Microglia have a variety of functions in the brain, including synaptic pruning, CNS repair and mediating the immune response against peripheral infection. Microglia rapidly become activated in ...response to CNS damage. Depending on the nature of the stimulus, microglia can take a number of activation states, which correspond to altered microglia morphology, gene expression and function. It has been reported that early microglia activation following traumatic brain injury (TBI) may contribute to the restoration of homeostasis in the brain. On the other hand, if they remain chronically activated, such cells display a classically activated phenotype, releasing pro-inflammatory molecules, resulting in further tissue damage and contributing potentially to neurodegeneration. However, new evidence suggests that this classification is over-simplistic and the balance of activation states can vary at different points. In this article, we review the role of microglia in TBI, analyzing their distribution, morphology and functional phenotype over time in animal models and in humans. Animal studies have allowed genetic and pharmacological manipulations of microglia activation, in order to define their role. In addition, we describe investigations on the
imaging of microglia using translocator protein (TSPO) PET and autoradiography, showing that microglial activation can occur in regions far remote from sites of focal injuries, in humans and animal models of TBI. Finally, we outline some novel potential therapeutic approaches that prime microglia/macrophages toward the beneficial restorative microglial phenotype after TBI.
Alzheimer's disease (AD) is characterized by memory loss and decline of cognitive function, associated with progressive neurodegeneration. While neuropathological processes like amyloid plaques and ...tau neurofibrillary tangles have been linked to neuronal death in AD, the precise role of glial activation on disease progression is still debated. It was suggested that neuroinflammation could occur well ahead of amyloid deposition and may be responsible for clearing amyloid, having a neuroprotective effect; however, later in the disease, glial activation could become deleterious, contributing to neuronal toxicity. Recent genetic and preclinical studies suggest that the different activation states of microglia and astrocytes are complex, not as polarized as previously thought, and that the heterogeneity in their phenotype can switch during disease progression. In the last few years, novel imaging techniques e.g., new radiotracers for assessing glia activation using positron emission tomography and advanced magnetic resonance imaging technologies have emerged, allowing the correlation of neuro-inflammatory markers with cognitive decline, brain function and brain pathology
. Here we review all new imaging technology in AD patients and animal models that has the potential to serve for early diagnosis of the disease, to monitor disease progression and to test the efficacy and the most effective time window for potential anti-inflammatory treatments.
Immune checkpoint inhibitor therapy targeting the PD-1/PD-L1 axis in cancer patients, is a promising oncological treatment. However, the number of non-responders remains high, causing a burden for ...the patient and the healthcare system. Consequently, a diagnostic tool to predict treatment outcomes would help with patient stratification. Molecular imaging provides said diagnostic tool by offering a whole-body quantitative assessment of PD-L1 expression, hence supporting therapy decisions. Four PD-L1 radioligand candidates containing a linker-chelator system for radiometalation, along with three hydrophilizing units—one sulfonic and two phosphonic acids—were synthesized. After labeling with 64Cu, log D7.4 values of less than −3.03 were determined and proteolytic stability confirmed over 94% intact compound after 48 h. Binding affinity was determined using two different assays, revealing high affinities up to 13 nM. µPET/CT imaging was performed in tumor-bearing mice to investigate PD-L1-specific tumor uptake and the pharmacokinetic profile of radioligands. These results yielded an unexpected in vivo distribution, such as low tumor uptake in PD-L1 positive tumors, high liver uptake, and accumulation in bone/bone marrow and potentially synovial spaces. These effects are likely caused by Ca2+-affinity and/or binding to macrophages. Despite phosphonic acids providing high water solubility, their incorporation must be carefully considered to avoid compromising the pharmacokinetic behavior of radioligands.
The 18 kDa translocator protein (TSPO) is increasingly used to study brain and spinal cord inflammation in degenerative diseases of the CNS such as multiple sclerosis. The enhanced TSPO PET signal ...that arises during disease is widely considered to reflect activated pathogenic microglia, although quantitative neuropathological data to support this interpretation have not been available. With the increasing interest in the role of chronic microglial activation in multiple sclerosis, characterising the cellular neuropathology associated with TSPO expression is of clear importance for understanding the cellular and pathological processes on which TSPO PET imaging is reporting. Here we have studied the cellular expression of TSPO and specific binding of two TSPO targeting radioligands (3H-PK11195 and 3H-PBR28) in tissue sections from 42 multiple sclerosis cases and 12 age-matched controls. Markers of homeostatic and reactive microglia, astrocytes, and lymphocytes were used to investigate the phenotypes of cells expressing TSPO. There was an approximate 20-fold increase in cells double positive for TSPO and HLA-DR in active lesions and in the rim of chronic active lesion, relative to normal appearing white matter. TSPO was uniformly expressed across myeloid cells irrespective of their phenotype, rather than being preferentially associated with pro-inflammatory microglia or macrophages. TSPO+ astrocytes were increased up to 7-fold compared to normal-appearing white matter across all lesion subtypes and accounted for 25% of the TSPO+ cells in these lesions. To relate TSPO protein expression to ligand binding, specific binding of the TSPO ligands 3H-PK11195 and 3H-PBR28 was determined in the same lesions. TSPO radioligand binding was increased up to seven times for 3H-PBR28 and up to two times for 3H-PK11195 in active lesions and the centre of chronic active lesions and a strong correlation was found between the radioligand binding signal for both tracers and the number of TSPO+ cells across all of the tissues examined. In summary, in multiple sclerosis, TSPO expression arises from microglia of different phenotypes, rather than being restricted to microglia which express classical pro-inflammatory markers. While the majority of cells expressing TSPO in active lesions or chronic active rims are microglia/macrophages, our findings also emphasize the significant contribution of activated astrocytes, as well as smaller contributions from endothelial cells. These observations establish a quantitative framework for interpretation of TSPO in multiple sclerosis and highlight the need for neuropathological characterization of TSPO expression for the interpretation of TSPO PET in other neurodegenerative disorders.
Small molecules offer some advantages for developing positron emission tomography (PET) tracers and are therefore a promising approach for imaging and therapy monitoring of programmed death ligand 1 ...(PD-L1) positive tumors. Here, we report six biphenyl PD-L1 radioligands using the NODA-GA-chelator for efficient copper-64 complexation. These radioligands contain varying numbers of sulfonic and/or phosphonic acid groups, serving as hydrophilizing units to lower the log D 7.4 value down to −4.28. The binding affinities of compounds were evaluated using saturation binding and a real-time binding assay, with a highest binding affinity of 21 nM. Small-animal PET imaging revealed vastly different pharmacokinetic profiles depending on the quantity and type of hydrophilizing units. Of the investigated radioligands, 64CuCu-3 showed the most favorable kinetics in vitro. This was also found in vivo, with a predominantly renal clearance and a specific uptake in the PD-L1-overexpressing tumor. With further modifications, this compound could be a promising candidate for the imaging of PD-L1 in the clinical setting.
Abstract
Neurovascular injury is often observed in traumatic brain injury (TBI). However, the relationship between mechanical forces and vascular injury is still unclear. A key question is whether ...the complex anatomy of vasculature plays a role in increasing forces in cerebral vessels and producing damage. We developed a high-fidelity multiscale finite element model of the rat brain featuring a detailed definition of the angioarchitecture. Controlled cortical impacts were performed experimentally and in-silico. The model was able to predict the pattern of blood–brain barrier damage. We found strong correlation between the area of fibrinogen extravasation and the brain area where axial strain in vessels exceeds 0.14. Our results showed that adjacent vessels can sustain profoundly different axial stresses depending on their alignment with the principal direction of stress in parenchyma, with a better alignment leading to larger stresses in vessels. We also found a strong correlation between axial stress in vessels and the shearing component of the stress wave in parenchyma. Our multiscale computational approach explains the unrecognised role of the vascular anatomy and shear stresses in producing distinct distribution of large forces in vasculature. This new understanding can contribute to improving TBI diagnosis and prevention.
Noninvasive molecular imaging of the PD-1/PD-L1 immune checkpoint is of high clinical relevance for patient stratification and therapy monitoring in cancer patients. Here we report nine ...small-molecule PD-L1 radiotracers with solubilizing sulfonic acids and a linker-chelator system, designed by molecular docking experiments and synthesized according to a new, convergent synthetic strategy. Binding affinities were determined both in cellular saturation and real-time binding assay (LigandTracer), revealing dissociation constants in the single digit nanomolar range. Incubation in human serum and liver microsomes proved in vitro stability of these compounds. Small animal PET/CT imaging, in mice bearing PD-L1 overexpressing and PD-L1 negative tumors, showed moderate to low uptake. All compounds were cleared primarily through the hepatobiliary excretion route and showed a long circulation time. The latter was attributed to strong blood albumin binding effects, discovered during our binding experiments. Taken together, these compounds are a promising starting point for further development of a new class of PD-L1 targeting radiotracers.
The relationship between biomechanical forces and neuropathology is key to understanding traumatic brain injury. White matter tracts are damaged by high shear forces during impact, resulting in ...axonal injury, a key determinant of long-term clinical outcomes. However, the relationship between biomechanical forces and patterns of white matter injuries, associated with persistent diffusion MRI abnormalities, is poorly understood. This limits the ability to predict the severity of head injuries and the design of appropriate protection. Our previously developed human finite element model of head injury predicted the location of post-traumatic neurodegeneration. A similar rat model now allows us to experimentally test whether strain patterns calculated by the model predicts in vivo MRI and histology changes. Using a controlled cortical impact, mild and moderate injuries (1 and 2 mm) were performed. Focal and axonal injuries were quantified with volumetric and diffusion 9.4 T MRI at 2 weeks post injury. Detailed analysis of the corpus callosum was conducted using multi-shell diffusion MRI and histopathology. Microglia and astrocyte density, including process parameters, along with white matter structural integrity and neurofilament expression were determined by quantitative immunohistochemistry. Linear mixed effects regression analyses for strain and strain rate with the employed outcome measures were used to ascertain how well immediate biomechanics could explain MRI and histology changes. The spatial pattern of mechanical strain and strain rate in the injured cortex shows good agreement with the probability maps of focal lesions derived from volumetric MRI. Diffusion metrics showed abnormalities in the corpus callosum, indicating white matter changes in the segments subjected to high strain, as predicted by the model. The same segments also exhibited a severity-dependent increase in glia cell density, white matter thinning and reduced neurofilament expression. Linear mixed effects regression analyses showed that mechanical strain and strain rate were significant predictors of in vivo MRI and histology changes. Specifically, strain and strain rate respectively explained 33% and 28% of the reduction in fractional anisotropy, 51% and 29% of the change in neurofilament expression and 51% and 30% of microglia density changes. The work provides evidence that strain and strain rate in the first milliseconds after injury are important factors in determining patterns of glial and axonal injury and serve as experimental validators of our computational model of traumatic brain injury. Our results provide support for the use of this model in understanding the relationship of biomechanics and neuropathology and can guide the development of head protection systems, such as airbags and helmets.
Axonal injury is a key determinant of long-term outcomes after traumatic brain injury (TBI) but has been difficult to measure clinically. Fluid biomarker assays can now sensitively quantify neuronal ...proteins in blood. Axonal components such as neurofilament light (NfL) potentially provide a diagnostic measure of injury. In the multicenter BIO-AX-TBI study of moderate-severe TBI, we investigated relationships between fluid biomarkers, advanced neuroimaging, and clinical outcomes. Cerebral microdialysis was used to assess biomarker concentrations in brain extracellular fluid aligned with plasma measurement. An experimental injury model was used to validate biomarkers against histopathology. Plasma NfL increased after TBI, peaking at 10 days to 6 weeks but remaining abnormal at 1 year. Concentrations were around 10 times higher early after TBI than in controls (patients with extracranial injuries). NfL concentrations correlated with diffusion MRI measures of axonal injury and predicted white matter neurodegeneration. Plasma TAU predicted early gray matter atrophy. NfL was the strongest predictor of functional outcomes at 1 year. Cerebral microdialysis showed that NfL concentrations in plasma and brain extracellular fluid were highly correlated. An experimental injury model confirmed a dose-response relationship of histopathologically defined axonal injury to plasma NfL. In conclusion, plasma NfL provides a sensitive and clinically meaningful measure of axonal injury produced by TBI. This reflects the extent of underlying damage, validated using advanced MRI, cerebral microdialysis, and an experimental model. The results support the incorporation of NfL sampling subacutely after injury into clinical practice to assist with the diagnosis of axonal injury and to improve prognostication.
Background
Programmed cell death ligand 1 (PD-L1) plays a critical role in the tumor microenvironment and overexpression in several solid cancers has been reported. This was associated with a ...downregulation of the local immune response, specifically of T-cells. Immune checkpoint inhibitors showed a potential to break this localized immune paralysis, but only 30% of patients are considered responders. New diagnostic approaches are therefore needed to determine patient eligibility. Small molecule radiotracers targeting PD-L1, may serve as such diagnostic tools, addressing the heterogeneous PD-L1 expression between and within tumor lesions, thus aiding in therapy decisions.
Results
Four biphenyl-based small-molecule PD-L1 ligands were synthesized using a convergent synthetic route with a linear sequence of up to eleven steps. As a chelator NODA-GA, CB-TE2A or DiAmSar was used to allow radiolabeling with copper-64 (
64
CuCu-
14
–
64
CuCu-
16
). In addition, a dimeric structure based on DiAmSar was synthesized (
64
CuCu-
17
). All four radioligands exhibited high proteolytic stability (> 95%) up to 48 h post-radiolabeling. Saturation binding yielded moderate affinities toward PD-L1, ranging from 100 to 265 nM. Real-time radioligand binding provided more promising
K
D
values around 20 nM for
64
CuCu-
14
and
64
CuCu-
15
. In vivo PET imaging in mice bearing both PC3 PD-L1 overexpressing and PD-L1-mock tumors was performed at 0–2, 4–5 and 24–25 h post injection (p.i.). This revealed considerably different pharmacokinetic profiles, depending on the substituted chelator.
64
CuCu-
14
, substituted with NODA-GA, showed renal clearance with low liver uptake, whereas substitution with the cross-bridged cyclam chelator CB-TE2A resulted in a primarily hepatobiliary clearance. Notably, the monomeric DiAmSar radioligand
64
CuCu-
16
demonstrated a higher liver uptake than
64
CuCu-
15
, but was still renally cleared as evidenced by the lack of uptake in gall bladder and intestines. The dimeric structure
64
CuCu-
17
showed extensive accumulation and trapping in the liver but was also cleared via the renal pathway. Of all tracer candidates and across all timepoints,
64
CuCu-
17
showed the highest accumulation at 24 h p.i. in the PD-L1-overexpressing tumor of all timepoints and all radiotracers, indicating drastically increased circulation time upon dimerization of two PD-L1 binding motifs.
Conclusions
This study shows that chelator choice significantly influences the pharmacokinetic profile of biphenyl-based small molecule PD-L1 radioligands. The NODA-GA-conjugated radioligand
64
CuCu-
14
exhibited favorable renal clearance; however, the limited uptake in tumors suggests the need for structural modifications to the binding motif for future PD-L1 radiotracers.
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