LCM), a new microscope technique for routine targeting and extraction of specific cells from tissue sections for subsequent multiplex molecular analysis. In LCM a polymer film placed in contact with the tissue is focally activated by a pulsed IR laser beam and is melted and bonded to adjacent targeted cells. A three-dimensional finite-element model is used to predict the thermal transients within the polymer, the captured tissue, and its macromolecules. The simulations allow a comparison of models for the physical process of LCM with the experimental data on the dependence of the transfer spot size on laser power. The validated physical model and the thermal simulations permit optimization of the complex LCM parameter space for a wide variety of configurations and applications.

Effects of multiple-passage probabilities on fluorescent signals from biological media.

By applying a random-walk model for photon migration, we find an exact expression for fluorescent signals emitted from a homogeneous, optically turbid medium containing a single fluorescent mass. In contrast to diffusion-theory-based models, our analysis accounts for multiple photon passages through the fluorophore sites and allows for a variable degree of fluorescent absorptivity. We particularly discuss effects on the amplitudes and phase shifts of frequency-domain fluorescent signals.

Laser capture microdissection.

Laser capture microdissection (LCM) under direct microscopic visualization permits rapid one-step procurement of selected human cell populations from a section of complex, heterogeneous tissue. In this technique, a transparent thermoplastic film (ethylene vinyl acetate polymer) is applied to the surface of the tissue section on a standard glass histopathology slide; a carbon dioxide laser pulse then specifically activates the film above the cells of interest. Strong focal adhesion allows selective procurement of the targeted cells. Multiple examples of LCM transfer and tissue analysis, including polymerase chain reaction amplification of DNA and RNA, and enzyme recovery from transferred tissue are demonstrated.

Protection from radiation-induced alopecia with topical application of nitroxides: fractionated studies.

PURPOSE: Hair loss resulting from irradiation of the head and neck or from whole brain irradiation often leads to cosmetic, social, and psychological problems for the radiotherapy patient. Few successful clinical interventions are available. We have shown that nitroxides (stable free radicals) afford radiation protection against single-dose radiation-induced alopecia in a guinea pig model. Here we determine if topical nitroxide application provides protection from fractionated radiation treatment. MATERIALS AND METHODS: Two symmetrical and contralateral areas (3 x 5 cm) of skin on the dorsal trunk of guinea pigs were shaved to a hair length of 0.25 cm. A 2 mL solution containing 70 mg/mL nitroxide (Tempo or Tempol) in 70% ethanol was topically applied to the skin surface of one side; 70% ethanol was applied to the contralateral (control) side 10 minutes before irradiation. Animals were placed in a special jig that held skin without decreasing blood flow to the treatment area and fractionated external beam radiation (7 Gy) was delivered daily for eight fractions over 10 days via a 4 MeV linear accelerator. Alopecia (hair density) was scored weekly for 13 to 14 weeks after radiotherapy, using a standardized reference with respect to hair loss and regrowth in the treatment field. RESULTS: After radiation treatment, dry desquamation and gradual hair loss were observed for both control and nitroxide-treated skin; however, over weeks 4 to 11 postirradiation hair loss was much more pronounced in control animals when compared with nitroxide-treated animals. Hair density measurements for Tempol treatment over weeks 9 to 13 were approximately 75% compared with measurements in controls of approximately 25%. Tempo-treated animals exhibited hair density values of approximately 90% compared with 12% in controls over weeks 11 to 14. Tempol and Tempo treatments resulted in significant radioprotection. Histologic evaluation showed that radiation treatment alone in ethanol controls resulted in a marked decrease in the number of hair follicles and poor development of remaining follicles; however, nitroxide pretreatment resulted in no appreciable decrease in hair follicles and hair follicles appeared mature. This was also observed in unirradiated ethanol controls. Electron paramagnetic resonance studies revealed that topical nitroxide application did not result in measurable systemic concentrations of either drug. CONCLUSIONS: The results of this study suggest that topical application of nitroxides may be useful in a clinical setting to reduce the undesirable toxicity of radiation-induced alopecia.