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Endogenous Two-Photon Excited Fluorescence Provides Label-Free Visualization of the Inflammatory Response in the Rodent Spinal Cord.

Uckermann O, Galli R, Beiermeister R, Sitoci-Ficici KH, Later R, Leipnitz E, Neuwirth A, Chavakis T, Koch E, Schackert G, Steiner G, Kirsch M - Biomed Res Int (2015)

Bottom Line: Iba1-positive microglia were found in areas lacking any TPEF signal.Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF.Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes.

View Article: PubMed Central - PubMed

Affiliation: Neurosurgery, Carl Gustav Carus University Hospital, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany.

ABSTRACT
Activation of CNS resident microglia and invasion of external macrophages plays a central role in spinal cord injuries and diseases. Multiphoton microscopy based on intrinsic tissue properties offers the possibility of label-free imaging and has the potential to be applied in vivo. In this work, we analyzed cellular structures displaying endogenous two-photon excited fluorescence (TPEF) in the pathologic spinal cord. It was compared qualitatively and quantitatively to Iba1 and CD68 immunohistochemical staining in two models: rat spinal cord injury and mouse encephalomyelitis. The extent of tissue damage was retrieved by coherent anti-Stokes Raman scattering (CARS) and second harmonic generation imaging. The pattern of CD68-positive cells representing postinjury activated microglia/macrophages was colocalized to the TPEF signal. Iba1-positive microglia were found in areas lacking any TPEF signal. In peripheral areas of inflammation, we found similar numbers of CD68-positive microglia/macrophages and TPEF-positive structures while the number of Iba1-positive cells was significantly higher. Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF. Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes.

No MeSH data available.


Related in: MedlinePlus

Comparison of endogenous TPEF after SCI and microglial markers at a cellular level. Magnification of the lesion border 21 d after SCI. (a) Multiphoton image of an unlabeled cryosection obtained by combining SHG (blue channel), TPEF (green channel), and CARS (red channel). (b) CARS channel shown in gray scale; the dotted line indicates the border between the strongly damaged tissue area and the more preserved contralateral white matter. (c) The TPEF channel shown after thresholding in black and white. (d) Immunohistochemical staining of the same tissue section as shown in (a–c). Overlay of DAPI staining (blue channel), CD68 (green channel), and Iba1 (red channel). (e) Iba1 immunoreactivity shown in black and white. (f) CD68 immunoreactivity shown in black and white. Scale bar: 100 μm.
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fig2: Comparison of endogenous TPEF after SCI and microglial markers at a cellular level. Magnification of the lesion border 21 d after SCI. (a) Multiphoton image of an unlabeled cryosection obtained by combining SHG (blue channel), TPEF (green channel), and CARS (red channel). (b) CARS channel shown in gray scale; the dotted line indicates the border between the strongly damaged tissue area and the more preserved contralateral white matter. (c) The TPEF channel shown after thresholding in black and white. (d) Immunohistochemical staining of the same tissue section as shown in (a–c). Overlay of DAPI staining (blue channel), CD68 (green channel), and Iba1 (red channel). (e) Iba1 immunoreactivity shown in black and white. (f) CD68 immunoreactivity shown in black and white. Scale bar: 100 μm.

Mentions: Figure 2 shows the lesion border 21 d after SCI to compare label-free multiphoton imaging (Figures 2(a)–2(c)) and the expression of microglial markers (Figures 2(d)–2(f)) on a cellular scale. More preserved tissue areas of white matter were identified by high CARS signal intensity that also enables assessing the disturbed axonal alignment (Figure 2(b), upper part). In these regions, only weak endogenous TPEF was found (Figure 2(c)) and immunohistochemistry revealed Iba1-positive cells with elongated cell bodies (Figure 2(e)) but no CD68-positive cells (Figure 2(f)). In strongly damaged tissue regions (Figure 2(b), lower part), Iba1-positive cells were detected which displayed enlarged cell bodies and a round morphology without processes (Figure 2(e)). Additionally, strong CD68 immunoreactivity and round structures exhibiting intense endogenous TPEF were observed (compare Figures 2(c) and 2(f)). Structures displaying endogenous fluorescence were assigned to originate from CD68-positive microglia/macrophages.


Endogenous Two-Photon Excited Fluorescence Provides Label-Free Visualization of the Inflammatory Response in the Rodent Spinal Cord.

Uckermann O, Galli R, Beiermeister R, Sitoci-Ficici KH, Later R, Leipnitz E, Neuwirth A, Chavakis T, Koch E, Schackert G, Steiner G, Kirsch M - Biomed Res Int (2015)

Comparison of endogenous TPEF after SCI and microglial markers at a cellular level. Magnification of the lesion border 21 d after SCI. (a) Multiphoton image of an unlabeled cryosection obtained by combining SHG (blue channel), TPEF (green channel), and CARS (red channel). (b) CARS channel shown in gray scale; the dotted line indicates the border between the strongly damaged tissue area and the more preserved contralateral white matter. (c) The TPEF channel shown after thresholding in black and white. (d) Immunohistochemical staining of the same tissue section as shown in (a–c). Overlay of DAPI staining (blue channel), CD68 (green channel), and Iba1 (red channel). (e) Iba1 immunoreactivity shown in black and white. (f) CD68 immunoreactivity shown in black and white. Scale bar: 100 μm.
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fig2: Comparison of endogenous TPEF after SCI and microglial markers at a cellular level. Magnification of the lesion border 21 d after SCI. (a) Multiphoton image of an unlabeled cryosection obtained by combining SHG (blue channel), TPEF (green channel), and CARS (red channel). (b) CARS channel shown in gray scale; the dotted line indicates the border between the strongly damaged tissue area and the more preserved contralateral white matter. (c) The TPEF channel shown after thresholding in black and white. (d) Immunohistochemical staining of the same tissue section as shown in (a–c). Overlay of DAPI staining (blue channel), CD68 (green channel), and Iba1 (red channel). (e) Iba1 immunoreactivity shown in black and white. (f) CD68 immunoreactivity shown in black and white. Scale bar: 100 μm.
Mentions: Figure 2 shows the lesion border 21 d after SCI to compare label-free multiphoton imaging (Figures 2(a)–2(c)) and the expression of microglial markers (Figures 2(d)–2(f)) on a cellular scale. More preserved tissue areas of white matter were identified by high CARS signal intensity that also enables assessing the disturbed axonal alignment (Figure 2(b), upper part). In these regions, only weak endogenous TPEF was found (Figure 2(c)) and immunohistochemistry revealed Iba1-positive cells with elongated cell bodies (Figure 2(e)) but no CD68-positive cells (Figure 2(f)). In strongly damaged tissue regions (Figure 2(b), lower part), Iba1-positive cells were detected which displayed enlarged cell bodies and a round morphology without processes (Figure 2(e)). Additionally, strong CD68 immunoreactivity and round structures exhibiting intense endogenous TPEF were observed (compare Figures 2(c) and 2(f)). Structures displaying endogenous fluorescence were assigned to originate from CD68-positive microglia/macrophages.

Bottom Line: Iba1-positive microglia were found in areas lacking any TPEF signal.Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF.Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes.

View Article: PubMed Central - PubMed

Affiliation: Neurosurgery, Carl Gustav Carus University Hospital, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany.

ABSTRACT
Activation of CNS resident microglia and invasion of external macrophages plays a central role in spinal cord injuries and diseases. Multiphoton microscopy based on intrinsic tissue properties offers the possibility of label-free imaging and has the potential to be applied in vivo. In this work, we analyzed cellular structures displaying endogenous two-photon excited fluorescence (TPEF) in the pathologic spinal cord. It was compared qualitatively and quantitatively to Iba1 and CD68 immunohistochemical staining in two models: rat spinal cord injury and mouse encephalomyelitis. The extent of tissue damage was retrieved by coherent anti-Stokes Raman scattering (CARS) and second harmonic generation imaging. The pattern of CD68-positive cells representing postinjury activated microglia/macrophages was colocalized to the TPEF signal. Iba1-positive microglia were found in areas lacking any TPEF signal. In peripheral areas of inflammation, we found similar numbers of CD68-positive microglia/macrophages and TPEF-positive structures while the number of Iba1-positive cells was significantly higher. Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF. Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes.

No MeSH data available.


Related in: MedlinePlus