Dual Contrast Molecular Imaging Allows Noninvasive Characterization of Myocardial Ischemia/Reperfusion Injury After Coronary Vessel Occlusion in Mice by mri running title
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Figure Legends: Figure 1. Static and flow chamber adhesion assays showing strong binding of LIBS-MPIOs on activated platelets. A: Representative microscopy images of MPIOs attachment under static conditions. Attachment of LIBS-MPIOs was significantly higher than for non-targeted control- MPIOs. (MPIO exemplarily depicted by arrows) B: Representative microscopy images of flow chamber adhesion assays demonstrating that more LIBS-MPIOs attach to the microthrombi under flow as compared to control-MPIOs. Time point assessment for optimal myocardial platelet imaging is demonstrated in C. For staining of platelets, a rat anti-mouse CD41 antibody was used, which then was detected by a biotinylated rabbit anti-rat IgG. Red staining was obtained by alkaline phosphate with a corresponding substrate kit. Platelet aggregates with bound LIBS-MPIO were quantified at different time points after reperfusion (10min, 2h, 4h, 6h, 8h, 10h, and 12h). Maximum amounts of absolute LIBS-MPIO-platelet-aggregates were found clinically relevant field strengths. Mol Imaging. 2008;7:59-67. Figure Legends: F Figu u gu re 1. Stat t ic ic a an nd nd f f f lo l w w w ch ch cham am am be be e r r r ad ad a h he he s sion
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A: R R Rep ep re e se se s nt nt nt at at at iv iv iv e e mi mi mi cr cr cr o os os co co opy py py i
i ma ma ma ge ge ges s s of o of M M MP PI PIOs Os Os a at tt tt ac ac chm hm men en nt
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conditions. At At t ta ta ta ch ch h me me m nt nt n o o f f LI LI LI BS BS BS -M -M M PI PI P Os Os Os w w w as as as s s s ig ig g ni ni n fi fi f ca ca c nt nt t ly ly ly h h h ig ig ig he he he r r r th h h an an an f f f or or or n n n on on o -t -t ar ar ar ge ge ge te te te d d control- by guest on December 22, 2017 http://circ.ahajournals.org/ Downloaded from
DOI: 10.1161/CIRCULATIONAHA.113.008157 28
2h after reperfusion, with significant differences compared to all other time points (p<0.05). A representative image of a LIBS-MPIO-platelet-aggregate is shown in the inlay (platelets = red, MPIOs = round/brown structure, see arrows, x100 magnification).
axis image from the medial ventricular areas are depicted. For animals injected with LIBS-MPIO (A, upper row) and control-MPIO (B, lower row), baseline scans are shown on the left. After contrast agent injection, a continuous signal decrease can be seen in ischemic areas of animals with LIBS-MPIO-injection (red arrows) as the typical susceptibility artifact induced by MPIOs. No signal effect is visible in animals with control-MPIO-injection. After imaging for 37min, gadolinium was injected to observe the LGE for detection of myocardial necrosis, which is present in both treatment groups (see yellow arrow).
was not influenced by the injection of LIBS-MPIO and control-MPIO (A). Simultaneous staining for platelets (CD41, red) and neutrophils (Gr1, black) shows platelet-neutrophil-conjugates (B, 20x magnification; C, negative control omitting primary antibody; D, 100x magnification, with an example of bound MPIOs (arrows)), with comparable amounts of such conjugates in both groups (E). For quantification and characterization of platelet accumulation (F) depicts a representative myocardial section with platelet aggregates stained by anti-CD41 immunohistochemistry and direct visualization of bound MPIOs (G; arrows). No platelet accumulation can be observed in non-ischemic myocardium such as the septal wall (H). Microthrombi are equally distributed in both groups (I), whereas MPIO-binding was No signal effect is visible in animals with control-MPIO-injection. After imagin n g g g fo
fo o r 37 37 37 mi mi min, n, n,
ff gadolinium was injected to observe the LGE for detection of myocardial necrosis, which is pr r
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E E was not influe ue e nc nc nc ed ed d b b b y y y th th h e in in in je je je ct t t io io io n n of o o L L L IB IB B S- S- S MP MP MP IO IO IO a a a nd nd c c c on on on tr tr tr ol ol ol -M -M M PI PI PI O O O ( ( ( A A A ). ). ). S S im m m ul ul ul ta ta ta ne ne ne ous stainin ng g by guest on December 22, 2017 http://circ.ahajournals.org/ Downloaded from
DOI: 10.1161/CIRCULATIONAHA.113.008157 29
significantly higher in animals injected with LIBS-MPIO than with control-MPIOs (J). A significant correlation between the amount of bound LIBS-MPIO and microthrombi in ischemic areas is evident (K).
myocardial necrosis. Signal quantification is pooled for the anterior and anterolateral segments, representing the areas of ischemia (A, arrows), as described in detail in “Methods”. After LIBS- MPIO-injection, a significant signal decrease as the typical MPIO-induced effect can be observed (B, red line), suggesting effective binding of LIBS-MPIO to platelets. After gadolinium-injection, the signal in LIBS-MPIO-injected mice increases above baseline (p<0.003). Mice injected with control-MPIO demonstrate no MPIO-induced signal decrease (B, blue line). CA = contrast agent; Gd = Gadolinium. Figure 5. P2Y 12 -/- mice demonstrate reduced platelet and neutrophil infiltration. No signal increase can be observed after injection of LIBS-MPIO (A), similar to animals with control- MPIO (B). However, the presence of myocardial necrosis can be confirmed after the injection of gadolinium in both groups (right, yellow arrows).
12 -/-
mice. The area of LGE is significantly smaller in P2Y
12 -/-
mice (A). As a potential consequence of the expected lower platelet accumulation in P2Y
12 -/-
mice, no significant difference in the MR signal after MPIO injection was observed between control-MPIO (green line) and LIBS-MPIO (yellow line) in these animals (B). Infarct size was significantly smaller in P2Y 12 -/- mice in TTC staining (C) and echocardiographic gadolinium-injection, the signal in LIBS-MPIO-injected mice increases above b b a
asel el l in in e e p<0.003). Mice injected with control-MPIO demonstrate no MPIO-induced signal decrease (B, bl l
ue e l l l in in in ) e) e). CA
CA CA = = c c c o on ontrast agent; Gd = Gadolinium um m. Fi Fi gu gu gure re re 5 5 . . P2 P2 P2Y Y Y 12 12 2 - -/-
mi mi m c ce ce d d d em em o on on st st st ra ra ra te te te r r e ed educ uc uced ed ed p p pla
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by guest on December 22, 2017 http://circ.ahajournals.org/ Downloaded from
DOI: 10.1161/CIRCULATIONAHA.113.008157 30
assessment demonstrated a trend towards better preservation of left ventricular ejection fraction in P2Y
12 -/-
mice (D) compared to WT.
12 -/-
mice. Only minimal platelet accumulation was found in ischemic/reperfused areas (A, anti-CD41 staining), and the number of platelet-neutrophil-aggregates decreased (B, CD41/Gr1-stain). Comparison of WT versus P2Y 12 -/-
mice confirms a significantly smaller number of microthombi (C), and the number of bound MPIOs was significantly reduced in all P2Y 12 -/-
mice, towards the level of WT mice (D). When adding the data obtained with the P2Y 12 -/-
mice to the data obtained with the WT-animals, correlation between bound MPIOs and microthrombi remains highly significant (E; orange = P2Y 12 -/- -animals), whereas the amount of platelet-neutrophil-aggregates was reduced in P2Y 12 -/-
animals (F). Figure 8. Quantification and correlation of myocardial necrosis, LIBS-MPIO and LGE effects: In H&E-staining, the proportion of myocardial necrosis was not significantly different between LIBS-MPIO and control-MPIO- injected animals, whereas the myocardial necrosis was lower in P2Y
12 -/-
mice (A/B). The extent of the area with a LIBS-MPIO-induced effect in MRI correlated well with the extent of the LGE area of the left ventricle (C), and with the area of necrosis in histology (D). When pooling these data with the results from the P2Y 12 -/- mice, the area of necrosis correlated well with the LGE-area (E).
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12 -/-
-animals), whereas the amount of platelet-neutrophil-aggregates was e e du du u ce ce e d d d in in in P P P 2 2 2Y 12 12 2 -/- -/-
a animals (F). Fi Fi gu gu gure re re 8 8 . . Qu Qu Qu an an t ti ifi i ica
ca ti ti i on on n a a nd nd d c c or or orre re re la la la ti ti on on on o o o f f f my my myoc
oc o ar ar d dia al al n
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si si s, s, L L LIB IB IB S S- S- M MP MPI I I O O O an
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n H&E-stain n in in in g, g, g t t t he he he p p p ro ro r po po o rt rt t io i i n n n of of o m m m yo yo yo ca ca ca rd rd d ia ia a l l ne ne e cr cr c os os o is s s w w w as as as n n n ot ot ot s s ig g g ni ni ni fi fi fi ca ca ca nt nt nt ly ly l d d d if if if fe fe fe re re re nt n n between by guest on December 22, 2017 http://circ.ahajournals.org/ Downloaded from Figure 1 10 μM 10 μM B A Control LIBS
0 200
400 600
800 Control
LIBS 0 200 400 600
*** ***
MPIO C
10 m 10 m
10 m MPIO 10 m
10 m p<0.05 20 40 60 80 100 120 0 LIBS-MPI Os bound to pla tel et s Time of reperfusion LIBS- MPIO control- MPIO 600 400 200 0 Numbe r of MPI Os p<0.0001 p<0.0001 800 600 400 200 Numbe r of MPI Os LIBS- MPIO control- MPIO B C C C 10 m 0 10 m
m m
< 0 05 05 p<0 05 5 p< p< 0.05 05 10 10 0 0 12 12 0 0 a a e te e le le le s ts MP MP IO IO MP MP MP IO IO O p< p< p 0. 0. 0. 00 00 00 01 01 01 80 0 0 0 600 400 0 20 20 0 0 0 Nu Nu u mb mb m er e of MPI Os Nu mb mb m er e of MPI Os Nu mb mb m er e of MPI Os LI LI S BS B - MP MP MP IO IO IO o co co nt o ro ro l- MP MP PIO IO IO by guest on December 22, 2017 http://circ.ahajournals.org/ Downloaded from control- MPIO
LIBS-MPIO pre control-MPIO 15‘ post control-MPIO 30‘ post Gd 22‘ post control-MPIO 37‘ post control-MPIO pre LIBS-MPIO 15‘ post LIBS-MPIO 22‘ post LIBS-MPIO 37‘ post LIBS-MPIO 30‘ post Gd O A B Figure 2
re LI LI BS BS BS -M -M -M PI PI PI O 1 O 5‘ post LIBS-MPIO 22‘ post t LI LI LI BS B B -MPIO 37‘ pos s t t LI LI LI BS B B -MPIO
30‘ post by guest on December 22, 2017 http://circ.ahajournals.org/ Downloaded from A B A B CD41/Gr1-IHC of ischemic areas; x20 p=0.0047 R 2 =0.8244 p=0.0047 R 2 =0.8244 CD41-IHC of ischemic areas; x20 100 m CD41-IHC of non-ischemic areas; x20 100 m C D E F G H I J K 100 m
100 m control-IHC of ischemic areas; x20 bound MPIOs p=0.0034 20 40 60 80 100 120 0 LGE-Area in % of left ve ntri cl e LG E n.s. CD41/Gr1-IHC of ischemic areas; x100 20 m
CD41-IHC of ischemic areas; x100 20 m
Figure 3
20 40 60 80 100 120 140 G G H H C of is is ch ch em em ic i a re re as; x100
20 m el et-ne u G G Pl at e 5 0 10 by guest on December 22, 2017 http://circ.ahajournals.org/ Downloaded from Figure 4 A B CA-injection * LIBS-MPIO vs. control-MPIO for all post CA-values: p<0.02 § signal increase of mean values in LIBS-MPIO before vs. after Gd-injection: p<0.03 Gd-injection WT control-MPIO WT LIBS-MPIO * § Download 1.2 Mb. Do'stlaringiz bilan baham: 
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