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STAR Protocols Jun 2021Noninvasive immunoimaging holds great potential for studying and stratifying disease as well as therapeutic efficacy. Radiolabeled single-domain antibody fragments...
Noninvasive immunoimaging holds great potential for studying and stratifying disease as well as therapeutic efficacy. Radiolabeled single-domain antibody fragments (i.e., nanobodies) are appealing probes for immune landscape profiling, as they display high stability, rapid targeting, and excellent specificity, while allowing extremely sensitive nuclear readouts. Here, we present a protocol for radiolabeling an anti-CD11b nanobody and studying its uptake in mice by a combination of positron emission tomography imaging, gamma counting, and autoradiography. Our protocol is applicable to nanobodies against other antigens. For complete details on the use and execution of this protocol, please see Priem et al. (2020), Senders et al. (2019), or Rashidian et al. (2017).
Topics: Animals; Histological Techniques; Immunologic Techniques; Mice; Molecular Imaging; Organ Specificity; Positron-Emission Tomography; Single-Domain Antibodies
PubMed: 33899016
DOI: 10.1016/j.xpro.2021.100434 -
Frontiers in Neurology 2021Autoradiography on brain tissue is used to validate binding targets of newly discovered radiotracers. The purpose of this study was to correlate quantification of...
Autoradiography on brain tissue is used to validate binding targets of newly discovered radiotracers. The purpose of this study was to correlate quantification of autoradiography signal using the novel next-generation tau positron emission tomography (PET) radiotracer [F]PI-2620 with immunohistochemically determined tau-protein load in both formalin-fixed paraffin-embedded (FFPE) and frozen tissue samples of patients with Alzheimer's disease (AD) and Progressive Supranuclear Palsy (PSP). We applied [F]PI-2620 autoradiography to postmortem cortical brain samples of six patients with AD, five patients with PSP and five healthy controls, respectively. Binding intensity was compared between both tissue types and different disease entities. Autoradiography signal quantification (CWMR = cortex to white matter ratio) was correlated with the immunohistochemically assessed tau load (AT8-staining, %-area) for FFPE and frozen tissue samples in the different disease entities. In AD tissue, relative cortical tracer binding was higher in frozen samples when compared to FFPE samples (CWMR vs. CWMR: 2.5-fold, < 0.001), whereas the opposite was observed in PSP tissue (CWMR vs. CWMR: 0.8-fold, = 0.004). In FFPE samples, [F]PI-2620 autoradiography tracer binding and immunohistochemical tau load correlated significantly for both PSP ( = 0.641, < 0.001) and AD tissue ( = 0.435, = 0.016), indicating a high agreement of relative tracer binding with underlying pathology. In frozen tissue, the correlation between autoradiography and immunohistochemistry was only present in AD ( = 0.417, = 0.014) but not in PSP tissue ( = -0.115, = n.s.). Our head-to-head comparison indicates that FFPE samples show superiority over frozen samples for autoradiography assessment of PSP tau pathology by [F]PI-2620. The [F]PI-2620 autoradiography signal in FFPE samples reflects AT8 positive tau in samples of both PSP and AD patients.
PubMed: 34276540
DOI: 10.3389/fneur.2021.684523 -
Neuropharmacology May 2022The protein alpha-synuclein (αSYN) plays a central role in synucleinopathies such as Parkinsons's disease (PD) and multiple system atrophy (MSA). Presently, there are...
The protein alpha-synuclein (αSYN) plays a central role in synucleinopathies such as Parkinsons's disease (PD) and multiple system atrophy (MSA). Presently, there are no selective αSYN positron emission tomography (PET) radioligands that do not also show affinity to amyloid-beta (Aβ). We have previously shown that radiolabeled antibodies, engineered to enter the brain via the transferrin receptor (TfR), is a promising approach for PET imaging of intrabrain targets. In this study, we used this strategy to visualize αSYN in the living mouse brain. Five bispecific antibodies, binding to both the murine TfR and αSYN were generated and radiolabeled with iodine-125 or iodine-124. All bispecific antibodies bound to αSYN and mTfR before and after radiolabelling in an ELISA assay, and bound to brain sections prepared from αSYN overexpressing mice as well as human PD- and MSA subjects, but not control tissues in autoradiography. Brain concentrations of the bispecific antibodies were between 26 and 63 times higher than the unmodified IgG format 2 h post-injection, corresponding to about 1.5% of the injected dose per gram brain tissue. Additionally, intrastriatal αSYN fibrils were visualized with PET in an αSYN deposition mouse model with one of the bispecific antibodies, [I]RmAbSynO2-scFv8D3. However, PET images acquired in αSYN transgenic mice with verified brain pathology injected with [I]RmAbSynO2-scFv8D3 and [I]RmAb48-scFv8D3 showed no increase in antibody retention compared to WT mice. Despite successful imaging of deposited extracellular αSYN using a brain-penetrating antibody-based radioligand with no cross-specificity towards Aβ, this proof-of-concept study demonstrates challenges in imaging intracellular αSYN inclusions present in synucleinopathies.
Topics: Amyloid beta-Peptides; Animals; Antibodies, Bispecific; Brain; Humans; Mice; Multiple System Atrophy; Parkinson Disease; Positron-Emission Tomography; Synucleinopathies; alpha-Synuclein
PubMed: 35149134
DOI: 10.1016/j.neuropharm.2022.108985 -
European Journal of Nuclear Medicine... Oct 2020Longitudinal mouse PET imaging is becoming increasingly popular due to the large number of transgenic and disease models available but faces challenges. These challenges...
UNLABELLED
Longitudinal mouse PET imaging is becoming increasingly popular due to the large number of transgenic and disease models available but faces challenges. These challenges are related to the small size of the mouse brain and the limited spatial resolution of microPET scanners, along with the small blood volume making arterial blood sampling challenging and impossible for longitudinal studies. The ability to extract an input function directly from the image would be useful for quantification in longitudinal small animal studies where there is no true reference region available such as TSPO imaging.
METHODS
Using dynamic, whole-body F-DPA-714 PET scans (60 min) in a mouse model of hippocampal sclerosis, we applied a factor analysis (FA) approach to extract an image-derived input function (IDIF). This mouse-specific IDIF was then used for 4D-resolution recovery and denoising (4D-RRD) that outputs a dynamic image with better spatial resolution and noise properties, and a map of the total volume of distribution (V) was obtained using a basis function approach in a total of 9 mice with 4 longitudinal PET scans each. We also calculated percent injected dose (%ID) with and without 4D-RRD. The V and %ID parameters were compared to quantified ex vivo autoradiography using regional correlations of the specific binding from autoradiography against V and %ID parameters.
RESULTS
The peaks of the IDIFs were strongly correlated with the injected dose (Pearson R = 0.79). The regional correlations between the %ID estimates and autoradiography were R = 0.53 without 4D-RRD and 0.72 with 4D-RRD over all mice and scans. The regional correlations between the V estimates and autoradiography were R = 0.66 without 4D-RRD and 0.79 with application of 4D-RRD over all mice and scans.
CONCLUSION
We present a FA approach for IDIF extraction which is robust, reproducible and can be used in quantification methods for resolution recovery, denoising and parameter estimation. We demonstrated that the proposed quantification method yields parameter estimates closer to ex vivo measurements than semi-quantitative methods such as %ID and is immune to tracer binding in tissue unlike reference tissue methods. This approach allows for accurate quantification in longitudinal PET studies in mice while avoiding repeated blood sampling.
Topics: Algorithms; Animals; Disease Models, Animal; Mice; Positron-Emission Tomography
PubMed: 32211931
DOI: 10.1007/s00259-020-04755-5 -
Frontiers in Plant Science 2021Due to its high sensitivity and specificity for tumor detection, positron emission tomography (PET) has become a standard and widely used molecular imaging technique....
Due to its high sensitivity and specificity for tumor detection, positron emission tomography (PET) has become a standard and widely used molecular imaging technique. Given the popularity of PET, both clinically and preclinically, its use has been extended to study plants. However, only a limited number of research groups worldwide report PET-based studies, while we believe that this technique has much more potential and could contribute extensively to plant science. The limited application of PET may be related to the complexity of putting together methodological developments from multiple disciplines, such as radio-pharmacology, physics, mathematics and engineering, which may form an obstacle for some research groups. By means of this manuscript, we want to encourage researchers to study plants using PET. The main goal is to provide a clear description on how to design and execute PET scans, process the resulting data and fully explore its potential by quantification via compartmental modeling. The different steps that need to be taken will be discussed as well as the related challenges. Hereby, the main focus will be on, although not limited to, tracing CO to study plant carbon dynamics.
PubMed: 34149742
DOI: 10.3389/fpls.2021.602550 -
Life (Basel, Switzerland) Aug 2023This article relates personal recollections and starts with the origin of electron microscopy in the sixties of the previous century at the University of Amsterdam.... (Review)
Review
This article relates personal recollections and starts with the origin of electron microscopy in the sixties of the previous century at the University of Amsterdam. Novel fixation and embedding techniques marked the discovery of the internal bacterial structures not visible by light microscopy. A special status became reserved for the freeze-fracture technique. By freeze-fracturing chemically fixed cells, it proved possible to examine the morphological effects of fixation. From there on, the focus switched from bacterial structure as such to their cell cycle. This invoked bacterial physiology and steady-state growth combined with electron microscopy. Electron-microscopic autoradiography with pulses of [H] Dap revealed that segregation of replicating DNA cannot proceed according to a model of zonal growth (with envelope-attached DNA). This stimulated us to further investigate the sacculus, the peptidoglycan macromolecule. In particular, we focused on the involvement of penicillin-binding proteins such as PBP2 and PBP3, and their role in division. Adding aztreonam (an inhibitor of PBP3) blocked ongoing divisions but not the initiation of new ones. A PBP3-independent peptidoglycan synthesis (PIPS) appeared to precede a PBP3-dependent step. The possible chemical nature of PIPS is discussed.
PubMed: 37629639
DOI: 10.3390/life13081782 -
Molecular Imaging and Biology Aug 2022The loss of viable cardiac cells and cell death by myocardial infarction (MI) is still a significant obstacle in preventing deteriorating heart failure. Imaging of...
PURPOSE
The loss of viable cardiac cells and cell death by myocardial infarction (MI) is still a significant obstacle in preventing deteriorating heart failure. Imaging of apoptosis, a defined cascade to cell death, could identify areas at risk.
PROCEDURES
Using 2-(5-[F]fluoropentyl)-2-methyl-malonic acid ([F]ML-10) in autoradiography and positron emission tomography (PET) visualized apoptosis in murine hearts after permanent ligation of the left anterior descending artery (LAD) inducing myocardial infarction (MI). 2-deoxy-2-[F]fluoro-D-glucose ([F]FDG) PET imaging localized the infarct area after MI. Histology by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining validated apoptosis in the heart.
RESULTS
Accumulation of [F]ML-10 was evident in the infarct area after permanent ligation of the LAD in autoradiography and PET imaging. Detection of apoptosis by [F]ML-10 is in line with the defect visualized by [F]FDG and the histological approach.
CONCLUSION
[F]ML-10 could be a suitable tracer for apoptosis imaging in a mouse model of permanent LAD ligation.
Topics: Animals; Apoptosis; Disease Models, Animal; Fluorodeoxyglucose F18; Heart; Mice; Myocardial Infarction; Positron-Emission Tomography
PubMed: 35352214
DOI: 10.1007/s11307-022-01718-0 -
ChemMedChem Oct 2020The nonselective Ca -permeable transient receptor potential channel subfamily member 5 (TRPC5) belongs to the transient receptor potential canonical (TRPC) superfamily...
The nonselective Ca -permeable transient receptor potential channel subfamily member 5 (TRPC5) belongs to the transient receptor potential canonical (TRPC) superfamily and is widely expressed in the brain. Compelling evidence reveals that TRPC5 plays crucial roles in depression and other psychiatric disorders. To develop a TRPC5 radioligand, following up on our previous effort, we synthesized the iodine compound TZ66127 and its iodine-125-labeled counterpart [ I]TZ66127. The synthesis of TZ66127 was achieved by replacing chloride with iodide in the structure of HC608, and the [ I]TZ66127 was radiosynthesized using its corresponding tributylstannylated precursor. We established a stable human TRPC5-overexpressed HEK293-hTRPC5 cell line and performed Ca imaging and a cell-binding assay study of TZ66127; these indicated that TZ66127 had good inhibition activity for TRPC5, and the inhibitory efficiency of TZ66127 toward TRPC5 presented in a dose-dependent manner. An in vitro autoradiography and immunohistochemistry study of rat brain sections suggested that [ I]TZ66127 had binding specificity toward TRPC5. Altogether, [ I]TZ66127 has high potential to serve as a radioligand for screening the binding activity of other new compounds toward TRPC5. The availability of [ I]TZ66127 might facilitate the development of therapeutic drugs and PET imaging agents that target TRPC5.
Topics: Animals; Brain; Calcium; Cells, Cultured; Dose-Response Relationship, Drug; HEK293 Cells; Humans; Iodine Radioisotopes; Molecular Structure; Positron-Emission Tomography; Radioligand Assay; Radiopharmaceuticals; Rats; TRPC Cation Channels
PubMed: 32717096
DOI: 10.1002/cmdc.202000339 -
Experimental Neurology Aug 2021Parkinson's disease (PD) is characterized by Lewy body and neurite pathology associated with dopamine terminal dysfunction. Clinically, it is associated with motor...
BACKGROUND
Parkinson's disease (PD) is characterized by Lewy body and neurite pathology associated with dopamine terminal dysfunction. Clinically, it is associated with motor slowing, rigidity, and tremor. Postural instability and pain are also features. Physical exercise benefits PD patients - possibly by promoting neuroplasticity including synaptic regeneration.
OBJECTIVES
In a parkinsonian rat model, we test the hypotheses that exercise: (a) increases synaptic density and reduces neuroinflammation and (b) lowers the nociceptive threshold by increasing μ-opioid receptor expression.
METHODS
Brain autoradiography was performed on rats unilaterally injected with either 6-hydroxydopamine (6-OHDA) or saline and subjected to treadmill exercise over 5 weeks. [H]UCB-J was used to measure synaptic vesicle glycoprotein 2A (SV2A) density. Dopamine D2/3 receptor and μ-opioid receptor availability were assessed with [H]Raclopride and [H]DAMGO, respectively, while neuroinflammation was detected with the 18kDA translocator protein (TSPO) marker [H]PK11195. The nociceptive threshold was determined prior to and throughout the exercise protocol.
RESULTS
We confirmed a dopaminegic deficit with increased striatal [H]Raclopride D2/3 receptor availability and reduced nigral tyrosine hydroxylase immunoreactivity in the ipsilateral hemisphere of all 6-OHDA-injected rats. Sedentary rats lesioned with 6-OHDA showed significant reduction of ipsilateral striatal and substantia nigra [H]UCB-J binding while [H]PK11195 showed increased ipsilateral striatal neuroinflammation. Lesioned rats who exercised had higher levels of ipsilateral striatal [H]UCB-J binding and lower levels of neuroinflammation compared to sedentary lesioned rats. Striatal 6-OHDA injections reduced thalamic μ-opioid receptor availability but subsequent exercise restored binding. Exercise also raised thalamic and hippocampal SV2A synaptic density in 6-OHDA lesioned rats, accompanied by a rise in nociceptive threshold.
CONCLUSION
These data suggest that treadmill exercise protects nigral and striatal synaptic integrity in a rat lesion model of PD - possibly by promoting compensatory mechanisms. Exercise was also associated with reduced neuroinflammation post lesioning and altered opioid transmission resulting in an increased nociceptive threshold.
Topics: Animals; Brain; Exercise Test; Male; Oxidopamine; Parkinsonian Disorders; Physical Conditioning, Animal; Rats; Rats, Wistar; Synapses
PubMed: 33965411
DOI: 10.1016/j.expneurol.2021.113741 -
European Journal of Nuclear Medicine... Aug 2023Hepatocellular carcinoma (HCC) is a highly vascularized solid carcinoma and tumor vessel-targeted molecular imaging might be effective for early diagnosis of HCC....
PURPOSE
Hepatocellular carcinoma (HCC) is a highly vascularized solid carcinoma and tumor vessel-targeted molecular imaging might be effective for early diagnosis of HCC. Herein, we developed a novel trimeric affibody (Z) with highly specific binding to the platelet-derived growth factor receptor beta (PDGFRβ). The aim of this study is to evaluate the feasibility of Ga-radiolabeled Z ([Ga]Ga-DOTA-Z) as PET tracer for diagnosis of HCC.
METHODS
The bioinformatics analysis of clinical database and immunoblotting of clinical specimens were performed to validate the potential of PDGFRβ as HCC biomarker. The trimeric affibody Z was conjugated with DOTA-NHS-ester and radiolabeled with Ga to produce [Ga]Ga-DOTA-Z conjugate. Immunoreactivity and specific uptake of [Ga]Ga-DOTA-Z were assessed by dose-dependent cell binding, autoradiography, and biodistribution analysis. [Ga]Ga-DOTA-Z PET/CT scanning of diethylnitrosamine (DEN)-induced primary HCC rats and a rare case of idiopathical HCC rhesus monkey was performed to evaluate the imaging capability and radiation dosimetry of [Ga]Ga-DOTA-Z in vivo.
RESULTS
Excessive PDGFRβ was validated as a representative biomarker of HCC neovascularization. The radiolabeling of [Ga]Ga-DOTA-Z was achieved at more than 95% radiochemical yield. In vitro assays showed specific uptake of [Ga]Ga-DOTA-Z in HCC tumor vessels by autoradiography. Animal PET/CT imaging with [Ga]Ga-DOTA-Z successfully visualized the tumor lesions in primary HCC rats and rhesus monkey, and indicated radiation absorbed dose of 2.03E-02 mSv/MBq for each scanning.
CONCLUSIONS
Our results demonstrated that [Ga]Ga-DOTA-Z conjugate could be applied as a promising PET tracer for early diagnosis of hepatocellular carcinoma.
Topics: Rats; Animals; Positron Emission Tomography Computed Tomography; Carcinoma, Hepatocellular; Gallium Radioisotopes; Tissue Distribution; Macaca mulatta; Cell Line, Tumor; Liver Neoplasms; Positron-Emission Tomography; Biomarkers
PubMed: 37256321
DOI: 10.1007/s00259-023-06260-x