ARTICLE |
Correspondence to: R.F. Tuma, Dept. of Physiology, Temple Univ. School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140.
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Summary |
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Alterations in leukocyte/endothelium interaction due to phototoxic effects of the fluorescent dyes acridine orange (AO) and rhodamine 6G (Rh6G) were studied by intravital microscopy using the dorsal skinfold model in awake Syrian golden hamsters. AO (0.5 mg/kg/min; constant IV infusion) and Rh6G (0.1 µmol/kg; bolus IV) were administered via an indwelling venous catheter. Five to seven arterioles (35-55 µm) and postcapillary venules (30-65 µm) were investigated in each animal. Vessels were exposed four times for 30 sec to continuous light of the appropriate excitation wavelength with a 10-15-min time interval between exposures. Animals were randomly assigned to five experimental groups (five distinct light energy levels). AO and Rh6G induced leukocyte rolling/sticking in postcapillary venules and arterioles when exposed to high light energy levels. AO, but not Rh6G, induced arteriolar vasospasm when exposed to high light energies. The potential phototoxic effect of AO and Rh6G is demonstrated, as assessed by the stimulation of leukocyte-endothelium interaction and arteriolar vasospasm in vivo. This study underscores the necessity to optimize microscopic set-ups for intravital microscopy, to reduce the excitation light energy level significantly, and to perform stringent control experiments, ruling out an artificial phototoxicity-induced stimulation of leukocyte adhesion. (J Histochem Cytochem 45:505-513, 1997)
Key Words: intravital fluorescence microscopy, leukocyte-endothelium interaction, acridine orange, rhodamine 6G, phototoxicity, microcirculation
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Introduction |
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Leukocyte-endothelium interaction is a key feature of inflammation (
That the illumination of fluorescently labeled cells or tissues with high energetic light of the appropriate excitation wavelength can cause structural changes or even cell death was suggested several years ago in in vitro cell culture experiments (
On the basis of these considerations, we have investigated whether these phototoxic effects can lead to in vivo activation of leukocytes labeled with AO or Rh6G and thereby influence their adhesion properties during intravital microscopic investigations in the dorsal skinfold chamber preparation in hamsters. A better understanding of the direct effects of photoexcitation during in vivo studies of leukocyte-endothelium interaction might also help to explain discrepancies among different laboratories investigating leukocyte adhesion to microvascular endothelium.
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Materials and Methods |
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Animal Model and Preparation
The experiments were performed in compliance with guidelines of the National Institute of Health for the use of laboratory animals. Forty female Syrian golden hamsters weighing 55-65 g were used for this study. The animals had free access to food (Purina lab chow 8604: vitamin E content 50.58 IU/kg) and water. This concentration of vitamin E in the laboratory chow is unlikely to exert inhibitory effects on leukocyte rolling and adhesion in response to fluorescent dyes (
The surgical technique has previously been described in more detail (
Intravital Microscopy: Technical Set-up
An epi-illuminiscence fluorescence microscope (Laborlux D 513 591; Leitz, Wetzlar, Germany) was used for the intravital microcirculatory investigations. A 100-W/2 HBO mercury lamp (Osram; Berlin, Germany) connected to a short arc lamp power supply (model 1230; Leitz, Rockleigh, NJ) served as a light source and emitted light with a relative intensity peak at 490 nm wavelength. The light leaving the lamp housing was filtered through a heat absorption filter (514-396 heat, thickness 2 mm; Leitz) and various neutrodensity filters (ND), i.e., ND6, ND12, ND25, ND50, to reduce light energy. The number specification of each of these neutrodensity filters ( 45 mm) indicates the amount of light transmitted through the filter given as percent of the total incident light. By applying and combining different neutrodensity filters, we were able to expose the tissue repeatedly to distinct light energy levels. A heat absorption filter was installed in all experiments to protect the neutrodensity filters from temperature damage. The light was further passed through excitation filters for AO (D480/x 30, 4039; Chroma, Brattleboro, VT or Rh6G (TRITC 31002, 506; D605/55m; Chroma) and then reflected by a dichroic mirror which reflected wavelengths >400 nm to the skin muscle tissue of the hamster, and passed emitted wavelengths >500 nm to the camera. The dichroic filter and the excitation and emission filters were parts of the microscope filterblock (H3 513673 and TRITC 31002, 506; Leitz). For the intravital fluorescence microscopic investigations, a x20 water-immersion objective (WI 20, 0.4; Olympus, Tokyo, Japan) was used. The final magnification was 660-fold. The output of the CCD camera was then displayed on a monitor (12 VM 968; Audiotronics, North Hollywood, CA). The images were captured with a videocassette recorder (Video Hi8, EV-C100, NTSC; Sony, Tokyo, Japan) at a video frame rate of 30 frames/sec. A video timer (VTG33; Fora Company, Tokyo, Japan) was connected to the video recorder.
Non-intensified Camera System
A non-intensified CCD camera [CCD72, horizontal 570 TVL (television lines), vertical 350 TVL, pickup area 8.8 x 6.6 mm; Dage-MTI, Michigan City, IN] was used for visualization of fluorescently labeled leukocytes at higher light energy levels because it required stronger fluorescence signals for detection of the cells. The light sensitivity of this camera 0.001-0.002 fc (footcandles), which was equivalent to the sensitivity of other camera systems such as COHU 4400, FK6990 (COHU; Prospective Measurements, San Diego, CA) employed in other intravital microscopic studies using the identical animal model (
Intensified Camera System
To detect weaker fluorescent signals at very low light levels, the CCD 72 camera was connected to an image intensifier; (GENIISYS Image intensifier; Dage-MTI, Michigan City, IN). This system enabled us to reduce the incident light energy level by a factor of 40-fold. The GENIISYS device uses an 18-mm GENII proximity-focused image intensifier as the light amplification device, with three basic components: a photocathode, a microchannel plate (MCP), and a phosphor screen. When light reaches the photocathode surface (P20), electrons are accelerated and are proximity-focused onto the microchannel plate (MCP). The MCP multiplies the number of electrons through secondary emission and the output of the MCP is focused onto a phosphor screen. This electron bombardment of the phosphor screen creates an image that is optically coupled to the camera system (CCD 72). The photocathode (S20) has a spectral emittance (blue and green portion of the visible spectrum) similar to the spectral response of the dark adapted-human eye. The maximal light sensitivity of the intensified camera system was 10-6 fc (lumen/feet2) which corresponds to 10-5 lx (lumen/m2) (lx stands for lux). This camera system is approximately 100-1000-fold more light sensitive compared to the CCD 72 camera alone.
Measurement of Light Energy at the Tissue Level
Before the experiments a photodiode (UV-100BQ; EG&G Electro-Optics, Salem, MA) was positioned horizontally beneath the 20-fold objective (20WI 20, NA 0.4; Tokyo, Japan). As in the intravital microscopic studies, a drop of water was placed beneath the objective. The diode was adjusted until the light beam was centered and focused in the middle of the diode's window. We determined experimentally that the light did not overfill the diode sensitive surface which was a 2.5-mm-diameter circle. The outlet of the diode was connected to an ammeter (Beckman Industrial 310; Brea, CA). The photodiode's readout, given in milli-amperes (mA), was multiplied by a constant factor K = [(3.333 mW x 60 sec) / 1 mA] to obtain the correct value of light energy in mJ/min, as stipulated by the diode specifications. The diameter of the field (d) that an objective illuminates is approximately the microscope tube diameter divided by the objective magnification. In our microscopic setup this was d = (16 mm / 20) = 0.8 mm, which corresponded to an illuminated area (A) of A = 5 x 10-3 cm2. This represented the area to which the previously computed energy was applied. The energy per unit area (mJ/min/cm2) was E = K / (5 x 10-3 cm2) = [(3.333 mW/mA) x 60 sec)] / 5 x 10-3 cm2 = (3.333 x 60) / 5 x 10-3 mJ/min/cm2. Our measurements revealed that the coverglass of the titanium chamber did not absorb any light, so that the above described measurements in fact represented the light energy at the tissue level.
Experimental Protocol
As described in previous publications, we exposed the skinfold microcirculation four times for 30 sec to the excitation light (
Leukocyte rolling and leukocyte sticking in postcapillary venules and arterioles were evaluated and quantified off-line. In addition, the occurrence of arteriolar vasospasm was assessed. In accordance with previous studies, sticking leukocytes were defined as the total number of leukocytes that were firmly attached to the microvascular endothelium and did not change their location throughout the entire 30-sec light exposure period. Sticking leukocytes were given as number of cells/per mm2 of vessel surface, calculated from the diameter and standardized length (200 µm) of the vessel segment under investigation.
Rolling leukocytes were defined as the total number of leukocytes rolling along the endothelial cell lining at a substantially slower velocity compared to the midstream blood cell velocity. Rolling leukocytes were given as number of cells passing an imaginary line perpendicular to the vessel's longitudinal axis over a period of 30 sec.
Vasospasm was defined as a complete closure of the vessel lumen with no signs of microvascular flow.
Statistical Analysis
The data in the figures are given as mean ± SD at each light energy level. Five animals were included in each of the investigated light energy level. p<0.05 was considered statistically significant and was calculated by single-factor, repeated-measures ANOVA.
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Results |
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Figure 1 demonstrates that the photodiode output (mAmp) responded in a linear fashion to the reduction of incident light energy level (%). Reduction of light energy was achieved by using various neutrodensity filters (ND).
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Acridine Orange
To obtain adequate images at the lower light energy levels (77.6 mJ/cm2/min, 192.1 mJ/cm2/min, 698.8 mJ/cm2/min), the intensified camera system had to be used because the fluorescence signal was too weak to be detected by the non-intensified camera alone (CCD 72). At higher light energy levels (1514.2 mJ/cm2/min, 3106.3 mJ/cm2/min), the experiments were executed with the CCD 72 without the intensifying system.
Figure 2 and Figure 3 demonstrate the effect of AO-induced phototoxicity on leukocyte rolling (Figure 2) and sticking to the endothelium in arterioles and postcapillary venules (Figure 3). In postcapillary venules, we observed a baseline leukocyte rolling value of 6-8 leukocytes/30 sec observation period. Repeated exposures at the lowest light energy level (77.6 mJ/cm2/min) caused no changes in leukocyte rolling (Figure 2). Leukocyte rolling increased at light energy levels equal to or greater than 192.1 mJ/cm2/min. Slightly less pronounced leukocyte rolling was observed in arterioles. At the lower light levels, ranging between 77.6 mJ/cm2/min and 192.1 mJ/cm2/min, arteriolar leukocyte rolling could not be evoked by repeated light exposures (Figure 2). Reproducible data for leukocyte-endothelium interaction could not be obtained at light levels equal to or greater than 698.8 mJ/cm2/min because of significant reductions in arteriolar diameters as well as complete vasospasm.
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No sticking leukocytes during our baseline measurements (first light exposure) were found in arterioles and postcapillary venules (Figure 3). No leukocyte adhesion could be detected at light energy levels at and below 192.1 mJ/cm2/min. At higher light levels, repeated light exposures of the same vessel segment resulted in a steady, almost linear increase in leukocyte sticking in postcapillary venules (Figure 3).
In a series of control experiments, no leukocyte-endothelium interaction or arteriolar diameter changes were observed when the tissue was repeatedly exposed to light of various energy levels but without previous injection of the photosensitizing dye (data not shown).
Acridine Orange-induced Vasospasm
At light energy levels of 77.6 and 192.1 mJ/cm2/min, no arteriole that was exposed to the incident light showed complete vasospasm (0%). At light energy levels of 698.8, 1514.2, and 3106.3 mJ/cm2/min, 66%, 100%, and 100% of all investigated arterioles, respectively, went into complete vasospasm. At light energy levels of 1514.2 mJ/cm2/min and 3106.3 mJ/cm2/min virtually all arterioles went into spasm after the second light exposure. Upstream and downstream from the site of light exposure, the afflicted arteriole exhibited normal vessel diameters. Observation of the identical vessels 24 hr after light exposures revealed in most cases that the arteriolar vasospasm was still present at the site of light epi-illumination. Vasospasms occurred after 1.9 ± 0.3, 2.1 ± 0.3, and 2.9 ± 0.2 exposures at light levels or 3106.3, 1514.2, and 698.8 mJ/cm2/min, respectively.
Rhodamine 6G
Because of the much weaker fluorescence signal from Rh6G- labeled leukocytes, we could not obtain satisfactory images at light levels of 77.6 mJ/cm2/min and 192.1 mJ/cm2/min, even using the intensified system. All the images were captured with the image-intensifying camera system.
At light levels beyond 698.8 mJ/cm2/min, leukocyte rolling increased slightly after each exposure in postcapillary venules, but not in arterioles (Figure 4). Similar results were obtained for sticking leukocytes in postcapillary venules and arterioles (Figure 5). No sticking leukocytes were evident during the baseline measurements (first exposure). In postcapillary venules, the number of sticking leukocytes increased slightly after each subsequent exposure to light levels of 1514.2 mJ/cm2/min and 3106.3 mJ/cm2/min, but not at 698.8 mJ/cm2/min.
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We did not observe any arteriolar vasospasm or arteriolar diameter changes at any of the light levels investigated when Rh6G was used as the fluorescent dye.
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Discussion |
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The purpose of this investigation was to test the hypothesis that the energy level of the incident light used for illumination of fluorescently labeled leukocytes influences leukocyte-endothelial interaction in vivo and to determine the most appropriate experimental methodology for direct in vivo microcirculatory studies to avoid this artifact. The principal findings of this investigation are as follows: Leukocyte rolling and adhesion to endothelial cells can be provoked by photoexcitation of these fluorescent dye molecules. The influence of these photochemical processes on cell activation is directly related to the amount of light energy used for illumination of these cells. In contrast to Rh6G, AO caused arteriolar vasospasm and diameter changes at high light energy levels. Finally, visualization of fluorescently labeled leukocytes by an intensified camera system allows significant reduction of incident light energy to levels at which phototoxic interference can be excluded.
Intravital Microscopic Set-up
The basic set-up for intravital fluorescence microscopy requires a high-energy light source (usually a mercury or xenon lamp), a microscope suited for epi- or transillumination, and a camera. The quality of the camera (light sensitivity) is the most important determinant of the amount of incident light energy that must be used during the experiments. In the present study, the light sensitivity of our non-intensified camera system (CCD72) was comparable to those used by us in previous studies, as well as by other investigators (
Cell Activation Through Photoexcited Acridine Orange
AO (MW 669.7) is a weak acidophilic dye that can readily diffuse through cell membranes. Because of its weak acidotropic properties, AO accumulates in acidic cell compartments on protonation, e.g., lysosomes, endosomes, and the Golgi apparatus (
There are a number of potential mechanisms through which radical-mediated cell activation could elicit leukocyte adhesion.
Cell Activation Through Photoexcited Rhodamine 6G
Rh6G is a cationic fluorescent dye (MW 479) that selectively accumulates in nuclei and mitochondria of living cells (
Phototoxicity-induced alterations of microvascular perfusion, vascular diameter, and cell function are common phenomena inherent not only to AO or Rh6G but also to other fluorescent dyes when excited at the appropriate wavelengths (
The results of the present study emphasize that intravital fluorescence microscopic approaches to study cell-cell interactions are susceptible to phototoxic artifacts. The amount of light needed to visualize leukocytes in vivo is determined by the quality of the camera system. Non-intensified cameras with limited light sensitivity options require significantly stronger fluorescence signals to obtain images with an adequate signal-to-noise ratio. To generate stronger signals, fluorescently labeled cells and tissues must be exposed to higher incident light energy levels, thus substantially increasing the risk for phototoxic effects. In contrast, intensified cameras, which are 100-1000-fold more light sensitive than non-intensified systems, can register much weaker fluorescent signals. When the amount of excitation light energy is reduced considerably, by use of an intensified camera system, the risk of phototoxicity is abolished.
Our study therefore emphasizes the need to optimize the microscopic set-up to obtain adequate imaging of the microcirculation with a minimum of light energy. This can be achieved (a) by using various optical filters and image intensified camera systems, (b) by applying the smallest amount of the fluorescent dye in a standardized fashion (constant infusion or single bolus), (c) by reducing observation time periods and, most importantly, (d) by performing stringent control experiments to exclude phototoxic activation of leukocytes. When these conditions are established, acridine orange and rhodamine 6G can continue to be of great help in the investigation of leukocyte-endothelial cell interaction by means of intravital microscopy.
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Acknowledgments |
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Supported by NIH grant no. HL-46022. The results were presented in part during the 43rd Annual Conference of the Microcirculatory Society, April 13-14, 1996, in Washington, DC.
Received for publication May 3, 1996; accepted November 27, 1996.
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