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. 2016 Aug 10;282(1):202–211. doi: 10.1148/radiol.2016152222

Spinal Cord Inflammation: Molecular Imaging after Thoracic Aortic Ischemia Reperfusion Injury

Hassan Albadawi 1,✉, John W Chen 1, Rahmi Oklu 1, Yue Wu 1, Gregory Wojtkiewicz 1, Benjamin Pulli 1, John D Milner 1, Richard P Cambria 1, Michael T Watkins 1
PMCID: PMC5207124  PMID: 27509542

The proinflammatory enzyme myeloperoxidase, which is secreted by activated myeloid cells, could represent a key imaging biomarker for tracking early inflammatory changes noninvasively in the spinal cords of patients after interventions in the thoracic aorta.

Abstract

Purpose

To evaluate whether noninvasive molecular imaging technologies targeting myeloperoxidase (MPO) can reveal early inflammation associated with spinal cord injury after thoracic aortic ischemia-reperfusion (TAR) in mice.

Materials and Methods

The study was approved by the institutional animal care and use committee. C57BL6 mice that were 8–10 weeks old underwent TAR (n = 55) or sham (n = 26) surgery. Magnetic resonance (MR) imaging (n = 6) or single photon emission computed tomography (SPECT)/computed tomography (CT) (n = 15) studies targeting MPO activity were performed after intravenous injection of MPO sensors (bis-5-hydroxytryptamide-tetraazacyclododecane [HT]-diethyneletriaminepentaacetic acid [DTPA]-gadolinium or indium 111-bis-5-HT-DTPA, respectively). Immunohistochemistry and flow cytometry were used to identify myeloid cells and neuronal loss. Proinflammatory cytokines, keratinocyte chemoattractant (KC), and interleukin 6 (IL-6) were measured with enzyme-linked immunosorbent assay. Statistical analyses were performed by using nonparametric tests and the Pearson correlation coefficient. P < .05 was considered to indicate a significant difference.

Results

Myeloid cells infiltrated into the injured cord at 6 and 24 hours after TAR. MR imaging confirmed the presence of ischemic lesions associated with mild MPO-mediated enhancement in the thoracolumbar spine at 24 hours compared with the sham procedure. SPECT/CT imaging of MPO activity showed marked MPO-sensor retention at 6 hours (P = .003) that continued to increase at 24 hours after TAR (P = .0001). The number of motor neurons decreased substantially at 24 hours after TAR (P < .01), which correlated inversely with in vivo inflammatory changes detected at molecular imaging (r = 0.64, P = .0099). MPO was primarily secreted by neutrophils, followed by lymphocyte antigen 6 complexhigh monocytes and/or macrophages. There were corresponding increased levels of proinflammatory cytokines KC (P = .0001) and IL-6 (P = .0001) that mirrored changes in MPO activity.

Conclusion

MPO is a suitable imaging biomarker for identifying and tracking inflammatory damage in the spinal cord after TAR in a mouse model.

© RSNA, 2016

Online supplemental material is available for this article.

Introduction

Despite a variety of surgical, technical, and pharmacologic protective adjuncts applied over time, spinal cord ischemic injury after otherwise successful open or endovascular thoracic aortic repair remains an unresolved clinical problem (1–3). Acute spinal cord injury can also be a comorbidity associated with trauma (4), spinal cord infarction (5), and epidural anesthesia (6). Temporal, pathologic, or biochemical factors leading to the development of spinal cord injury in humans and in animal models after thoracic aortic ischemia-reperfusion (TAR) are being actively investigated. Several clinically relevant models have been developed to study spinal cord injury and the provoked inflammatory response (7–10) using tissue harvested from animals after the onset of neurologic deficits (11–13). However, this tissue is not attainable in the clinical setting. Therefore, a noninvasive tool that can identify early those at risk for developing neurologic deficits and allow preventative, timely interventions is needed.

Myeloperoxidase (MPO) is one of the most abundant enzymes secreted by activated myeloid cells (14). MPO is a key downstream effector molecule in the inflammatory cascade that has been implicated in exacerbating myelin and neuronal injury in acute or degenerative central nervous system conditions (15,16). MPO generates highly reactive molecular moieties (17,18) that can cause damage and further exacerbate the inflammatory response (19). MPO also degrades protective endothelial-derived nitric oxide to promote endothelial dysfunction (20) and upregulates inducible nitric oxide synthase that can exacerbate inflammation (21). Thus, we hypothesize that MPO, as a potent proinflammatory enzyme, is an important biomarker for spinal cord ischemic injury. MPO activity can be tracked noninvasively by using activatable molecular imaging agents (22,23) that act as substrates for MPO. MPO can oxidize these substrates in the inflamed tissue, causing the substrates to form oligomers and bind to local proteins, thereby increasing the retention of the sensors in areas of elevated MPO activity (22,24), with imaging signal directly proportional to in vivo MPO activity (24). The specificity of these sensors for reporting MPO activity has been demonstrated in genetically deficient MPO (MPO−/−) mice in models of myocardial infarction (25), stroke (26), and heart transplantation (27). Therefore, the purpose of this study was to evaluate whether noninvasive molecular imaging technologies targeting MPO can reveal early inflammation associated with spinal cord injury after TAR in mice.

Materials and Methods

Please refer to Appendix E1 (online) for detailed description of the study methods.

Inducing Spinal Cord Ischemic Injury after TAR

The protocol for animal experiments was approved by the institutional animal care and use committee at the Massachusetts General Hospital. TAR surgery was performed to induce spinal cord ischemic injury, manifested by paraplegia of the hind limbs, as previously described (28). This study was begun in November 2011, and was completed in March 2015. Briefly, 10–12-week-old male C57BL6 mice (n = 81) and homozygote MPO genetically deficient (MPO−/−) mice (n = 5) underwent mediastinotomy with isoflurane anesthesia, followed by thoracic aortic and left subclavian artery cross clamping for 8.5 minutes (n = 55) to induce spinal cord ischemic injury. The clamps were removed to start reperfusion, and the animals were allowed to recover from anesthesia and to survive for 6 or 24 hours. Paraplegia, which persisted until 6 or 24 hours, was confirmed in the TAR group immediately after surgery. Sham-procedure animals (n = 26) underwent mediastinotomy without aortic cross clamping of the vessels and did not experience neurologic deficit at any time. H.A. and M.T.W. had more than 13 years of experience in performing this surgical technique. Separate subgroups of C57BL6 or MPO−/− mice that had undergone either sham or TAR surgery then underwent a specific imaging protocol for magnetic resonance (MR) imaging or single photon emission computed tomography (SPECT)/computed tomography (CT), as indicated in the study flow diagram (Fig 1). These groups of mice had their tissues harvested and processed according to each type of analysis, as indicated (Fig 1).

Figure 1:

Figure 1:

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Study flow diagram shows schematic summary of the C57BL6 and MPO genetically deficient (MPO−/−) mice enrolled in the different experimental groups. A subset of mice in each group underwent either sham or TAR surgery and was allowed to survive for a predetermined time point of 6 or 24 hours after surgery. Separate groups of mice underwent a specific imaging protocol for MR imaging or SPECT/CT as indicated. Spinal cord tissues were harvested from subset groups of mice and were processed according to each type of analysis, as indicated. DWI = diffusion-weighted (DW ) imaging; ELISA = enzyme-linked immunosorbent assay; IL-6 = interleukin 6; KC = keratinocyte chemoattractant; Ly6B = lymphocyte antigen 6 complex (Ly6), locus B; Mac3 = macrophage antigen-3; T1w = T1-weighted imaging.

Spinal Cord MR Imaging

Mice subjected to sham surgery (n = 3) and mice 24 hours after TAR surgery (n = 3) received an intravenous injection of 0.3 mmol bis-5-hydroxytryptamide-tetraazacyclododecane (HT)-diethyneletriaminepentaacetic acid (DTPA)-gadolinium (MPO-Gd) per kilogram of body weight 24 hours after surgery. MR imaging was performed by using a 4.7-T MR imaging unit (Bruker Pharmascan; Bruker Daltronics; Billerica; Mass). We assessed only with MR imaging at 24 hours because our prior experience with MPO MR imaging in stroke showed that prior to 24 hours after ischemia, MR imaging did not have enough sensitivity to detect the small amount of MPO present (26). J.W.C. and G.W. had more than 10 years of experience with this imaging technique in animals.

Spinal Cord SPECT/CT Imaging

Separate groups of sham mice (n = 5) or TAR mice (C57BL6 mice: 6 hours after, n = 5; 24 hours after, n = 5; MPO−/− mice: 24 hours after, n = 5) received approximately 700 μCi (25.9 MBq of indium 111 (111In)-bis-5-HT-DTPA through tail vein injection. The sensor was allowed to wash out for 90 minutes, and the mice were re-anesthetized and the retention of the MPO sensor was visualized by using a X-SPECT imaging system (GammaMedica, Northridge, Calif). J.W.C. and G.W. had more than 10 years of experience with this imaging technique in rodents. After imaging, spinal cord tissues were harvested for NeuN immunohistochemistry to enable us to identify and count motor neurons in the anterior horn, as indicated in the immunohistochemistry detection method.

Ex Vivo MPO Activity Assay

So that we could correlate MPO activity with MPO imaging findings, spinal cord tissues were harvested from separate groups of mice to measure MPO activity at 24 hours after the sham procedure (n = 5), or at 6 hours (n = 5) and 24 hours (n = 5) after TAR. Spinal cord MPO was captured with a monoclonal anti-MPO antibody (Hycult Biotech, Plymouth Meeting, Pa), followed by detection of MPO activity with 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP; AAT Bioquest, Sunnyvale, Calif) (29).

Assessing Local Protein Levels of KC and IL-6

Separate groups of mice were subjected to sham surgery (n = 5) or TAR surgeries and then allowed to recover for 6 hours (n = 10) or 24 hours (n = 6). Quantitative analysis for KC and IL-6 proteins were performed in spinal cord tissue extracts by using an enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minn).

Spinal Cord Analysis with Flow Cytometry

Spinal cord tissues were harvested from separate groups of sham-procedure mice and mice at 6 and 24 hours after TAR and were processed for flow cytometry (30). The cell numbers of different cell populations were calculated as the total number of cells multiplied by the percentage within the respective cell population gate. MPO-positive cell populations were normalized to the level in resting microglia in sham-procedure mice, which did not express MPO (n = 3 in each group). J.W.C. and B.P. had more than 10 years of experience with flow cytometric analysis.

Immunohistochemical Detection of Myeloid Cells and Motor Neurons in the Spinal Cord Tissue

So that we could identify myeloid cells and motor neurons in the anterior horns of the thoracolumbar segment (T11–L5), we incubated slices obtained in separate groups of mice with rabbit anti–macrophage antigen-3 immunoglobulin G (IgG), which identifies mononuclear phagocytes, or with anti-Ly6, locus B IgG, which identifies leukocytes. For identifying motor neurons, spinal cord tissue slices harvested from the mice groups subjected to the sham procedure or 6 or 24 hours after TAR followed by SPECT/CT imaging were incubated with the antineuronal nuclear antigen NeuN, a specific marker of motor neurons, and IgG (Cell Signaling, Danvers, Mass), followed by incubation with goat antirabbit horseradish peroxidase conjugated IgG. The specific protein signal was visualized with 3,3ʹ-Diaminobenzidine substrate (Vector Laboratories, Burlingame, Calif). The number of NeuN-positive cells per field in the anterior horns of the thoracolumbar segment in each mouse was counted and averaged by a blinded observer (n = 5 in each group). J.W.C., H.A., and M.T.W. had more than 10 years of experience in performing this technique in neural tissue.

Statistical Analysis

Comparisons between different groups were performed by using the nonparametric Kruskal-Wallis test, followed by the Dunnett multiple-comparison post-hoc test. The relationship between variables was studied by using the Pearson correlation coefficient with a two-tailed test (GraphPad Prism software). Data are means ± standard errors of the mean. P < .05 was considered to indicate a significant difference.

Results

Myeloid Inflammatory Cells Infiltrate the Injured Spinal Cord after TAR

We first investigated the innate immune response after TAR. Using immunohistochemistry (Fig 2), we found an infiltration of neutrophils (with Ly6B) and monocytes, macrophages, and/or microglia (with macrophage antigen-3) starting at 6 hours after TAR that increased at 24 hours after TAR. We further evaluated different leukocyte subpopulations after TAR with flow cytometry. We found that neutrophils (P = .012) and Ly6Chigh monocytes and/or macrophages (P = .0028) infiltrated the spinal cord most abundantly at 24 hours after TAR compared with sham (Fig 3). In contrast, lymphocyte and microglia populations did not change during the first 6 or 24 hours after TAR (Fig 3) and were similar to the levels in sham-procedure mice. Flow cytometry data details can be found in Table E1 (online).

Figure 2:

Figure 2:

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Myeloid cells in spinal cord tissue after TAR. Immunohistochemistry staining for Ly6, locus B (Ly6B), or macrophage antigen-3 (Mac3) was performed on paraffin cross sections from the spinal cords of mice that underwent the sham procedure (top) and of mice 6 hours (middle) or 24 hours (bottom) after TAR. Representative photomicrographs from five mice per group are shown. Arrows = positive cells. Ly6B staining (left row) revealed no positive cells in sham-procedure mice. However, there were few detectable Ly6B-positive cells at 6 hours after TAR and a marked increase in Ly6B-positive cells at 24 hours. Similarly, detection of macrophage antigen-3 revealed a slight increase in positive cells at 6 hours that markedly increased at 24 hours after TAR. (Original magnification, ×100; scale bar = 100 μm.)

Figure 3:

Figure 3:

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Identification of myeloid cell infiltration with flow cytometry. Flow cytometric analysis was performed on spinal cord tissues from mice that underwent the sham procedure and from mice 6 and 24 hours after TAR to identify leukocyte subpopulations. Data as summarized in the graphs showed that neutrophils (N, red) infiltrated the spinal cord and were most abundant at 24 hours after TAR. Microglia/monocytes (MM, blue) were subdivided into Ly6Chigh monocytes (red), which also accumulated in the spinal cord at 24 hours, and Ly6Clow microglia (orange), which were unchanged. Also unchanged were lymphocytes (L, green).

MR Imaging of the Spinal Cord after TAR

To assess spinal cord injury after TAR noninvasively, we imaged the mice after TAR with MR imaging. DW imaging findings confirmed the presence of ischemic injury in TAR mice but not in sham-procedure mice (Fig 4, top row). Because MPO is a major product of myeloid cells, we performed T1-weighted imaging in TAR and sham-procedure mice after intravenous injection of MPO-Gd at 15 minutes (Fig 4, middle row) and 60 minutes (Fig 3, bottom row). At 24 hours after TAR, there was mildly elevated delayed MPO-Gd enhancement in the thoracolumbar spinal cord on the 60-minute images, consistent with increased MPO-mediated activation and retention of the MPO-Gd sensor. In contrast, there was no detectable signal in the sham-procedure mice at 60 minutes after MPO-Gd injection. Gray-scale DW and MR images are included in Figure E1 (online).

Figure 4:

Figure 4:

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MR imaging of the spinal cord after TAR. Representative DW MR image in one sham-procedure mouse (left column) and two mice subjected to 24 hours TAR (middle and right columns). DW imaging revealed no spinal cord lesion in the sham-procedure mouse. However, a clearly visible lesion (white arrows) was detected in the spinal cord 24 hours after TAR in both mice. T1-weighted MR images obtained 15 minutes after MPO-Gd administration (middle row) in the corresponding planes of the DW images showed no visible MPO-Gd enhancement in any of the groups. However, at 60 minutes after agent injection (bottom row), while the sham-procedure mouse had no MPO-Gd enhancement, mice 24 hours after TAR (yellow arrows), showed mild but clearly visible MPO-Gd enhancement in the corresponding planes of the same spinal cord segment, consistent with increased MPO in vivo. Images are representative of three mice in each group.

SPECT/CT Imaging of Spinal Cord after TAR

Owing to the relatively subtle findings at MPO-Gd MR imaging, we explored a more sensitive method to detect MPO activity in vivo. Nuclear imaging methods such as SPECT or positron emission tomography (PET) are more than 100 000 times as sensitive as MR imaging. The MPO sensor can be also used for either SPECT or PET depending on the radioisotope used (31). For this study, we chose to label the sensor with 111In because it is a readily available radioisotope. Corresponding to the increased sensitivity, there was a marked increase in MPO radiosensor uptake in the thoracolumbar cord at 6 hours (P = .003) and 24 hours (P = .0001) after TAR, increasing over time (Fig 5, Table E2 [online]) (sham: 3.2 cpm ± 5.3 vs 6 hours after TAR: 262.5 cpm ± 44.6 vs 24 hours after TAR: 422.2 cpm ± 160.5; P < .0087; n = 5 in each group). To further validate the specificity of the imaging probe, we also performed imaging in MPO−/− mice, and, as expected, found no significant sensor activation and uptake in these mice.

Figure 5:

Figure 5:

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Detection of MPO activity with SPECT/CT and ex vivo MPO assay after TAR. SPECT/CT sagittal images (SPECT images overlaid onto volume CT reconstructions) acquired, A, in mice 6 hours after TAR, B, in mice 24 hours after TAR, C, in mice treated with the sham procedure, and, D, in MPO genetically deficient (MPO−/−) mice show a large amount of 111In-bis-5-HT-DTPA retention 90 minutes after injection in the thoracolumbar segment at 6 and 24 hours after TAR. The sham-procedure and MPO−/− mice had no detectable radiotracer uptake. E, Counts normalized to background after the sham procedure and 6 and 24 hours after TAR. There was virtually no tracer retention in the sham-procedure mice. However, there was marked MPO sensor retention at 6 hours (*P = .0035) and at 24 hours after TAR (*P = .0001). F, Ex vivo MPO activity in spinal cord tissue after TAR. There was marked increase in MPO activity at 24 hours compared with sham (*P = .0485). Results are representative of five independent experiments in each group.

MPO Activity in Spinal Cord Tissue after TAR

Spinal cord tissue was harvested for ex vivo MPO activity assay to corroborate imaging findings. There was a trend toward but not significant MPO activity increase at 6 hours compared with sham. However, at 24 hours after TAR, there was an increase in MPO activity (P = .046; sham: 0.025 U/mg protein ± 0.014 vs 6 hours after TAR: 0.032 U/mg protein ± 0.016 vs 24 hours after TAR: 0.089 U/mg protein ± 0.022; n = 5 in each group; Fig 5, F; Table E2 [online]). This paralleled the enhanced MPO activity at MR imaging and SPECT/CT after TAR.

Cellular Contribution to MPO Activity in Spinal Cord Tissue after TAR

We next investigated the individual cellular contributions to MPO activity in the injured spinal cord with flow cytometry (Fig 6). We found that at 24 hours after TAR, MPO was predominately expressed in neutrophils (approximately 65%) (1391 cells ± 85.46), and Ly6Chigh monocytes and/or macrophages (approximately 35%) (744.3 cells ± 247.1). All other cell types such as lymphocytes and microglia contributed negligibly to the MPO signal (n = 3 in each group, Fig 5). Results of detailed flow cytometric analysis of the MPO-expressing inflammatory cells are given in Table E3 (online).

Figure 6:

Figure 6:

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Graph shows cellular sources of MPO in spinal cord tissue after TAR. Flow cytometric evaluation of spinal cord leukocyte subpopulations at 24 hours after TAR showed that MPO is predominately expressed in neutrophils (approximately 65%) and Ly6Chigh monocytes and/or macrophages (approximately 35%). All other cell types, such as lymphocytes and microglia, did not contribute considerably to the MPO signal (n = 3 in each group).

Local Protein Levels of KC and IL-6

KC protein started to increase at 6 hours (n = 10) compared with sham (n = 5), but it did not reach statistical significance until 24 hours (n = 6) after TAR (sham: 1.1 pg/mg total protein ± 0.2 vs 6 hours after TAR: 28.6 pg/mg total protein ± 4.7 vs 24 hours after TAR: 88.9 pg/mg total protein ± 11.4; P = .0001; Fig 7, A; Table E2 [online]). IL-6 levels in spinal cord tissue increased at 6 hours after TAR compared with sham, but this did not reach statistical significance. However, at 24 hours, the IL-6 level was markedly higher than that in the sham group and the 6-hour TAR group (sham: 1.20 pg/mg protein ± 0.52 vs 6 hours after TAR: 15.40 pg/mg protein ± 3.15 vs 24 hours after TAR: 111.39 pg/mg protein ± 16.80; P = .0001 for 24 hours compared with sham; Fig 7, B; Table E2 [online]). These data indicate that TAR triggers local proinflammatory cytokines and chemokine responses that corresponded with in vivo MPO activity observed with molecular imaging.

Figure 7:

Figure 7:

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Graphs show, A, KC and, B, IL-6 in the spinal cord tissue after TAR. The levels of KC protein started to increase at 6 hours (n = 10) after TAR and became higher than those from sham-procedure mice (n = 5) by 24 hours (n = 6) (***P = .0001). KC levels were higher at 24 hours (n = 6) than at 6 hours (n = 10) after TAR (*P = .0376). IL-6 levels were marginally increased at 6 hours and became significantly increased by 24 hours compared with sham (***P = .0001). IL-6 was markedly higher at 24 hours after TAR than after 6 hours (*P = .037).

Motor Neuron Count in Spinal Cord Tissue after TAR

The average number of motor neurons found in the anterior horns of the thoracolumbar segment decreased at 6 hours after TAR compared with sham, but this was not significant. However, by 24 hours, motor neuron count decreased substantially compared with sham (sham: 10.9 average motor neurons per field ± 0.7 vs 6 hours after TAR: 5.8 average motor neurons per field ± 0.4 vs 24 hours after TAR: 3.8 average motor neurons per field ± 0.6; P = .0043; n = 5 in each group; Fig 8, Table E2 [online]). We found an inverse correlation between the number of motor neurons and the in vivo MPO signal with SPECT at 24 hours after TAR (r = −0.64; P = .0099; Fig 8, E).

Figure 8:

Figure 8:

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Motor neuron counts in spinal cord tissue after TAR. Spinal cord slices obtained from the thoracolumbar cord segments of, A, mice treated with the sham procedure, B, mice 6 hours after TAR, and, C, mice 24 hours after TAR were stained with a specific neuronal marker, NeuN (five animals in each group). (Original magnification, ×100; scale bar = 100 μm.) D, Graph shows the average motor neuron counts. There was a decrease in motor neurons counted at 6 hours after TAR, but this was not significant. Motor neuron counts markedly declined at 24 hours after TAR compared with sham (**P = .0043), mirroring MPO activity imaging results. E, Correlation analysis for motor neuron counts and MPO-sensor retention count (CPM) with SPECT/CT in the spinal cord tissue of the mouse cohort showed that in vivo MPO activity was inversely proportional to the motor neuron loss in the anterior horns of the thoracolumbar segment (Pearson r = −0.64, P = .0099).

Discussion

In our study, we showed that there was an increase in the number of myeloid cells in the injured spinal cord after TAR. MPO, a major product of myeloid cells, was elevated in vivo after ischemic cord injury, increasing from 6 hours to 24 hours after TAR. Both MR imaging and SPECT/CT molecular imaging revealed focal elevated MPO activity in vivo in the thoracolumbar cord after TAR. The elevated MPO activity was confirmed with ex vivo biochemical assay. Corresponding to the imaging findings, analysis of tissue specimens showed similarly elevated markers of inflammatory activity, including KC and IL-6. Neutrophils and Ly6Chigh monocytes and/or macrophages were the main sources of MPO. Similar to the histopathologic markers, these proinflammatory cells increased over time, corresponding to the imaging results and demonstrating that MPO imaging reflected the underlying immunologic and inflammatory changes. Importantly, the degree of motor neuron loss correlated inversely with in vivo MPO activity, demonstrating that molecular imaging of MPO activity is a suitable biomarker for post-TAR inflammation and tissue injury.

In our study, the inflammatory response paralleled a marginal decrease in viable motor neurons in the lumbar and thoracic spinal cord segments as early as 6 hours after TAR, with significant decline by 24 hours. Earlier studies have implicated inflammation in enhancing the ischemic lesion expansion that causes neuronal cell death in models of spinal cord ischemia reperfusion injury (32). For example, a study by Izumi et al (33) found that reduced postoperative neurologic deficit was associated with decrease in MPO activity and increased motor neuron viability after spinal cord ischemia-reperfusion in rabbits. The temporal pattern of viable motor neuron decrease in our study is also in agreement with the immediate and delayed motor neuron death in a porcine model of thoracoabdominal aortic occlusion (34).

The imaging sensors bis-5-HT-DTPA-Gd and 111In-bis-5-HT-DTPA were first reported in 2005 and 2006, respectively (22,35). These agents are injected intravenously and at sites of inflammation; secreted MPO oxidizes and radicalizes the 5-hydroxytryptamide moiety on these imaging sensors (22,24). The oxidized sensors can combine with other radicalized molecules (another sensor or phenolic/indolic moieties in proteins) to form a polymeric molecule and bind to proteins. The combination of polymerization and protein binding cause local retention of the activated agents at the site of inflammation, resulting in an increase in T1-weighted signal enhancement at MR imaging or an increase in radiotracer signal at SPECT.

Conventional agents without molecular specificity such as DTPA-Gd can also indirectly reveal inflammation due to leakage across an impaired blood-brain or blood-spinal barrier. However, this is not specific, and these agents cannot help ascertain whether the inflammation is ongoing or happened some time ago (36). On the other hand, MPO imaging has molecular specificity for a key enzyme expressed during active inflammation, as well as increased sensitivity due to signal amplification. Thus, MPO imaging can reveal ongoing inflammation at an earlier time point than conventional agents such as DTPA-Gd (30). Indeed, we found that both imaging sensors, bis-5-HT-DTPA-Gd (MPO-Gd, for MR imaging) and 111In-bis-5-HT-DTPA (for SPECT/CT), revealed increased MPO activity after TAR. The findings at MR imaging were more subtle and detectable only at 24 hours after TAR. However, DW images confirmed the presence of ischemic injury in TAR mice but not in sham-procedure mice. DW images revealed ischemic changes, which represent nonviable tissue, while MPO activity imaging revealed inflammation, which results from ischemia. Such inflammation can further cause injury and propagate tissue damage (37).

Key advantages of MR imaging are detailed structural imaging and more advanced sequences such as DW imaging. However, while MR imaging has the advantage of higher spatial resolution, its spatial coverage is limited. On the other hand, while nuclear imaging has lower spatial resolution, it can survey the entire spinal cord in one imaging session and offers orders of magnitude higher sensitivity. Indeed, we found SPECT/CT to be able to show early inflammatory changes at 6 hours after TAR. Both modalities are widely used clinically, and this sensor can be radiolabeled with copper 64 for PET imaging. We did not find a statistically significant increase in MPO activity with biochemical assay or of MPO-expressing cells with flow cytometry at 6 hours. With imaging, focal abnormalities can be appreciated that may become undetectable when the entire spinal cord, including both normal and abnormal areas, is homogenized for further analysis. This underscores the importance of using in vivo imaging to diagnose and track subtle focal changes.

Our study had several limitations. While our study demonstrated the sensitivity of the 111In-bis-5-HT-DTPA sensor in detecting early inflammation associated with immediate spinal cord injury after TAR, the clinical scenario is more often a delayed onset. Thus, future work will further validate this technology in animal models of delayed spinal cord injury. Our results showed that MPO activity paralleled the decrease in viable motor neurons in the spinal cord thoracolumbar segment after TAR. However, given the deleterious roles of MPO and its products in the pathogenesis of different diseases (37,38), it is possible MPO not only is a bystander reporter but also directly participates in causing motor neuron injury. While our data included MPO−/− mice to validate the specificity of the imaging, we did not assess whether motor neuron injury was improved in this model. Thus, to elucidate the role MPO activity plays in motor neuron death, we will focus future experiments on treating the animals with an MPO inhibitor and further investigating neuronal injury in MPO−/− mice.

In conclusion, the proinflammatory enzyme MPO secreted by activated myeloid cells could represent a key imaging biomarker to noninvasively track early inflammatory changes associated with TAR in the spinal cord. SPECT/CT imaging with an MPO sensor can be useful in identifying patients at risk for developing neurologic deficit after thoracic aortic reconstruction surgery and allowing preventive interventions.

Practical application: Immediate or delayed paraplegia or paraparesis remain unpredictable complications after otherwise successful surgical interventions in the thoracic aorta. There are several risk factors for developing ischemic spinal cord injury, including extensive aortic repair; prior aortic repair; spinal cord malperfusion; clinical presentations with systemic hypotension, acute anemia, and prolonged aortic clamping; and the presence of vascular steal. It is now possible to detect the presence and magnitude of spinal cord inflammation noninvasively during the early period after aortic intervention by using molecular imaging techniques designed to track MPO activity. When risk factors exist, the patient will be a candidate for molecular imaging to interrogate the spinal cord for evidence of increased MPO activity. Early detection of spinal cord inflammation with MPO imaging will help clinicians implement aggressive intervention before treatable injury evolves into irreversible neurologic deficit.

Advances in Knowledge

  • ■ Nuclear imaging specific to the activity of a major product of inflammation, myeloperoxidase (MPO), can reveal and help pinpoint inflammation in the spinal cord tissue within 6 hours after thoracic aortic ischemia-reperfusion in mice (P = .003).

  • ■ We confirmed the imaging results with tissue activity assays and showed that changes in in vivo MPO-mediated imaging results mirrored changes in proinflammatory cytokines and were inversely correlated with motor neuron loss in the spinal cord after ischemia (Pearson r = 0.64, P = .0099).

Implication for Patient Care

  • ■ When translated, molecular imaging targeting MPO can potentially be useful in identifying patients at risk for developing neurologic deficits after otherwise successful thoracic aortic interventions and allow preventive therapies to be used.

APPENDIX

Appendix E1 and Tables E1–E3 (PDF)
ry152222suppa1.pdf (274KB, pdf)

SUPPLEMENTAL FIGURE

Figure E1:
ry152222suppf1.jpg (177.7KB, jpg)

Received October 8, 2015; revision requested November 25; revision received March 25, 2016; revision accepted April 25; final version accepted May 24.

Supported by the National Institutes of Health (R01-NS070835, R01-NS072167) and by Massachusetts General Hospital (Division of Vascular and Endovascular Surgery, Henry and Nod Meyer Sundry Fund).

Disclosures of Conflicts of Interest: H.A. disclosed no relevant relationships. J.W.C. Activities related to the present article: none to disclose. Activities not related to the present article: institution receives grants from Pfizer; is on the speakers bureau of Novartis; receives royalties from Elsevier. Other relationships: disclosed no relevant relationships.. R.O. disclosed no relevant relationships. Y.W. disclosed no relevant relationships. G.W. disclosed no relevant relationships. B.P. disclosed no relevant relationships. J.D.M. disclosed no relevant relationships. R.P.C. disclosed no relevant relationships. M.T.W. disclosed no relevant relationships.

Abbreviations:

DTPA
diethyneletriaminepentaacetic acid
DW
diffusion weighted
HT
hydroxytryptamide-tetraazacyclododecane
IL-6
interleukin 6
KC
keratinocyte chemoattractant
Ly6
lymphocyte antigen 6 complex
MPO
myeloperoxidase
TAR
thoracic aortic ischemia-reperfusion

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Appendix E1 and Tables E1–E3 (PDF)
ry152222suppa1.pdf (274KB, pdf)
Figure E1:
ry152222suppf1.jpg (177.7KB, jpg)

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