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Confocal fluorescence imaging enables noninvasive quantitative assessment of host cell populations in vivo following photodynamic therapy.

Mitra S, Mironov O, Foster TH - Theranostics (2012)

Bottom Line: The maximum accumulation of Gr1(+) cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point.Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1(+ )cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status.Co-localization analysis reveals an increase in the fraction of Gr1(+) cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Imaging Sciences, University of Rochester Medical Center, Rochester, NY 14642, USA.

ABSTRACT
We report the use of optical imaging strategies to noninvasively examine photosensitizer distribution and physiological and host responses to 2-[1-hexyloxyethyl]-2 devinyl pyropheophorbide-a (HPPH)-mediated photodynamic therapy (PDT) of EMT6 tumors established in the ears of BALB/c mice. 24 h following intravenous (IV) administration of 1 μmol kg(-1) HPPH, wide-field fluorescence imaging reveals tumor selectivity with an approximately 2-3-fold differential between tumor and adjacent normal tissue. Confocal microscopy demonstrates a relatively homogeneous intratumor HPPH distribution. Labeling of host cells using fluorophore-conjugated antibodies allowed the visualization of Gr1(+)/CD11b(+) leukocytes and major histocompatibility complex class II (MHC-II)(+) cells in vivo. Imaging of the treated site at different time-points following irradiation shows significant and rapid increases in Gr1(+) cells in response to therapy. The maximum accumulation of Gr1(+) cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point. Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1(+ )cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status. Dual color confocal imaging experiments demonstrates that about 90% of the anti-Gr1 cell population co-localized with anti-CD11b labeling, thus indicating that majority of the Gr1-labeled cells were neutrophils. At 24 h post-PDT, an approximately 2-fold increase in MHC-II+ cells relative to untreated control is also observed. Co-localization analysis reveals an increase in the fraction of Gr1(+) cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype.

No MeSH data available.


Related in: MedlinePlus

(a-c) Series of stereofluorescence images illustrating vessel perfusion status up to 48 h in an individual ear tumor treated with 100 J cm-2. (a) Untreated control, (b) 24 h post HPPH-PDT and (c) 48 h post-irradiation. These images were acquired from the same mouse tumor for 3 consecutive days. The dark vascular morphology in (c) represents tumor vessels that have lost their perfusion status. The FOV in these images is 3.6 mm x 3.2 mm. (d-f) Representative in vivo confocal fluorescence images of perfusion (green) in CD31+ vessels (red) in (d) untreated control, (e) 24 h and (f) 48 h post HPPH-PDT irradiation. The FOV in these images is 500μm x 500μm.
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Figure 4: (a-c) Series of stereofluorescence images illustrating vessel perfusion status up to 48 h in an individual ear tumor treated with 100 J cm-2. (a) Untreated control, (b) 24 h post HPPH-PDT and (c) 48 h post-irradiation. These images were acquired from the same mouse tumor for 3 consecutive days. The dark vascular morphology in (c) represents tumor vessels that have lost their perfusion status. The FOV in these images is 3.6 mm x 3.2 mm. (d-f) Representative in vivo confocal fluorescence images of perfusion (green) in CD31+ vessels (red) in (d) untreated control, (e) 24 h and (f) 48 h post HPPH-PDT irradiation. The FOV in these images is 500μm x 500μm.

Mentions: Perfused tumor blood vessels have important functions as the source of local tumor oxygenation and as a route for host cell trafficking 24. With this motivation, bulk tumor perfusion in vivo was imaged with the stereofluorescence microscope, and microscopic imaging of small numbers of individual vessels was performed using the confocal microscope. Perfusion in live mice was visualized at pre-, 24 h, and 48 h post-irradiation using IV-injected FITC-conjugated high molecular weight dextran as an optical perfusion marker. Figures 4(a-c) are representative stereofluorescence images of perfusion status in an EMT6 ear tumor in the same mouse, followed up to 48 h post-PDT. As illustrated in figures 4(a) and 4(b), there was no detectable difference in perfusion status between the control and treated tumor at 24 h post-treatment. However, at the 48 h time-point a severe perfusion deficit is observed (figure 4(c)). Figures 4(d-f) are confocal images of microscopic patterns of perfusion level (green) in CD31+ labeled vessels (red). Consistent with the stereofluorescence images, we find that relative to the highly perfused vessels observed in control tissue, most of the CD31-positive vasculature in the treated region at 48 h post-irradiation exhibits an absence or low levels of the perfusion marker. This observation of perfusion loss at the 48 h correlates positively with the reduced Gr1+ population density at the same time-point. It is well established that vascular damage may be mediated by neutrophil secretion products such as chemokines, heparin-binding protein and arachidionic acid 25, 26. Therefore, the reduced perfusion status at 48 h post-PDT is likely triggered by the large accumulation of neutrophils in the tumor tissue. This mode of vascular response is in contrast to the vascular shutdown induced by Visudyne-PDT, where direct damage to ECs leads to rapid loss of vascular barrier function 27. Further, recent evidence has shown that neutrophils release proteolytic enzymes like MMP-9 upon activation 28. Therefore, degradation of the extracellular matrix and damage to blood vessels following HPPH-PDT may also be mediated in part by the release of MMPs from the accumulated neutrophil population.


Confocal fluorescence imaging enables noninvasive quantitative assessment of host cell populations in vivo following photodynamic therapy.

Mitra S, Mironov O, Foster TH - Theranostics (2012)

(a-c) Series of stereofluorescence images illustrating vessel perfusion status up to 48 h in an individual ear tumor treated with 100 J cm-2. (a) Untreated control, (b) 24 h post HPPH-PDT and (c) 48 h post-irradiation. These images were acquired from the same mouse tumor for 3 consecutive days. The dark vascular morphology in (c) represents tumor vessels that have lost their perfusion status. The FOV in these images is 3.6 mm x 3.2 mm. (d-f) Representative in vivo confocal fluorescence images of perfusion (green) in CD31+ vessels (red) in (d) untreated control, (e) 24 h and (f) 48 h post HPPH-PDT irradiation. The FOV in these images is 500μm x 500μm.
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Related In: Results  -  Collection

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Figure 4: (a-c) Series of stereofluorescence images illustrating vessel perfusion status up to 48 h in an individual ear tumor treated with 100 J cm-2. (a) Untreated control, (b) 24 h post HPPH-PDT and (c) 48 h post-irradiation. These images were acquired from the same mouse tumor for 3 consecutive days. The dark vascular morphology in (c) represents tumor vessels that have lost their perfusion status. The FOV in these images is 3.6 mm x 3.2 mm. (d-f) Representative in vivo confocal fluorescence images of perfusion (green) in CD31+ vessels (red) in (d) untreated control, (e) 24 h and (f) 48 h post HPPH-PDT irradiation. The FOV in these images is 500μm x 500μm.
Mentions: Perfused tumor blood vessels have important functions as the source of local tumor oxygenation and as a route for host cell trafficking 24. With this motivation, bulk tumor perfusion in vivo was imaged with the stereofluorescence microscope, and microscopic imaging of small numbers of individual vessels was performed using the confocal microscope. Perfusion in live mice was visualized at pre-, 24 h, and 48 h post-irradiation using IV-injected FITC-conjugated high molecular weight dextran as an optical perfusion marker. Figures 4(a-c) are representative stereofluorescence images of perfusion status in an EMT6 ear tumor in the same mouse, followed up to 48 h post-PDT. As illustrated in figures 4(a) and 4(b), there was no detectable difference in perfusion status between the control and treated tumor at 24 h post-treatment. However, at the 48 h time-point a severe perfusion deficit is observed (figure 4(c)). Figures 4(d-f) are confocal images of microscopic patterns of perfusion level (green) in CD31+ labeled vessels (red). Consistent with the stereofluorescence images, we find that relative to the highly perfused vessels observed in control tissue, most of the CD31-positive vasculature in the treated region at 48 h post-irradiation exhibits an absence or low levels of the perfusion marker. This observation of perfusion loss at the 48 h correlates positively with the reduced Gr1+ population density at the same time-point. It is well established that vascular damage may be mediated by neutrophil secretion products such as chemokines, heparin-binding protein and arachidionic acid 25, 26. Therefore, the reduced perfusion status at 48 h post-PDT is likely triggered by the large accumulation of neutrophils in the tumor tissue. This mode of vascular response is in contrast to the vascular shutdown induced by Visudyne-PDT, where direct damage to ECs leads to rapid loss of vascular barrier function 27. Further, recent evidence has shown that neutrophils release proteolytic enzymes like MMP-9 upon activation 28. Therefore, degradation of the extracellular matrix and damage to blood vessels following HPPH-PDT may also be mediated in part by the release of MMPs from the accumulated neutrophil population.

Bottom Line: The maximum accumulation of Gr1(+) cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point.Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1(+ )cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status.Co-localization analysis reveals an increase in the fraction of Gr1(+) cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Imaging Sciences, University of Rochester Medical Center, Rochester, NY 14642, USA.

ABSTRACT
We report the use of optical imaging strategies to noninvasively examine photosensitizer distribution and physiological and host responses to 2-[1-hexyloxyethyl]-2 devinyl pyropheophorbide-a (HPPH)-mediated photodynamic therapy (PDT) of EMT6 tumors established in the ears of BALB/c mice. 24 h following intravenous (IV) administration of 1 μmol kg(-1) HPPH, wide-field fluorescence imaging reveals tumor selectivity with an approximately 2-3-fold differential between tumor and adjacent normal tissue. Confocal microscopy demonstrates a relatively homogeneous intratumor HPPH distribution. Labeling of host cells using fluorophore-conjugated antibodies allowed the visualization of Gr1(+)/CD11b(+) leukocytes and major histocompatibility complex class II (MHC-II)(+) cells in vivo. Imaging of the treated site at different time-points following irradiation shows significant and rapid increases in Gr1(+) cells in response to therapy. The maximum accumulation of Gr1(+) cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point. Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1(+ )cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status. Dual color confocal imaging experiments demonstrates that about 90% of the anti-Gr1 cell population co-localized with anti-CD11b labeling, thus indicating that majority of the Gr1-labeled cells were neutrophils. At 24 h post-PDT, an approximately 2-fold increase in MHC-II+ cells relative to untreated control is also observed. Co-localization analysis reveals an increase in the fraction of Gr1(+) cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype.

No MeSH data available.


Related in: MedlinePlus