Male Brown Norway (BN; RT1ⁿ) rats were obtained from Harlan. Animal protocols were approved by the Institutional Animal Care and Use Committee of Carnegie Mellon University. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals.

We employed a transient left anterior descending (LAD) artery occlusion model for creating IRI in rats [27] . Male inbred Brown Norway (BN; RT1ⁿ) rats, 2 to 3 months of age, weighing 250 ± 30 g, were subjected to general anesthesia with isoflorane. After induction via a nose cone, the animal was intubated and ventilated with 2% isoflorane in oxygen and nitrogen gas mixture with a tidal volume of 1.0 ml/100g body weight at a rate of 55 - 60 strokes per minute. A rectal temperature was maintained at 37˚C with a heating pad.

Thoracotomy through the fourth intercostal space was performed followed by pericardiotomy, to gain access to the heart and LAD. The LAD was temporarily ligated with 6-0 Prolene suture (Novafil). After 45 min, the suture was removed to re-perfuse the heart, the chest wall was closed, and the animal was recovered from anesthesia and returned to its cage; or to be subjected to in-vivo CMRI.

At the onset of re-perfusion, Intralipid^® (20%, purchased from Fresenius Kabi) was intravenously administered at a dose of 2 g/kg. Control animals received saline. 16 rats were given Intralipid^® and 14 were given saline (untreated). For in-vivo MRI procedures, animals were subjected to the same anesthesia procedure, with the core body temperature maintained at 36.8˚C with warm air, along with constant ECG and respiration monitoring (SA Instruments).

The micrometer-sized iron-oxide (MPIO) particles (4.5 mg/kg, from Bangs Laboratories) were administered by a direct intra-venous injection through a penal vein 1 - 2 days prior to the ischemic-reperfusion procedure to label monocytes/macrophages in vivo, to assess the inflammation status of the myocardium by cellular MRI.

In-vivo MR imaging was carried out on a Bruker Biospec AV3 7T/21-cm system equipped with 12-cm, 50 Gauss/cm shielded gradients with a 72-mm bird-cage volume coil.

In-vivo Cellular MRI: -weighted imaging was acquired with an ECG- and respiratory-gated-FLASH sequence, with 10 to 12 short-axis slices used to cover the whole volume of the heart with the following parameters: FOV = 4 cm; slice thickness = 1.1 mm; in-plane resolution = 156 μm; TE = 5.5 ms, and TR = 1 respiratory cycle, about 1 sec.

In-vivo Functional MRI: An INTRAGATE sequence was used to evaluate global systolic cardiac function, anatomical changes, and wall thinning over time. Cine MRI was acquired with the following parameters: TE/TR = 3.2 ms/5.6ms; equivalent temporal resolution for the cine loops was about 16.5 - 19.5 ms per frame, with 20 to 26 cardiac phases per cardiac cycle with in-plane resolution = 156 μm.

Regional wall motion and strain were assessed by tagging MRI followed by strain analysis with HARP (Diagnosoft Inc.). Tagging was achieved by a modified SPAMM sequence, with both ECG and respiration gating; TR = one cardiac cycle (about 180 ms); TE = 3.2 ms; with 20 to 26 frames per cardiac cycle, tag distance = 1 mm.

Myocardial perfusion and infarction were measured by first-pass contrast-enhance dynamic and late-Gadolilium enhancement (LGE) with a single bolus Gd (0.1 mmol/kg of ProHance, Bracco Diagnostics, Inc.) administration. Myocardial first-pass perfusion was evaluated using cine MRI acquired with an Intragate sequence with the following parameters: TE/TR = 3.2 ms/5.6ms; FOV = 4 cm; slice thickness = 2 mm; in-plane resolution = 156 μm; frame rate = 18 sec per 256 × 256 image frame. 80 end-diastolic (ED) time-frames were acquired over the time course of 24 min. A single-bolus Gd was injected at 5 min after the onset of the time series acquisition. Myocardial infarction was evaluated by the LGE from the same time series of the first-pass myocardial perfusion.

At the end point of the study, the animals were euthanized, the hearts were harvested and fixed in 4% paraformaldehyde. Fixed exercised hearts were imaged using a Bruker AVANCE DRX 11.7-T system with a Micro2.5 gradient insert. High-resolution 3D - weighted MRM images were acquired with a TR/TE = 500/5.5ms, and a 36-μm isotropic resolution.

Myocardial strain analysis was performed with HARP (http://www.diagnosoft.com/), based on the harmonic phase method in Fourier domain. Four consecutive mid-level short-axis slices out of the 10-slice stack were subjected to strain analysis. For regional strain analysis, each short-axis slice was divided into 6 regions: R1, anterioseptal; R2, inferiospetal; R3, inferior; R4, inferiolateral; R5, anteriolateral; and R6, anterior walls. The anterior attachment of the right ventricle (RV) to left ventricle (LV) was always chosen as the landmark for segment numbering. Strain values were presented as unit-less percentile of deformation compared to the reference phase (ED).

Flow cytometry (FACSVantage, Becton Dickinson) was used to determine the percentage of MØs, lymphocytes, and NK cells in peripheral blood. Blood samples were taken from untreated IRI rats and Intralipid^®-treated IRI rats on day 1 and day 2 prior to sacrifice. The following antibodies and reagents were purchased from AbD Serotec: ED1, anti-rat CD68:AlexaFluor488 antibody labels MØs; W3/25:FITC/Ox-8:RPE, anti-rat CD4/CD8; Ox-6, anti-rat major histocompatibility complex class II, RT1.B:FITC (H2A- like) molecule; and BUF09. Ox-39, anti-rat CD25:PE was purchased from eBioscience. Data were processed with the FlowJo software (Tree Star, Inc.).

Excised hearts were fixed in 4% paraformaldehyde for 1 - 2 weeks and embedded in paraffin. 5-μm transverse heart tissue sections were taken at the ischemic regions, and H&E-stained slides were obtained. Immunohistochemistry was also conducted to label ED1⁺ macrophages using 1:200 anti-rat ED1⁺ monoclonal purified IgG (AbD SeroTec). The H&E-stained slides and immunohistochemistry slides were scanned on a whole slide scanner (Nanozoomer 2.0-HT, Hamamatsu, Japan) to acquire WSI at 20x magnification. The whole-slide images were then processed by WS-Recognizer (http://ws-recognizer.labsolver.org) [28] . The cell nuclei in the H&E-stained slides were recognized to calculate the cell density across the entire tissue section, whereas the ED1⁺ macrophages were recognized to calculate the spatial distribution of macrophages.

Descriptive statistics were summarized as mean ± standard errors or as median (interquartile range) as appropriate. Examination of normal distribution assumption for continuous data was determined by q-q plots, histograms and Shapiro-Wilk test. Two-sample independent t test or Wilcoxon Mann-Whitney Rank Sum test was used to determine the differences between groups for normally or non-normally distributed continuous data, respectively. Adjustment for a multiple comparison for Wilcoxon Mann-Whitney test was performed using Sidak adjustment method. For Figure 7 data, adjustment for multiple hypotheses testing (six hypotheses) was performed using the Holm’s method. P-values and adjusted p-values (p_a) are presented.

Cine MRI was used to quantify the ejection fraction (EF) from Intralipid^®-treated and control rats in the acute phase following IRI. Figure 1 shows the representative end-diastolic (ED) and end-systolic (ES) images from an un-treated rat (Figure 1(A) & Figure 1(B), respectively) and an Intralipid^®-treated rat (Figure 1(C) & Figure 1(D), respectively). A plot of the temporal changes in the left-ventricular volume (LVV) for 6

subjects (3 untreated animals and 3 Intralipid^®-treated animals) (Figure 1(E)) demonstrates a clear improvement in the LV function following the acute Intralipid^® treatment. Over all, immediate treatment with Intralipid^® following a 45-min LAD artery occlusion and reperfusion is found to significantly enhance the EF performance by over 17%, from 52.4% ± 9.1% to 61.6% ± 8.4% (p = 0.0086).

Myocardial tagging [29] - [31] and strain analysis [32] [33] were used to quantify the regional myocardial function following IRI and the acute response to Intralipid^® treatment. Figure 2 shows the representative images of the tagging-MRI for the same subjects shown in Figure 1. ED and ES tagging images for an untreated rat are shown in Figure 2(A) and Figure 2(B), respectively, and the Intralipid^®-treated subject in Figure 2(D) and Figure 2(E), respectively. Color encoded circumferential strain (Ecc) maps are shown in Figure 2(C) and Figure 2(F) for the untreated and the Intralipid^®-treated rats, respectively.

After transient ischemic insult followed by re-perfusion, although the coronary blood flow is restored, and the hearts with IRI regain good EF (Figure 1), the myocardium is injured due to IRI (Figure 2). The untreated IRI heart (Figure 2(A), Figure 2(B)) shows impaired wall motion of the ischemic injury site. The injured anterial lateral wall (Figure 2(C), and Figure 2(G) gray bars) exhibits greatly reduced Ecc. With the Intralipid^® treatment (Figure 2(D), Figure 2(E) and Figure 2(G) gray bars), in addition to restored EF, the Intralipid^®-treated IRI heart shows preserved ventricular wall motion (Figure 2(D), Figure 2(E)) and improved strain (Figure 2(F), Figure 2(G)). The wall-motion abnormality appears to be centered around the injury site (Figure 2(A), Figure 2(B), Figure 2(G)) and the remote myocardium on the septal wall seems to be intact (Figure 2(A), Figure 2(B), Figure 2(G)). Some wall abnormality is observed in the peri-infarct regions next to the ischemic injury site (Figure 2(A), Figure 2(B), Figure 2(G)).

Strain analysis [32] [33] has been used to quantify the regional wall motion detected by tagging MRI (Figure 2(C), Figure 2(F), Figure 2(G)). Conventional 6-segment model of a short-axis mid-ventricular level is used to quantify the strain. After ischemic-reperfusion insult, both untreated and Intralipid^®-treated hearts show impaired and reduced strain at the ischemic site (Figure 2(G), “affected” segments). Intralipid^®- treated hearts preserve some wall-motion capacity and exhibit better strain than the untreated group. The differences in Ecc between untreated (9.64 ± 0.85) and intralipid^®-treated groups (15.49 ± 0.92) at the ischemic site (2G, “affected” segments), and at the peri-injury myocardial areas (2G, “neighboring” segments) between the untreated (15.23 ± 0.98) and the Intralipid^®-treated (20.94 ± 1.13), were found to be statistically significant (p < 0.01). The regional wall motion of the remote myocardium near septal and inferio-septal wall regions (2G, “remote” segments) show less affected wall motion.

Free-breathing no-gating first-pass contrast enhanced dynamic MRI with a single Gd bolus (Figure 3) and the LGE is used to access the myocardial perfusion and to evaluate the extent of infarction in vivo (Figure 3(G)). The untreated IRI hearts shows impaired myocardial perfusion at the IRI site (Figures 3(A)-(C)) and the areas with LGE (Figure 3(B), Figure 3(C), and Figure 3(G)). With Intralipid^® treatment (Figures 3(D)-(F)), the myocardial perfusion is improved and the infarct size is reduced (Figure 3(G)). There is a statistically significant difference between the underlying distributions of the infarct size for the untreated and the infarct size for the intralipid^®-treated groups (Wilcoxon statistic = 264, n₁ = 16, n₂ = 13, p = 0.0056).

After a transient ischemia, although blood flow is fully restored, inflammation has already set in. MØs infiltrated to the injured myocardium can be detected in-vivo as the hypointensity area on the -weighted MRI with iron-oxide particle labeling [13] [16] [17] . After the in-vivo MPIO labeling, the hypointensity can be detected in the IRI site of myocardium (Figures 4(A)-(C) and Figure 4(G)) with the in-vivo -weighted MRI, indicating the accumulation of MPIO-labeled MØs. With Intralipid^® treatment, the myocardial hypointensity is greatly decreased (Figures 4(D)-(F) and Figure 4(G)), indicating a reduced in-situ myocardial inflammation.

Percentage of the myocardium that contains image hypointensity (Figure 4(G)), obtained by in-vivo MRI, is calculated to quantify the degree MPIO-labeled MØ infiltration in the heart, as a measurement of in-situ inflammation. In-vivo cellular MRI evaluated on the day of ischemic surgery (“day 0” groups) as well as after 1 to 5 days (“<1 week” groups). The difference between the underlying distributions of the area for the untreated and the areas for the intralipid^®-treated groups both for “day 0” (Wilcoxon statistic = 39.50, n₁ = 7, n₂ = 14, p = 0.0112, p_a = 0.0223) and a few days after ischemic insult (Wilcoxon statistic = 45.00, n₁ = 6, n₂ = 19, p = 0.0494, p_a = 0.0964) were found to be statistically significant.

The hearts were harvested at the end-point of the study for high-resolution ex-vivo MRM (Figure 5). At this imaging resolution, single MPIO-labeled MØs can be visualized as individually resolved dark spots [16] . All hearts examined showed low-level sparse distribution of dark spots throughout the heart, which are non-specific background infiltration of MPIO-labeled MØs. This sparse background infiltration of MØs may function as a surveillance for inflammation. In addition to this background of sparse infiltration, the untreated heart (Figure 5(A)) showed concentrated hypointensity in the ischemic site, which is composed of individual dark spots, as well as dark streaks. Individual dark spots are MPIO-labeled MØs [16] infiltrated in the ischemic site, whereas the dark streaks are likely to be hemorrhage in the infarct core. On the other hand, Intralipid^®-treated IRI hearts showed reduced hypointensity. In the example shown in Figure 5(B), both infiltration of MPIO-labeled MØs and hemorrhage in

the infarct core are reduced.

Figure 6 shows the pathology images of the untreated (Figures 6(A)-(D)) and Intralipid^®-treated group (Figures 6(E)-(H)). The untreated group presents a substantial increase of the immune cells in the ischemic myocardium. Figure 6(A) shows the cell-density distribution of the tissue sections on the H&E slide. The distribution reveals a prominent increase of the cell density at the ischemic injury region in the sham group. Figure 6(B) shows the distribution of ED1⁺ MØs of the same animal. The distribution also reveals a prominent increase of the MØ infiltration in the ischemic tissue. Figure 6(C) illustrates a view in the H&E slide, showing the infiltration of immune cells in the myocardium. The immunohistochemistry shown in Figure 6(D) confirms that the infiltrating cells are ED1⁺ MØs. The Intralipid^® group also presents an increase of the immune cells in the ischemic myocardium, but the cell density is substantially less than the sham group. Figure 6(E) shows the cell-density distribution of the tissue sections on the H&E slide, whereas Figure 6(F) shows the distribution of ED1⁺ MØs of the same animal. The density of the infiltrating cells is substantially less than the sham group. Figure 6(G) illustrates a view in the H&E slide, whereas Figure 6(H) illustrates a view in the ED1⁺ stain slides. The density of the immune cells is less than the sham group, suggesting that Intralipid^® reduces the amount of MØs infiltrating into the ischemic myocardium.

One limitation of the cellular MRI is that both hemorrhage and MPIO-labeled MØs show hypointensity in -weighted MRI. We cannot distinguish the blood clots from the infiltrated labeled MØs in the heart in vivo. Ex-vivo high-resolution -weighted MRM (Figure 5) revealed that the hypointensed area detected in the in-vivo - weighted MRI are composed of a “core” with continuous dark area, probably the hemorrhage of the infarct core, and the individual dark “dots”, are probably MPIO-la- beled MØs. One single dot is likely to be one labeled MØ [16] . Pathological examinations (Figure 6) have confirmed that these are ED1⁺ MØs and blood. Although in-vivo -weighted MRI cannot distinguish labeled MØs and hemorrhage, both are reduced with Intralipid^® treatment after IRI.

On day 1 after IRI, we have found that Intralipid^® can decrease the circulating ED1⁺ MØs dramatically in the peripheral blood (Figure 7(A) and Figure 7(B)). On day 2, although the in-situ MØ infiltration is greatly reduced by the Intralipid^® treatment (Figure 4 and Figure 6), the composition of MØ and other types of immune cells, including helper T-lymphocytes (CD4), cytotoxic T-cells (CD8), TL-2α receptor containing cells (CD25), Class II antigen-presenting cells (RT1B), NK cells (CD 161), in the peripheral blood show no different with or without Intralipid^® treatment (Figure 7(C) and Figure 7(D)). No statistically significant differences in CD4 (p = 0.4162, p_a = 1.000), CD8 (p = 0.3024, p_a = 1.000), CD25 (p = 0.3024, p_a = 1.000), RT1B (p = 0.1234, p_a = 1.000), CD161 (p = 0.5557, p_a = 1.000), and ED1 (p = 0.8119, p_a = 1.000) were found between the two groups.