Multiplex Multimodal Imaging of Infiltrating Cells in vivo- Mouse Burn Wound Model

Rao V.L. Papineni*, William Mclaughlin, Douglas Vizard, and Sean Orton.

Carestream Health, Inc.

*E.Mail: rao.papineni1@carestreamhealth.com

Infiltration of macrophages occurs in NIRF Imaging of Burn Injury

atherosclerotic plaque, cancer, and inflammatory EndogenousMacrophages as Carriers of NIRF Contrast Agent
pathways. Regulatory processes involved in

Mouse
peritoneum
Inject KODAK X-Sight
infiltration have been identified in the recent past. To 650 and 761 Nanospheres
i.p
aid drug discovery, Imaging and estimation of the in + Time Lapse Imaging
vivo sequestration dynamics of differentially treated Burn injury LPS
cell culture population would be a valuable KODAK in-Vivo Multispectral FX system.
experimental asset. Mouse model of thermal injury Image X-ray, NIRF EM:700, and EM 790
was used to demonstrate the infiltration of both
endogenous cells and the exogenous murine cells at
the site of injury. Macrophages laden with different
commercially available near-IR fluorescence dye
encapsulated nanoparticles, KODAK X-Sight
Nanospheres, was used for multimodal imaging of
burn wound in a non-invasive manner.

Using a commercially available imaging system,

KODAK in-Vivo Multispectral FX system, X-ray
and near infrared fluorescence (NIRF) images were
obtained at different time intervals. This allowed
determination of different cell populations at the site
of injury non-invasively. The fluorescent signals on 30 min 120 min
anatomical overlays were used to obtain anatomical EX:630 EM:700 EX:730 EM:790 EX:630 EM:700 EX:730 EM:790
localization of fluorescent murine cells in the mice. 10sec; f-stop 2.8 10sec; f-stop 2.8 10sec; f-stop 2.8 10sec; f-stop 2.8

The outcome provides ample evidence for adaptation 17 Hrs

Left paw Right paw
of this approach in other inflammatory process, and
high throughput drug discovery that not only targets
infiltrating macrophages but also other pathological
NIRF signals from KODAK X-
events that encompass cell migratory process. Sight Nanospheres at the burn
wound were extracted in cell
EX:635 EM:700 cultures. Both the X-Sight 650 and
NIRF Imaging of Burn Injury X-Sight 761 are sequestered
putatively by the peritoneal
macrophages. The lower section
Animal Studies: All animal experiments were confirms the migratory role of
based on approved methodologies and well these macrophages.
documented animal protocols (Padfield et. al. PNAS 2005)
EX:735 EM:790
Swiss albino mice were anesthetized using
isoflurane and denuded hours before the thermal
injury.
Cell Culture Macrophages as Carriers of NIRF Contrast Agent
Thermal injury model: A non lethal scald injury was Inject exogenous macrophages laden with
induced to the left hind limb by immersion in 90oC i.p
KODAK X-Sight 650 Nanospheres
peritoneum
Mouse

water for 10 sec. This model is clinically relevant to + Time Lapse Imaging
common burn injuries. LPS

Burn injury KODAK in-Vivo Multispectral FX system.

Image X-ray and NIRF EX:630 EM:700
Inject exogenous macrophages laden with
KODAK X-Sight 650 or 761 Nanospheres

EX:735 EM:790
Injected macrophages laden with
Or KODAK X-Sight Nanospheres
peritoneum
Mouse

infiltrate other organs as well.

Burn injury 30 min 17 Hr Representative images of spleen
Inject KODAK X-Sight and liver are shown here.
i.p 650 and 761 Nanospheres

Mouse
peritoneum

Injected macrophages laden with KODAK X-Sight Nanospheres were

in circulation even after 20 hrs. The peritoneal extraction was subjected
to cell culture O/N and imaged using 40X NIRF microscopy.
NIRF Imaging- Autofluorescence (Left panel) and NIRF (right Panel).
KODAK in-Vivo Multispectral FX system.
Conclusions
•NIRF imaging of the burn wound or any inflammation can be facilitated by either external and internal source of
macrophages laden with NIRF contrast agents. It remains to be seen if the intra-peritoneal development of the
Mouse Ex Vivo Imaging imaging probe would maintain the phenotype integrity of the macrophages that potentially are compromised with
peritoneum targeting currently carried out.
•Non-invasive multimodal imaging approaches shown here delineates different cell populations that were
Cell Harvest exogenously laden with fluorescence contrast agents. This paves way aiding drug discovery - estimate the in vivo
Culture and Image sequestration dynamics of differentially treated cell culture populations.

"Molecular Imaging - Wisdom To See For Maladies To Flee"

Dr. Rao V. L. Papineni