Longitudinal positron emission tomography and postmortem analysis reveals widespread neuroinflammation in SARS-CoV-2 infected rhesus macaques.

BACKGROUND: Coronavirus disease 2019 (COVID-19) patients initially develop respiratory symptoms, but they may also suffer from neurological symptoms. People with long-lasting effects after acute infections with severe respiratory syndrome coronavirus 2 (SARS-CoV-2), i.e., post-COVID syndrome or long COVID, may experience a variety of neurological manifestations. Although we do not fully understand how SARS-CoV-2 affects the brain, neuroinflammation likely plays a role. METHODS: To investigate neuroinflammatory processes longitudinally after SARS-CoV-2 infection, four experimentally SARS-CoV-2 infected rhesus macaques were monitored for 7 weeks with 18-kDa translocator protein (TSPO) positron emission tomography (PET) using [18F]DPA714, together with computed tomography (CT). The baseline scan was compared to weekly PET-CTs obtained post-infection (pi). Brain tissue was collected following euthanasia (50 days pi) to correlate the PET signal with TSPO expression, and glial and endothelial cell markers. Expression of these markers was compared to brain tissue from uninfected animals of comparable age, allowing the examination of the contribution of these cells to the neuroinflammatory response following SARS-CoV-2 infection. RESULTS: TSPO PET revealed an increased tracer uptake throughout the brain of all infected animals already from the first scan obtained post-infection (day 2), which increased to approximately twofold until day 30 pi. Postmortem immunohistochemical analysis of the hippocampus and pons showed TSPO expression in cells expressing ionized calcium-binding adaptor molecule 1 (IBA1), glial fibrillary acidic protein (GFAP), and collagen IV. In the hippocampus of SARS-CoV-2 infected animals the TSPO+ area and number of TSPO+ cells were significantly increased compared to control animals. This increase was not cell type specific, since both the number of IBA1+TSPO+ and GFAP+TSPO+ cells was increased, as well as the TSPO+ area within collagen IV+ blood vessels. CONCLUSIONS: This study manifests [18F]DPA714 as a powerful radiotracer to visualize SARS-CoV-2 induced neuroinflammation. The increased uptake of [18F]DPA714 over time implies an active neuroinflammatory response following SARS-CoV-2 infection. This inflammatory signal coincides with an increased number of TSPO expressing cells, including glial and endothelial cells, suggesting neuroinflammation and vascular dysregulation. These results demonstrate the long-term neuroinflammatory response following a mild SARS-CoV-2 infection, which potentially precedes long-lasting neurological symptoms.

Long-acting reversible contraception with etonogestrel implants in female macaques (Macaca mulatta and Macaca fascicularis).

Introduction: Contraception is often required for management and population control purposes in group-housed and free-roaming non-human primates. Long-acting reversible contraceptives, including subdermal progestin-releasing implants, are preferred as they eliminate challenges associated with frequent administration. Etonogestrel (ENG)-releasing subdermal implants are reversible and long-acting for a minimum of 3 years, and are commercially available for human use as Implanon® or Nexplanon®. Methods: A retrospective analysis was performed detailing the contraceptive effectiveness and reversibility of subdermal placement of one-fourth or one-third of an ENG implant (68 mg/implant) in 129 female rhesus macaques (Macaca mulatta) and 67 cynomolgus macaques (Macaca fascicularis) at the Biomedical Primate Research Centre (Rijswijk, Netherlands). Furthermore, single cross-sectional ENG serum concentrations were measured for 16 rhesus and 10 cynomolgus macaques, and hemoglobin and blood chemistry pre-ENG and at timepoints >0.5, >1.5, and > 2.5 years post-ENG insertion were evaluated for 24 rhesus macaques. Finally, data were obtained using trans-abdominal ultrasound regarding the influence of ENG on uterine volume and endometrial thickness in 14 rhesus and 11 cynomolgus macaques. Results: As a contraceptive ENG was in 99.80% (CI 93.50-99.99) and 99.95% (CI 99.95-100) effective in rhesus and cynomolgus macaques, respectively. Prolonged ENG durations of implant use in 14 rhesus macaques (range 3.1-5.0 years) and eight cynomolgus macaques (range 3.2-4.0 years) resulted in no unintended pregnancies. A total of 17 female macaques were allowed to breed after ENG removal, and among them, 14 female macaques (82%) had an uneventful delivery. Serum ENG concentrations with a median ENG duration of 1.2 years (range 0.1-6.0 years) and 1.9 years (range 0.6-4.7 years) resulted in median concentrations of 112 pg./mL (range 0-305 pg./mL) and 310 pg./mL (range 183-382 pg./mL) for rhesus and cynomolgus macaques, respectively. ENG had no clinical effect on hemoglobin and blood chemistry parameters nor on the thickness of the endometrial lining or uterus volume. Conclusion: This study indicates that both one-fourth and one-third of the ENG implants are effective, long-acting, reversible, and safe contraceptive to use in macaques.

Novel application of [18F]DPA714 for visualizing the pulmonary inflammation process of SARS-CoV-2-infection in rhesus monkeys (Macaca mulatta).

RATIONALE: The aim of this study was to investigate the application of [18F]DPA714 to visualize the inflammation process in the lungs of SARS-CoV-2-infected rhesus monkeys, focusing on the presence of pulmonary lesions, activation of mediastinal lymph nodes and surrounded lung tissue. METHODS: Four experimentally SARS-CoV-2 infected rhesus monkeys were followed for seven weeks post infection (pi) with a weekly PET-CT using [18F]DPA714. Two PET images, 10 min each, of a single field-of-view covering the chest area, were obtained 10 and 30 min after injection. To determine the infection process swabs, blood and bronchoalveolar lavages (BALs) were obtained. RESULTS: All animals were positive for SARS-CoV-2 in both the swabs and BALs on multiple timepoints pi. The initial development of pulmonary lesions was already detected at the first scan, performed 2-days pi. PET revealed an increased tracer uptake in the pulmonary lesions and mediastinal lymph nodes of all animals from the first scan obtained after infection and onwards. However, also an increased uptake was detected in the lung tissue surrounding the lesions, which persisted until day 30 and then subsided by day 37-44 pi. In parallel, a similar pattern of increased expression of activation markers was observed on dendritic cells in blood. PRINCIPAL CONCLUSIONS: This study illustrates that [18F]DPA714 is a valuable radiotracer to visualize SARS-CoV-2-associated pulmonary inflammation, which coincided with activation of dendritic cells in blood. [18F]DPA714 thus has the potential to be of added value as diagnostic tracer for other viral respiratory infections.

Brain Inflammation and Intracellular α-Synuclein Aggregates in Macaques after SARS-CoV-2 Infection.

SARS-CoV-2 causes acute respiratory disease, but many patients also experience neurological complications. Neuropathological changes with pronounced neuroinflammation have been described in individuals after lethal COVID-19, as well as in the CSF of hospitalized patients with neurological complications. To assess whether neuropathological changes can occur after a SARS-CoV-2 infection, leading to mild-to-moderate disease, we investigated the brains of four rhesus and four cynomolgus macaques after pulmonary disease and without overt clinical symptoms. Postmortem analysis demonstrated the infiltration of T-cells and activated microglia in the parenchyma of all infected animals, even in the absence of viral antigen or RNA. Moreover, intracellular α-synuclein aggregates were found in the brains of both macaque species. The heterogeneity of these manifestations in the brains indicates the virus' neuropathological potential and should be considered a warning for long-term health risks, following SARS-CoV-2 infection.

Poxvirus MVA Expressing SARS-CoV-2 S Protein Induces Robust Immunity and Protects Rhesus Macaques From SARS-CoV-2.

Novel safe, immunogenic, and effective vaccines are needed to control the COVID-19 pandemic, caused by SARS-CoV-2. Here, we describe the safety, robust immunogenicity, and potent efficacy elicited in rhesus macaques by a modified vaccinia virus Ankara (MVA) vector expressing a full-length SARS-CoV-2 spike (S) protein (MVA-S). MVA-S vaccination was well tolerated and induced S and receptor-binding domain (RBD)-binding IgG antibodies and neutralizing antibodies against SARS-CoV-2 and several variants of concern. S-specific IFNγ, but not IL-4, -producing cells were also elicited. After SARS-CoV-2 challenge, vaccinated animals showed a significant strong reduction of virus loads in bronchoalveolar lavages (BAL) and decreased levels in throat and nasal mucosa. Remarkably, MVA-S also protected macaques from fever and infection-induced cytokine storm. Computed tomography and histological examination of the lungs showed reduced lung pathology in MVA-S-vaccinated animals. These findings favor the use of MVA-S as a potential vaccine for SARS-CoV-2 in clinical trials.

Medical imaging of pulmonary disease in SARS-CoV-2-exposed non-human primates.

Chest X-ray (CXR), computed tomography (CT), and positron emission tomography-computed tomography (PET-CT) are noninvasive imaging techniques widely used in human and veterinary pulmonary research and medicine. These techniques have recently been applied in studies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-exposed non-human primates (NHPs) to complement virological assessments with meaningful translational readouts of lung disease. Our review of the literature indicates that medical imaging of SARS-CoV-2-exposed NHPs enables high-resolution qualitative and quantitative characterization of disease otherwise clinically invisible and potentially provides user-independent and unbiased evaluation of medical countermeasures (MCMs). However, we also found high variability in image acquisition and analysis protocols among studies. These findings uncover an urgent need to improve standardization and ensure direct comparability across studies.

A Comparative Study of Chest CT With Lung Ultrasound After SARS-CoV-2 Infection in the Assessment of Pulmonary Lesions in Rhesus Monkeys (Macaca Mulatta).

Lung ultrasound (LUS) is a fast and non-invasive modality for the diagnosis of several diseases. In humans, LUS is nowadays of additional value for bedside screening of hospitalized SARS-CoV-2 infected patients. However, the diagnostic value of LUS in SARS-CoV-2 infected rhesus monkeys, with mild-to-moderate disease, is unknown. The aim of this observational study was to explore correlations of the LUS appearance of abnormalities with COVID-19-related lesions detected on computed tomography (CT). There were 28 adult female rhesus monkeys infected with SARS-CoV-2 included in this study. Chest CT and LUS were obtained pre-infection and 2-, 7-, and 14-days post infection. Twenty-five animals were sub-genomic PCR positive in their nose/throat swab for at least 1 day. CT images were scored based on the degree of involvement for lung lobe. LUS was scored based on the aeration and abnormalities for each part of the lungs, blinded to CT findings. Most common lesions observed on CT were ground glass opacities (GGOs) and crazy paving patterns. With LUS, confluent or multiple B-lines with or without pleural abnormalities were observed which is corresponding with GGOs on CT. The agreement between the two modalities was similar over the examination days. Pleural line abnormalities were clearly observed with LUS, but could be easily missed on CT. Nevertheless, due to the air interface LUS was not able to examine the complete volume of the lung. The sensitivity of LUS was high though the diagnostic efficacy for mild-to-moderate disease, as seen in macaques, was relatively low. This leaves CT the imaging modality of choice for diagnosis, monitoring, and longitudinal assessment of a SARS-CoV-2 infection in macaques.

The Post-Acute Phase of SARS-CoV-2 Infection in Two Macaque Species Is Associated with Signs of Ongoing Virus Replication and Pathology in Pulmonary and Extrapulmonary Tissues.

The post-acute phase of SARS-CoV-2 infection was investigated in rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis). During the acute phase of infection, SARS-CoV-2 was shed via the nose and throat, and viral RNA was occasionally detected in feces. This phase coincided with a transient change in systemic immune activation. Even after the alleged resolution of the infection, computed tomography (CT) and positron emission tomography (PET)-CT revealed pulmonary lesions and activated tracheobronchial lymph nodes in all animals. Post-mortem histological examination of the lung tissue revealed mostly marginal or resolving minimal lesions that were indicative of SARS-CoV-2 infection. Evidence for SARS-CoV-2-induced histopathology was also found in extrapulmonary tissue samples, such as conjunctiva, cervical, and mesenteric lymph nodes. However, 5-6 weeks after SARS-CoV-2 exposure, upon necropsy, viral RNA was still detectable in a wide range of tissue samples in 50% of the macaques and included amongst others the heart, the respiratory tract and surrounding lymph nodes, salivary gland, and conjunctiva. Subgenomic messenger RNA was detected in the lungs and tracheobronchial lymph nodes, indicative of ongoing virus replication during the post-acute phase. These results could be relevant for understanding the long-term consequences of COVID-19 in humans.

Effect of Housing Conditions on Cortisol and Body Fat Levels in Female Rhesus Macaques.

Macaques are among the most commonly used non-human primates in biomedical research. They are highly social animals, yet biomedical studies often require group-living animals to be pair-housed in a controlled environment. A change in environment causes only short-term stress in adapting individuals, while non-adapting animals may experience long-term stress that can adversely affect study results. Individuals likely differ in their ability to adapt depending on individual characteristics. Changes in cortisol and body fat levels may reflect these different individual responses. Here, we investigate the long-term effect of a change from group- to pair-housing on cortisol and body fat levels in 32 female rhesus macaques, exploring whether age, dominance rank, original cortisol, and body fat levels are related to long-term stress in pair-housing. Hair samples were analyzed for cortisol levels, while anthropometric measurements and computed tomography were performed to quantify body fat. Monkeys served as their own control with a 7.5-month period between the measurements. Cortisol levels increased, while average body fat levels did not differ when individuals were moved from group- to pair-housing. Cortisol and body fat levels were not significantly correlated. Changes in cortisol were independent of age and dominance rank, whereas individual variation in body fat alterations was related to the group-housed body fat level and dominance rank. Although this study did not identify individual characteristics related to long-term stress in pair-housing, the individual variation confirms that some individuals are more resilient to change than others and provides possibilities for future refinement studies.

Refined acquisition of high-resolution chest CTs in macaques by free breathing.

The use of medical imaging as a non-invasive or minimally invasive method to assess disease or treatment response continues to grow. A similar trend is observed in pre-clinical research, in general, and more specifically in macaques, enabling longitudinal assessment of disease in individual animals. Computed tomography (CT) is such an imaging technique used to obtain clinically applicable data. To acquire a chest CT using a cone beam tomography system, some kind of respiration control is needed. A commonly used technique for this is endotracheal intubation and mechanical ventilation. However, although routinely performed this can increase the risk of impact on welfare in comparison with non-invasive imaging. Therefore, we studied the option of retrospectively gated CTs: acquiring high resolution chest CTs in freely breathing macaques. For this, we compared 748 CTs obtained during free breathing with 881 CTs obtained with mechanical ventilation in combination with a breath-hold procedure predominantly on the appearance of misregistration artifacts. The scans were obtained during different stages of multiple experimentally induced respiratory diseases. The comparison shows that although there are still streaking artifacts present in the retrospective gated scans, the amount of shading artifacts is reduced to such a level that it possibly dominates underlying lesions, causing misdiagnosis. Our data reveal that the use of retrospective gating in high resolution CTs for macaques can be successfully applied. With the use of this technique, artifacts due to free breathing are reduced to a diagnostically appropriate level. Most importantly, this technique makes chest CTs with this instrumentation a non-invasive modality.

Bronchoalveolar lavage affects thorax computed tomography of healthy and SARS-CoV-2 infected rhesus macaques (Macaca mulatta).

Medical imaging as method to assess the longitudinal process of a SARS-CoV-2 infection in non-human primates is commonly used in research settings. Bronchoalveolar lavage (BAL) is regularly used to determine the local virus production and immune effects of SARS-CoV-2 in the lower respiratory tract. However, the potential interference of those two diagnostic modalities is unknown in non-human primates. The current study investigated the effect and duration of BAL on computed tomography (CT) in both healthy and experimentally SARS-CoV-2-infected female rhesus macaques (Macaca mulatta). In addition, the effect of subsequent BALs was reviewed. Thorax CTs and BALs were obtained from four healthy animals and 11 experimentally SARS-CoV-2-infected animals. From all animals, CTs were obtained just before BAL, and 24 hours post-BAL. Additionally, from the healthy animals, CTs immediately after, and four hours post-BAL were obtained. Thorax CTs were evaluated for alterations in lung density, measured in Hounsfield units, and a visual semi-quantitative scoring system. An increase in the lung density was observed on the immediately post-BAL CT but resolved within 24 hours in the healthy animals. In the infected animals, a significant difference in both the lung density and CT score was still found 24 hours after BAL. Furthermore, the differences between time points in CT score were increased for the second BAL. These results indicate that the effect of BAL on infected lungs is not resolved within the first 24 hours. Therefore, it is important to acknowledge the interference between BAL and CT in rhesus macaques.

Immunogenicity and efficacy of one and two doses of Ad26.COV2.S COVID vaccine in adult and aged NHP.

Safe and effective coronavirus disease-19 (COVID-19) vaccines are urgently needed to control the ongoing pandemic. While single-dose vaccine regimens would provide multiple advantages, two doses may improve the magnitude and durability of immunity and protective efficacy. We assessed one- and two-dose regimens of the Ad26.COV2.S vaccine candidate in adult and aged nonhuman primates (NHPs). A two-dose Ad26.COV2.S regimen induced higher peak binding and neutralizing antibody responses compared with a single dose. In one-dose regimens, neutralizing antibody responses were stable for at least 14 wk, providing an early indication of durability. Ad26.COV2.S induced humoral immunity and T helper cell (Th cell) 1-skewed cellular responses in aged NHPs that were comparable to those in adult animals. Aged Ad26.COV2.S-vaccinated animals challenged 3 mo after dose 1 with a SARS-CoV-2 spike G614 variant showed near complete lower and substantial upper respiratory tract protection for both regimens. Neutralization of variants of concern by NHP sera was reduced for B.1.351 lineages while maintained for the B.1.1.7 lineage independent of Ad26.COV2.S vaccine regimen.

Aerosolized Exposure to H5N1 Influenza Virus Causes Less Severe Disease Than Infection via Combined Intrabronchial, Oral, and Nasal Inoculation in Cynomolgus Macaques.

Infection with highly pathogenic avian H5N1 influenza virus in humans often leads to severe respiratory disease with high mortality. Experimental infection in non-human primates can provide additional insight into disease pathogenesis. However, such a model should recapitulate the disease symptoms observed in humans, such as pneumonia and inflammatory cytokine response. While previous studies in macaques have demonstrated the occurrence of typical lesions in the lungs early after infection and a high level of immune activation, progression to severe disease and lethality were rarely observed. Here, we evaluated a routinely used combined route of infection via intra-bronchial, oral, and intra-nasal virus inoculation with aerosolized H5N1 exposure, with or without the regular collection of bronchoalveolar lavages early after infection. Both combined route and aerosol exposure resulted in similar levels of virus replication in nose and throat and similar levels of immune activation, cytokine, and chemokine release in the blood. However, while animals exposed to H5N1 by combined-route inoculation developed severe disease with high lethality, aerosolized exposure resulted in less lesions, as measured by consecutive computed tomography and less fever and lethal disease. In conclusion, not virus levels or immune activation, but route of infection determines fatal outcome for highly pathogenic avian H5N1 influenza infection.

Recommendations for Standardizing Thorax PET-CT in Non-Human Primates by Recent Experience from Macaque Studies.

Despite the possibilities of routine clinical measures and assays on readily accessible bio-samples, it is not always essential in animals to investigate the dynamics of disease longitudinally. In this regard, minimally invasive imaging methods provide powerful tools in preclinical research. They can contribute to the ethical principle of gathering as much relevant information per animal as possible. Besides, with an obvious parallel to clinical diagnostic practice, such imaging platforms are potent and valuable instruments leading to a more refined use of animals from a welfare perspective. Non-human primates comprise highly relevant species for preclinical research to enhance our understanding of disease mechanisms and/or the development of improved prophylactic or therapeutic regimen for various human diseases. In this paper, we describe parameters that critically affect the quality of integrated positron emission tomography and computed tomography (PET-CT) in non-human primates. Lessons learned are exemplified by results from imaging experimental infectious respiratory disease in macaques; specifically tuberculosis, influenza, and SARS-CoV-2 infection. We focus on the thorax and use of 18F-fluorodeoxyglucose as a PET tracer. Recommendations are provided to guide various stages of PET-CT-supported research in non-human primates, from animal selection, scan preparation, and operation, to processing and analysis of imaging data.

Evaluation of heterologous prime-boost vaccination strategies using chimpanzee adenovirus and modified vaccinia virus for TB subunit vaccination in rhesus macaques.

Tuberculosis (TB) still is the principal cause of death from infectious disease and improved vaccination strategies are required to reduce the disease burden and break TB transmission. Here, we investigated different routes of administration of vectored subunit vaccines based on chimpanzee-derived adenovirus serotype-3 (ChAd3) for homologous prime-boosting and modified vaccinia virus Ankara (MVA) for heterologous boosting with both vaccine vectors expressing the same antigens from Mycobacterium tuberculosis (Ag85B, ESAT6, Rv2626, Rv1733, RpfD). Prime-boost strategies were evaluated for immunogenicity and protective efficacy in highly susceptible rhesus macaques. A fully parenteral administration regimen was compared to exclusive respiratory mucosal administration, while parenteral ChAd3-5Ag prime-boosting and mucosal MVA-5Ag boosting were applied as a push-and-pull strategy from the periphery to the lung. Immune analyses corroborated compartmentalized responses induced by parenteral versus mucosal vaccination. Despite eliciting TB-specific immune responses, none of the investigational regimes conferred a protective effect by standard readouts of TB compared to non-vaccinated controls, while lack of protection by BCG underpinned the stringency of this non-human primate test modality. Yet, TB manifestation after full parenteral vaccination was significantly less compared to exclusive mucosal vaccination.

Fluorescence- and multispectral optoacoustic imaging for an optimized detection of deeply located tumors in an orthotopic mouse model of pancreatic carcinoma.

A crucial point for the management of pancreatic ductal adenocarcinoma (PDAC) is the decrease of R1 resections. Our aim was to evaluate the combination of multispectral optoacoustic tomography (MSOT) with fluorescence guided surgery (FGS) for diagnosis and perioperative detection of tumor nodules and resection margins in a xenotransplant mouse model of human pancreatic cancer. The peptide cRGD, conjugated with the near infrared fluorescent (NIRF) dye IRDye800CW and with a trans-cyclooctene (TCO) tag for future click chemistry (cRGD-800CW-TCO), was applied to PDAC bearing immunodeficient nude mice; 27 days after orthotopic transplantation of human AsPC-1 cells into the head of the pancreas, mice were injected with cRGD-800CW-TCO and imaged with fluorescence- and optoacoustic devices before and 2, 6 and 24 hr after injection, before they were sacrificed and dissected with a guidance of FGS imaging system. Fluorescence imaging of cRGD-800CW-TCO allowed detection of the tumor area but without information about the depth, whereas MSOT allowed high resolution 3 D identification of the tumor area, in particular of small tumor nodules. Highly sensitive delineation of tumor burden was achieved during FGS in all mice. Imaging of whole-mouse cryosections, histopathological analysis and NIRF microscopy confirmed the localization of cRGD-800CW-TCO within the tumor tissue. In principle, all imaging modalities applied here were able to detect PDAC in vivo. However, the combination of MSOT and FGS provided detailed spatial information of the signal and achieved a complete overview of the distribution and localization of cRGD-800CW-TCO within the tumor before and during surgical intervention.

Evaluation of EphA2 and EphB4 as Targets for Image-Guided Colorectal Cancer Surgery.

Targeted image-guided oncologic surgery (IGOS) relies on the recognition of cell surface-associated proteins, which should be abundantly present on tumor cells but preferably absent on cells in surrounding healthy tissue. The transmembrane receptor tyrosine kinase EphA2, a member of the A class of the Eph receptor family, has been reported to be highly overexpressed in several tumor types including breast, lung, brain, prostate, and colon cancer and is considered amongst the most promising cell membrane-associated tumor antigens by the NIH. Another member of the Eph receptor family belonging to the B class, EphB4, has also been found to be upregulated in multiple cancer types. In this study, EphA2 and EphB4 are evaluated as targets for IGOS of colorectal cancer by immunohistochemistry (IHC) using a tissue microarray (TMA) consisting of 168 pairs of tumor and normal tissue. The IHC sections were scored for staining intensity and percentage of cells stained. The results show a significantly enhanced staining intensity and more widespread distribution in tumor tissue compared with adjacent normal tissue for EphA2 as well as EphB4. Based on its more consistently higher score in colorectal tumor tissue compared to normal tissue, EphB4 appears to be a promising candidate for IGOS of colorectal cancer. In vitro experiments using antibodies on human colon cancer cells confirmed the possibility of EphB4 as target for imaging.

Traumatic Brain Injury: Preclinical Imaging Diagnostic(s) and Therapeutic Approaches.

BACKGROUND: Traumatic brain injury (TBI) is the result of an external physical force to the head that harms the brain. TBI is a major public health problem worldwide and mainly results from falls, vehicle accidents and violence. Clinical problem: The management of TBI, causing a wide spectrum of possible health outcomes, has barely changed over the years as encouraging outcomes from many pre-clinical therapeutic and pharmacological studies have only rarely been translated to the clinical situation. New management options: In the last decades management of TBI is rapidly advancing and new innovative imaging modalities with sophisticated treatment options by using nanomedicine based drug delivery systems are under investigation. Nano formulations such as PLGA, exosomes and liposomes have the advantage of a targeted and controlled delivery of their cargo, such as diagnostic probes and/or therapeutic drugs. SUMMARY: Here we provide an overview of new promising pre-clinical developments in TBI management that may find their way to the clinic in the near future. Nanotechnology and nanomedicine in TBI intervention may establish new platforms for targeted drug delivery to the traumatized brain to improve the quality of life and survival of TBI patients.

The Necrosis-Avid Small Molecule HQ4-DTPA as a Multimodal Imaging Agent for Monitoring Radiation Therapy-Induced Tumor Cell Death.

PURPOSE: Most effective antitumor therapies induce tumor cell death. Non-invasive, rapid and accurate quantitative imaging of cell death is essential for monitoring early response to antitumor therapies. To facilitate this, we previously developed a biocompatible necrosis-avid near-infrared fluorescence (NIRF) imaging probe, HQ4, which was radiolabeled with 111Indium-chloride (111In-Cl3) via the chelate diethylene triamine pentaacetic acid (DTPA), to enable clinical translation. The aim of the present study was to evaluate the application of HQ4-DTPA for monitoring tumor cell death induced by radiation therapy. Apart from its NIRF and radioactive properties, HQ4-DTPA was also tested as a photoacoustic imaging probe to evaluate its performance as a multimodal contrast agent for superficial and deep tissue imaging. MATERIALS AND METHODS: Radiation-induced tumor cell death was examined in a xenograft mouse model of human breast cancer (MCF-7). Tumors were irradiated with three fractions of 9 Gy each. HQ4-DTPA was injected intravenously after the last irradiation, NIRF and photoacoustic imaging of the tumors were performed at 12, 20, and 40 h after injection. Changes in probe accumulation in the tumors were measured in vivo, and ex vivo histological analysis of excised tumors was performed at experimental endpoints. In addition, biodistribution of radiolabeled [111In]DTPA-HQ4 was assessed using hybrid single-photon emission computed tomography-computed tomography (SPECT-CT) at the same time points. RESULTS: In vivo NIRF imaging demonstrated a significant difference in probe accumulation between control and irradiated tumors at all time points after injection. A similar trend was observed using in vivo photoacoustic imaging, which was validated by ex vivo tissue fluorescence and photoacoustic imaging. Serial quantitative radioactivity measurements of probe biodistribution further demonstrated increased probe accumulation in irradiated tumors. CONCLUSION: HQ4-DTPA has high specificity for dead cells in vivo, potentiating its use as a contrast agent for determining the relative level of tumor cell death following radiation therapy using NIRF, photoacoustic imaging and SPECT in vivo. Initial preclinical results are promising and indicate the need for further evaluation in larger cohorts. If successful, such studies may help develop a new multimodal method for non-invasive and dynamic deep tissue imaging of treatment-induced cell death to quantitatively assess therapeutic response in patients.

Pre-clinical Evaluation of a Cyanine-Based SPECT Probe for Multimodal Tumor Necrosis Imaging.

PURPOSE: Recently we showed that a number of carboxylated near-infrared fluorescent (NIRF) cyanine dyes possess strong necrosis avid properties in vitro as well as in different mouse models of spontaneous and therapy-induced tumor necrosis, indicating their potential use for cancer diagnostic- and prognostic purposes. In the previous study, the detection of the cyanines was achieved by whole body optical imaging, a technique that, due to the limited penetration of near-infrared light, is not suitable for investigations deeper than 1 cm within the human body. Therefore, in order to facilitate clinical translation, the purpose of the present study was to generate a necrosis avid cyanine-based NIRF probe that could also be used for single photon emission computed tomography (SPECT). For this, the necrosis avid NIRF cyanine HQ4 was radiolabeled with 111indium, via the chelate diethylene triamine pentaacetic acid (DTPA). PROCEDURES: The necrosis avid properties of the radiotracer [111In]DTPA-HQ4 were examined in vitro and in vivo in different breast tumor models in mice using SPECT and optical imaging. Moreover, biodistribution studies were performed to examine the pharmacokinetics of the probe in vivo. RESULTS: Using optical imaging and radioactivity measurements, in vitro, we showed selective accumulation of [111In]DTPA-HQ4 in dead cells. Using SPECT and in biodistribution studies, the necrosis avidity of the radiotracer was confirmed in a 4T1 mouse breast cancer model of spontaneous tumor necrosis and in a MCF-7 human breast cancer model of chemotherapy-induced tumor necrosis. CONCLUSIONS: The radiotracer [111In]DTPA-HQ4 possessed strong and selective necrosis avidity in vitro and in various mouse models of tumor necrosis in vivo, indicating its potential to be clinically applied for diagnostic purposes and to monitor anti-cancer treatment efficacy.