pet-imaging-neuroinflammation-using-11cdpa-713-mouse-model-ischemic

PET Imaging of Neuroinflammation Using [<sup>11</sup>C]DPA-713 in a Mouse Model of Ischemic Stroke

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide ...

imaging-cd19-b-cells-an-experimental-autoimmune-encephalomyelitis

Imaging CD19<sup>+</sup> B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

Stanford University

The cystine/glutamate antiporter (xc-) has been implicated in several neurological disorders and, specifically, in multiple sclerosis (MS) as a mediator of glutamate excitotoxicity and proinflammatory immune responses. We aimed to evaluate an xc-specific positron emission tomography (PET) radiotracer, (4S)-4-(3-[F]fluoropropyl)-L-glutamate ([F]FSPG), for its ability to allow non-invasive monitoring of xc- activity in a mouse model of MS.

[F]FSPG-PET reveals increased cystine/glutamate antiporter (xc-) activity in a mouse model of multiple sclerosis.

Neuroinflammation plays a key role in neuronal injury after ischemic stroke. PET imaging of translocator protein 18 kDa (TSPO) permits longitudinal, noninvasive visualization of neuroinflammation in both preclinical and clinical settings. Many TSPO tracers have been developed, however, it is unclear which tracer is the most sensitive and accurate for monitoring the in vivo spatiotemporal dynamics of neuroinflammation across applications. Hence, there is a need for head-to-head comparisons of promising TSPO PET tracers across different disease states. Accordingly, the aim of this study was to directly compare 2 promising second-generation TSPO tracers, C-DPA-713 and F-GE-180, for the first time at acute and chronic time points after ischemic stroke.  After distal middle cerebral artery occlusion or sham surgery, mice underwent consecutive PET/CT imaging with C-DPA-713 and F-GE-180 at 2, 6, and 28 d after stroke. T2-weighted MR images were acquired to enable delineation of ipsilateral (infarct) and contralateral brain regions of interest (ROIs). PET/CT images were analyzed by calculating percentage injected dose per gram in MR-guided ROIs. SUV ratios were determined using the contralateral thalamus (SUV) as a pseudoreference region. Ex vivo autoradiography and immunohistochemistry were performed to verify in vivo findings.  Significantly increased tracer uptake was observed in the ipsilateral compared with contralateral ROI (SUV, 50-60 min summed data) at acute and chronic time points using C-DPA-713 and F-GE-180. Ex vivo autoradiography confirmed in vivo findings demonstrating increased TSPO tracer uptake in infarcted versus contralateral brain tissue. Importantly, a significant correlation was identified between microglial/macrophage activation (cluster of differentiation 68 immunostaining) and C-DPA-713- PET signal, which was not evident with F-GE-180. No significant correlations were observed between TSPO PET and activated astrocytes (glial fibrillary acidic protein immunostaining). C-DPA-713 and F-GE-180 PET enable detection of neuroinflammation at acute and chronic time points after cerebral ischemia in mice. C-DPA-713 PET reflects the extent of microglial activation in infarcted distal middle cerebral artery occlusion mouse brain tissue more accurately than F-GE-180 and appears to be slightly more sensitive. These results highlight the potential of C-DPA-713 for tracking microglial activation in vivo after stroke and warrant further investigation in both preclinical and clinical settings.

C-DPA-713 Versus F-GE-180: A Preclinical Comparison of Translocator Protein 18 kDa PET Tracers to Visualize Acute and Chronic Neuroinflammation in a Mouse Model of Ischemic Stroke.

Complex regional pain syndrome (CRPS) is a severely disabling disease characterized by pain, temperature changes, motor dysfunction, and edema that most often occurs as an atypical response to a minor surgery or fracture. Inflammation involving activation and recruitment of innate immune cells, including both peripheral and central myeloid cells (ie, macrophages and microglia, respectively), is a key feature of CRPS. However, the exact role and time course of these cellular processes relative to the known acute and chronic phases of the disease are not fully understood. Positron emission tomography (PET) of translocator protein-18 kDa (TSPO) is a method for noninvasively tracking these activated innate immune cells. Here, we reveal the temporal dynamics of peripheral and central inflammatory responses over 20 weeks in a tibial fracture/casting mouse model of CRPS through longitudinal TSPO-PET using [F]GE-180. Positron emission tomography tracer uptake quantification in the tibia revealed increased peripheral inflammation as early as 2 days after fracture and lasting 7 weeks. Centralized inflammation was detected in the spinal cord and brain of fractured mice at 7 and 21 days after injury. Spinal cord tissue immunofluorescent staining revealed TSPO expression in microglia (CD11b+) at 7 days but was restricted mainly to endothelial cells (PECAM1+) at baseline and 7 weeks. Our data suggest early and persistent peripheral myeloid cell activation and transient central microglial activation are limited to the acute phase of CRPS. Moreover, we show that TSPO-PET can be used to noninvasively monitor the spatiotemporal dynamics of myeloid cell activation in CRPS progression with potential to inform disease phase-specific therapeutics.

Longitudinal translocator protein-18 kDa-positron emission tomography imaging of peripheral and central myeloid cells in a mouse model of complex regional pain syndrome.

Acidosis is a key driver for many diseases, including cancer, sepsis, and stroke. The spatiotemporal dynamics of dysregulated pH across disease remain elusive, and current diagnostic strategies do not provide localization of pH alterations. We sought to explore if PET imaging using hydrophobic cyclic peptides that partition into the cellular membrane at low extracellular pH (denoted as pH [low] insertion cycles, or pHLIC) can permit accurate in vivo visualization of acidosis.  Acid-sensitive cyclic peptide c[EWC] pHLIC was conjugated to bifunctional maleimide-NO2A and radiolabeled with Cu (half-life, 12.7 h). C57BL/6J mice were administered lipopolysaccharide (15 mg/kg) or saline (vehicle) and serially imaged with [Cu]Cu-c[EWC] over 24 h. Ex vivo autoradiography was performed on resected brain slices and subsequently stained with cresyl violet to enable high-resolution spatial analysis of tracer accumulation. A non-pH-sensitive cell-penetrating control peptide (c[RWC]) was used to confirm specificity of [Cu]Cu-c[EWC]. CD11b (macrophage/microglia) and TMEM119 (microglia) immunostaining was performed to correlate extent of neuroinflammation with [Cu]Cu-c[EWC] PET signal.  [Cu]Cu-c[EWC] radiochemical yield and purity were more than 95% and more than 99%, respectively, with molar activity of more than 0.925 MBq/nmol. Significantly increased [Cu]Cu-c[EWC] uptake was observed in lipopolysaccharide-treated mice (vs. vehicle) within peripheral tissues, including blood, lungs, liver, and small intestines ( < 0.001-0.05). Additionally, there was significantly increased [Cu]Cu-c[EWC] uptake in the brains of lipopolysaccharide-treated animals. Autoradiography confirmed increased uptake in the cerebellum, cortex, hippocampus, striatum, and hypothalamus of lipopolysaccharide-treated mice (vs. vehicle). Immunohistochemical analysis revealed microglial or macrophage infiltration, suggesting activation in brain regions containing increased tracer uptake. [Cu]Cu-c[RWC] demonstrated significantly reduced uptake in the brain and periphery of lipopolysaccharide mice compared with the acid-mediated [Cu]Cu-c[EWC] tracer.  Here, we demonstrate that a pH-sensitive PET tracer specifically detects acidosis in regions associated with sepsis-driven proinflammatory responses. This study suggests that [Cu]Cu-pHLIC is a valuable tool to noninvasively assess acidosis associated with both central and peripheral innate immune activation.

Demarcation of Sepsis-Induced Peripheral and Central Acidosis with pH (Low) Insertion Cycle Peptide.

The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability. Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins. Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery.

Physiological blood-brain transport is impaired with age by a shift in transcytosis.

B cells play a central role in multiple sclerosis (MS) through production of injurious antibodies, secretion of pro-inflammatory cytokines, and antigen presentation. The therapeutic success of monoclonal antibodies (mAbs) targeting B cells in some but not all individuals suffering from MS highlights the need for a method to stratify patients and monitor response to treatments in real-time. Herein, we describe the development of the first CD19 positron emission tomography (PET) tracer, and its evaluation in a rodent model of MS, experimental autoimmune encephalomyelitis (EAE).

Haley C. Cropper

Department of Radiology

Stanford University

PET Imaging of Neuroinflammation Using [¹¹C]DPA-713 in a Mouse Model of Ischemic Stroke

Imaging CD19⁺ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Haley C. Cropper

Department of Radiology

Stanford University

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

PET Imaging of Neuroinflammation Using [¹¹C]DPA-713 in a Mouse Model of Ischemic Stroke

Imaging CD19⁺ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography