Imaging Lymphoid Cell Death In Vivo During Polymicrobial Sepsis.
OBJECTIVE
Cell death in lymphatic organs, such as the spleen, is in part responsible for immunosuppression and contributes to mortality during sepsis. An early and noninvasive detection of lymphoid cell death could thus have significant clinical implications. Here, we tested in vivo imaging of lymphoid cell death using a near-infrared annexin V (AV-750).


METHODS
Animal study.


METHODS
Laboratory investigation.


METHODS
C57BL/6J wild-type and toll-like receptor 3 knockout mice.


METHODS
Mild and severe polymicrobial sepsis was induced with cecum ligation and puncture. Serum cytokines and acute kidney injury markers were tested by immunoassay and quantitative reverse transcription-polymerase chain reaction, respectively. Sepsis-induced lymphoid cell death was detected by fluorescent AV-750 accumulation in the thorax and abdomen (in vivo), in isolated organs (ex vivo), and in isolated cells (flow cytometry). Caspase-3 cleavage/activity and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining were tested for apoptosis.


RESULTS
Severe sepsis induced marked apoptosis in the thymus, spleen, and liver as demonstrated by cleaved caspase-3 and an increase in caspase-3 activity and terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells. A significant increase in fluorescent AV-750 signal was seen in the thoracic and upper abdominal fields and associated with the severity of sepsis. The in vivo thoracic and abdominal AV-750 fluorescent signal was attributed to the thymus, liver, and spleen as determined by ex vivo imaging and highly correlated with the levels of cell death in thymocytes and splenocytes, respectively, as measured by flow cytometry. Compared with wild-type septic mice, toll-like receptor 3 septic mice had attenuated abdominal AV-750 fluorescent signal, reduced ex vivo fluorescence in the spleen, and decreased splenocyte cell death.


CONCLUSIONS
In vivo AV-750 fluorescent imaging provides spatially resolved and organ-specific detection of lymphoid cell death during polymicrobial sepsis. The AV-750 fluorescent intensity in the thoracic and abdominal fields is associated with sepsis severity and well correlated with sepsis-induced cell death in the thymus and spleen, respectively.
1Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA. 2Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA. 3Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Los Angeles, CA.
Lin
L
Zou
Howard H
HH
Chen
Dan
D
Li
Ganqiong
G
Xu
Yan
Y
Feng
Chan
C
Chen
Larry
L
Wang
David E
DE
Sosnovik
Wei
W
Chao
D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D016470:Bacteremia / Q000382:microbiology* / Q000503:physiopathology; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death; D060085:Coinfection / Q000175:diagnosis / Q000382:microbiology*; D004195:Disease Models, Animal; D005260:Female; D005434:Flow Cytometry; D020287:In Situ Nick-End Labeling / Q000379:methods; D008214:Lymphocytes / Q000166:cytology* / Q000502:physiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011897:Random Allocation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D018709:Statistics, Nonparametric
D053148:Caspase 3