Neural stem cells secreting bispecific T cell engager to induce selective antiglioma activity.

Glioblastoma (GBM) is the most lethal primary brain tumor in adults. No treatment provides durable relief for the vast majority of GBM patients. In this study, we've tested a bispecific antibody comprised of single-chain variable fragments (scFvs) against T cell CD3ε and GBM cell interleukin 13 receptor alpha 2 (IL13Rα2). We demonstrate that this bispecific T cell engager (BiTE) (BiTELLON) engages peripheral and tumor-infiltrating lymphocytes harvested from patients' tumors and, in so doing, exerts anti-GBM activity ex vivo. The interaction of BiTELLON with T cells and IL13Rα2-expressing GBM cells stimulates T cell proliferation and the production of proinflammatory cytokines interferon γ (IFNγ) and tumor necrosis factor α (TNFα). We have modified neural stem cells (NSCs) to produce and secrete the BiTELLON (NSCLLON). When injected intracranially in mice with a brain tumor, NSCLLON show tropism for tumor, secrete BiTELLON, and remain viable for over 7 d. When injected directly into the tumor, NSCLLON provide a significant survival benefit to mice bearing various IL13Rα2+ GBMs. Our results support further investigation and development of this therapeutic for clinical translation.

CAMSAP3 facilitates basal body polarity and the formation of the central pair of microtubules in motile cilia.

Synchronized beating of cilia on multiciliated cells (MCCs) generates a directional flow of mucus across epithelia. This motility requires a "9 + 2" microtubule (MT) configuration in axonemes and the unidirectional array of basal bodies of cilia on the MCCs. However, it is not fully understood what components are needed for central MT-pair assembly as they are not continuous with basal bodies in contrast to the nine outer MT doublets. In this study, we discovered that a homozygous knockdown mouse model for MT minus-end regulator calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), Camsap3tm1a/tm1a , exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination of Camsap3tm1a/tm1a mice revealed severe nasal airway blockage and abnormal ciliary morphologies in nasal MCCs. MCCs from different tissues exhibited defective synchronized beating and ineffective generation of directional flow likely underlying the PCD-like phenotypes. In normal mice, CAMSAP3 localized to the base of axonemes and at the basal bodies in MCCs. However, in Camsap3tm1a/tm1a , MCCs lacked CAMSAP3 at the ciliary base. Importantly, the central MT pairs were missing in the majority of cilia, and the polarity of the basal bodies was disorganized. These phenotypes were further confirmed in MCCs of Xenopus embryos when CAMSAP3 expression was knocked down by morpholino injection. Taken together, we identified CAMSAP3 as being important for the formation of central MT pairs, proper orientation of basal bodies, and synchronized beating of motile cilia.

Kidney-intrinsic factors determine the severity of ischemia/reperfusion injury in a mouse model of delayed graft function.

Delayed graft function due to transplant ischemia/reperfusion injury adversely affects up to 50% of deceased-donor kidney transplant recipients. However, key factors contributing to the severity of ischemia/reperfusion injury remain unclear. Here, using a clinically relevant mouse model of delayed graft function, we demonstrated that donor genetic background and kidney-intrinsic MyD88/Trif-dependent innate immunity were key determinants of delayed graft function. Functional deterioration of kidney grafts directly corresponded with the duration of cold ischemia time. The graft dysfunction became irreversible after cold ischemia time exceeded six hours. When cold ischemia time reached four hours, kidney grafts displayed histological features reflective of delayed graft function seen in clinical kidney transplantation. Notably, kidneys of B6 mice exhibited significantly more severe histological and functional impairment than kidneys of C3H or BALB/c mice, regardless of recipient strains or alloreactivities. Furthermore, allografts of B6 mice also showed an upregulation of IL-6, neutrophil gelatinase-associated lipocalin, and endoplasmic reticulum stress genes, as well as an increased influx of host neutrophils and memory CD8 T-cells. In contrast, donor MyD88/Trif deficiency inhibited neutrophil influx and decreased the expression of IL-6 and endoplasmic reticulum stress genes, along with improved graft function and prolonged allograft survival. Thus, kidney-intrinsic factors involving genetic characteristics and innate immunity serve as critical determinants of the severity of delayed graft function. This preclinical murine model allows for further investigations of the mechanisms underlying delayed graft function.

Cardiac MRI Myocardial Functional and Tissue Characterization Detects Early Cardiac Dysfunction in a Mouse Model of Chemotherapy-Induced Cardiotoxicity.

BACKGROUND: Doxorubicin and doxorubicin-trastuzumab combination chemotherapy have been associated with cardiotoxicity that eventually leads to heart failure and may limit dose-effective cancer treatment. Current diagnostic strategies rely on decreased ejection fraction (EF) to diagnose cardiotoxicity. PURPOSE: The aim of this study is to explore the potential of cardiac MR (CMR) imaging to identify imaging biomarkers in a mouse model of chemotherapy-induced cardiotoxicity. METHODS: A cumulative dose of 25 mg/kg doxorubicin was administered over three weeks using subcutaneous pellets (n = 9, Dox). Another group (n = 9) received same dose of Dox and a total of 10 mg/kg trastuzumab (DT). Mice were imaged at baseline, 5/6 weeks and 10 weeks post-treatment on a 7T MRI system. The protocol included short-axis cine MRI covering the left ventricle (LV) and mid-ventricular short-axis tissue phase mapping (TPM), pre- and post-contrast T1 mapping, T2 mapping and Displacement Encoding with Stimulated Echoes (DENSE) strain encoded MRI. EF, peak myocardial velocities, native T1, T2, extracellular volume (ECV), and myocardial strain were quantified. N = 7 mice were sacrificed for histopathologic assessment of apoptosis at 5/6 weeks. RESULTS: Global peak systolic longitudinal velocity was reduced at 5/6 weeks in Dox (0.6 ± 0.3 vs 0.9 ± 0.3, p = 0.02). In the Dox group, native T1 was reduced at 5/6 weeks (1.3 ± 0.2 ms vs 1.6 ± 0.2 ms, p = 0.02), and relatively normalized at week 10 (1.4 ± 0.1 ms vs 1.6 ± 0.2 ms, p > 0.99). There was no change in EF and other MRI parameters and histopathologic results demonstrated minimal apoptosis in all mice (~1-2 apoptotic cell/high power field), suggesting early-stage cardiotoxicity. CONCLUSIONS: In a mouse model of chemotherapy-induced cardiotoxicity using doxorubicin and trastuzumab, advanced CMR shows promise in identifying treatment-related decrease in myocardial velocity and native T1 prior to the onset of cardiomyocyte apoptosis and reduction of EF.

BOLD temporal variability differentiates wakefulness from anesthesia-induced unconsciousness.

Even though a number of findings, based on information content or information integration, are shown to define neural underpinnings characteristic of a conscious experience, the neurophysiological mechanism of consciousness is still poorly understood. Here, we investigated the brain activity and functional connectivity changes that occur in the isoflurane-anesthetized unconscious state in contrast to the awake state in rats (awake and/or anesthetized, n = 68 rats). We examined nine information measures previously shown to distinguish between conscious states: blood oxygen level-dependent (BOLD) variability, functional connectivity strength, modularity, weighted modularity, efficiency, clustering coefficient, small-worldness, and spatial and temporal Lempel-Ziv complexity measure. We also identified modular membership, seed-based network connectivity, and absolute and normalized power spectrums to assess the integrity of the BOLD functional networks between awake and anesthesia. fMRI BOLD variability and related absolute power were the only information measures significantly higher during the awake state compared with isoflurane anesthesia across animals, and with varying levels of anesthesia, after correcting for motion and respiration confounds. Thus, we conclude that, at least under the specific conditions examined here, global measures of information integration/sharing do not properly distinguish the anesthetized state from wakefulness, and heightened overall, global and local, BOLD variability is the most reliable determinant of conscious brain activity relative to isoflurane anesthesia. NEW & NOTEWORTHY Multiple metrics previously suggested to be able to distinguish between states of consciousness were compared, within and across rats in awake and isoflurane anesthesia-induced unconsciousness. All measures tested showed sensitivity to confounds, correcting for motion and for respiration changes due to anesthesia. Resting state local BOLD variability and the related absolute power were the only information measures that robustly differentiated wakefulness states. These results caution against the general applicability of global information measures in identifying levels of consciousness, thus challenging the popular concept that these measures reflect states of consciousness, and also pointing to local signal variability as a more reliable indicator of states of wakefulness.

Antigen-loaded dendritic cell migration: MR imaging in a pancreatic carcinoma model.

PURPOSE: To test the following hypotheses in a murine model of pancreatic cancer: (a) Vaccination with antigen-loaded iron-labeled dendritic cells reduces T2-weighted signal intensity at magnetic resonance (MR) imaging within peripheral draining lymph nodes ( LN lymph node s) and (b) such signal intensity reductions are associated with tumor size changes after dendritic cell vaccination. MATERIALS AND METHODS: The institutional animal care and use committee approved this study. Panc02 cells were implanted into the flanks of 27 C57BL/6 mice bilaterally. After tumors reached 10 mm, cell viability was evaluated, and iron-labeled dendritic cell vaccines were injected into the left hind footpad. The mice were randomly separated into the following three groups (n = 9 in each): Group 1 was injected with 1 million iron-labeled dendritic cells; group 2, with 2 million cells; and control mice, with 200 mL of phosphate-buffered saline. T1- and T2-weighted MR imaging of labeled dendritic cell migration to draining LN lymph node s was performed before cell injection and 6 and 24 hours after injection. The signal-to-noise ratio ( SNR signal-to-noise ratio ) of the draining LN lymph node s was measured. One-way analysis of variance ( ANOVA analysis of variance ) was used to compare Prussian blue-positive dendritic cell measurements in LN lymph node s. Repeated-measures ANOVA analysis of variance was used to compare in vivo T2-weighted SNR signal-to-noise ratio LN lymph node measurements between groups over the observation time points. RESULTS: Trypan blue assays showed no significant difference in mean viability indexes (unlabeled vs labeled dendritic cells, 4.32% ± 0.69 [standard deviation] vs 4.83% ± 0.76; P = .385). Thirty-five days after injection, the mean left and right flank tumor sizes, respectively, were 112.7 mm(2) ± 16.4 and 109 mm(2) ± 24.3 for the 1-million dendritic cell group, 92.2 mm(2) ± 9.9 and 90.4 mm(2) ± 12.8 for the 2-million dendritic cell group, and 193.7 mm(2) ± 20.9 and 189.4 mm(2) ± 17.8 for the control group (P = .0001 for control group vs 1-million cell group; P = .00007 for control group vs 2-million cell group). There was a correlation between postinjection T2-weighted SNR signal-to-noise ratio decreases in the left popliteal LN lymph node 24 hours after injection and size changes at follow-up for tumors in both flanks (R = 0.81 and R = 0.76 for left and right tumors, respectively). CONCLUSION: MR imaging approaches can be used for quantitative measurement of accumulated iron-labeled dendritic cell-based vaccines in draining LN lymph node s. The amount of dendritic cell-based vaccine in draining LN lymph node s correlates well with observed protective effects.

MR imaging enables measurement of therapeutic nanoparticle uptake in rat N1-S1 liver tumors after nanoablation.

PURPOSE: To test the hypothesis that magnetic resonance (MR) imaging can quantify intratumoral superparamagnetic iron oxide (SPIO) nanoparticle uptake after nanoablation. MATERIALS AND METHODS: SPIO nanoparticles functionalized with doxorubicin were synthesized. N1-S1 hepatomas were successfully induced in 17 Sprague-Dawley rats distributed into three dosage groups. Baseline tumor R2* values (the reciprocal of T2*) were determined using 7-tesla (T) MR imaging. After intravenous injection of SPIO nanoparticles, reversible electroporation (1,300 V/cm, 8 pulses, 100-µs pulse duration) was applied. Imaging of rats was performed to determine tumor R2* values after the procedure, and change in R2* (ΔR2*) was calculated. Inductively coupled plasma mass spectrometry was used to determine intratumoral iron (Fe) concentration after the procedure, which served as a proxy for SPIO nanoparticle uptake. Mean tumor Fe concentration [Fe] and ΔR2* for each subject were assessed for correlation with linear regression, and mean [Fe] for each dosage group was compared with analysis of variance. RESULTS: ΔR2* significantly correlated with tumor SPIO nanoparticle uptake after nanoablation (r = 0.50, P = .039). On average, each 0.1-ms(-1) increase in R2* corresponded to a 0.1394-mM increase in [Fe]. There was no significant difference in mean SPIO nanoparticle uptake among dosage groups (P = .57). CONCLUSIONS: Intratumoral SPIO nanoparticle uptake after nanoablation can be successfully quantified noninvasively with 7-T MR imaging. Imaging can be used as a method to estimate localized drug delivery after nanoablation.

Age-related loss of Notch3 underlies brain vascular contractility deficiencies, glymphatic dysfunction, and neurodegeneration in mice.

Vascular aging affects multiple organ systems, including the brain, where it can lead to vascular dementia. However, a concrete understanding of how aging specifically affects the brain vasculature, along with molecular readouts, remains vastly incomplete. Here, we demonstrate that aging is associated with a marked decline in Notch3 signaling in both murine and human brain vessels. To clarify the consequences of Notch3 loss in the brain vasculature, we used single-cell transcriptomics and found that Notch3 inactivation alters regulation of calcium and contractile function and promotes a notable increase in extracellular matrix. These alterations adversely impact vascular reactivity, manifesting as dilation, tortuosity, microaneurysms, and decreased cerebral blood flow, as observed by MRI. Combined, these vascular impairments hinder glymphatic flow and result in buildup of glycosaminoglycans within the brain parenchyma. Remarkably, this phenomenon mirrors a key pathological feature found in brains of patients with CADASIL, a hereditary vascular dementia associated with NOTCH3 missense mutations. Additionally, single-cell RNA sequencing of the neuronal compartment in aging Notch3-null mice unveiled patterns reminiscent of those observed in neurodegenerative diseases. These findings offer direct evidence that age-related NOTCH3 deficiencies trigger a progressive decline in vascular function, subsequently affecting glymphatic flow and culminating in neurodegeneration.

Brain activity studied with magnetic resonance imaging in awake rabbits.

We reviewed fMRI experiments from our previous work in conscious rabbits, an experimental preparation that is advantageous for measuring brain activation that is free of anesthetic modulation and which can address questions in a variety of areas in sensory, cognitive, and pharmacological neuroscience research. Rabbits do not struggle or move for several hours while sitting with their heads restrained inside the horizontal bore of a magnet. This greatly reduces movement artifacts in magnetic resonance (MR) images in comparison to other experimental animals such as rodents, cats, and monkeys. We have been able to acquire high-resolution anatomic as well as functional images that are free of movement artifacts during several hours of restraint. Results from conscious rabbit fMRI studies with whisker stimulation are provided to illustrate the feasibility of this conscious animal model for functional MRI and the reproducibility of data gained with it.

Fecal Microbiota Transfer Attenuates Gut Dysbiosis and Functional Deficits After Traumatic Brain Injury.

BACKGROUND: Traumatic brain injury (TBI) is an underrecognized public health threat. Survivors of TBI often suffer long-term neurocognitive deficits leading to the progressive onset of neurodegenerative disease. Recent data suggests that the gut-brain axis is complicit in this process. However, no study has specifically addressed whether fecal microbiota transfer (FMT) attenuates neurologic deficits after TBI. HYPOTHESIS: We hypothesized that fecal microbiota transfer would attenuate neurocognitive, anatomic, and pathologic deficits after TBI. METHODS: C57Bl/6 mice were subjected to severe TBI (n = 20) or sham-injury (n = 20) via an open-head controlled cortical impact. Post-injury, this cohort of mice underwent weekly oral gavage with a slurry of healthy mouse stool or vehicle alone beginning 1 h post-TBI followed by behavioral testing and neuropathologic analysis. 16S ribosomal RNA sequencing of fecal samples was performed to characterize gut microbial community structure pre- and post-injury. Zero maze and open field testing were used to evaluate post-traumatic anxiety, exploratory behavior, and generalized activity. 3D, contrast enhanced, magnetic resonance imaging was used to determine differences in cortical volume loss and white matter connectivity. Prior to euthanasia, brains were harvested for neuropathologic analysis. RESULTS: Fecal microbiome analysis revealed a large variance between TBI, and sham animals treated with vehicle, while FMT treated TBI mice had restoration of gut dysbiosis back to levels of control mice. Neurocognitive testing demonstrated a rescue of normal anxiety-like and exploratory behavior in TBI mice treated with FMT. FMT treated TBI mice spent a greater percentage of time (22%, P = 0.0001) in the center regions of the Open Field as compared to vehicle treated TBI mice (13%). Vehicle-treated TBI animals also spent less time (19%) in the open areas of zero maze than FMT treated TBI mice (30%, P = 0.0001). Comparing in TBI mice treated with FMT, MRI demonstrated a marked attenuation in ventriculomegaly (P < 0.002) and a significant change in fractional anisotropy (i.e., loss of white matter connectivity) (P < 0.0001). Histologic analysis of brain sections revealed a FMT- injury dependent interaction in the microglia/macrophage-specific ionized calcium-binding protein, Iba1 (P = 0.002). CONCLUSION: These data suggest that restoring a pre-injury gut microbial community structure may be a promising therapeutic intervention after TBI.

Diet-induced Alzheimer's-like syndrome in the rabbit.

INTRODUCTION: Although mouse models of Alzheimer's disease (AD) have increased our understanding of the molecular basis of the disease, none of those models represent late-onset Alzheimer's Disease which accounts for >90% of AD cases, and no therapeutics developed in the mouse (with the possible exceptions of aduhelm/aducanumab and gantenerumab) have succeeded in preventing or reversing the disease. This technology has allowed much progress in understanding the molecular basis of AD. To further enhance our understanding, we used wild-type rabbit (with a nearly identical amino acid sequence for amyloid as in humans) to model LOAD by stressing risk factors including age, hypercholesterolemia, and elevated blood glucose levels (BGLs), upon an ε3-like isoform of apolipoprotein. We report a combined behavioral, imaging, and metabolic study using rabbit as a non-transgenic model to examine effects of AD-related risk factors on cognition, intrinsic functional connectivity, and magnetic resonance-based biomarkers of neuropathology. METHODS: Aging rabbits were fed a diet enriched with either 2% cholesterol or 10% fat/30% fructose. Monthly tests of novel object recognition (NOR) and object location memory (OLM) were administered to track cognitive impairment. Trace eyeblink conditioning (EBC) was administered as a final test of cognitive impairment. Magnetic resonance imaging (MRI) was used to obtain resting state connectivity and quantitative parametric data (R2*). RESULTS: Experimental diets induced hypercholesterolemia or elevated BGL. Both experimental diets induced statistically significant impairment of OLM (but not NOR) and altered intrinsic functional connectivity. EBC was more impaired by fat/fructose diet than by cholesterol. Whole brain and regional R2* MRI values were elevated in both experimental diet groups relative to rabbits on the control diet. DISCUSSION: We propose that mechanisms underlying LOAD can be assessed by stressing risk factors for inducing AD and that dietary manipulations can be used to assess etiological differences in the pathologies and effectiveness of potential therapeutics against LOAD. In addition, non-invasive MRI in awake, non-anesthetized rabbits further increases the translational value of this non-transgenic model to study AD.

POSTINJURY FECAL MICROBIOME TRANSPLANT DECREASES LESION SIZE AND NEUROINFLAMMATION IN TRAUMATIC BRAIN INJURY.

ABSTRACT: Background: Traumatic brain injury (TBI) is an underrecognized public health threat. The constitutive activation of microglia after TBI has been linked to long-term neurocognitive deficits and the progression of neurodegenerative disease. Evolving evidence indicates a critical role for the gut-brain axis in this process. Specifically, TBI has been shown to induce the depletion of commensal gut bacteria. The resulting gut dysbiosis is associated with neuroinflammation and disease. Hypothesis: We hypothesized that fecal microbiota transplantation would attenuate microglial activation and improve neuropathology after TBI. Methods: C57Bl/6 mice were subjected to severe TBI (n = 10) or sham injury (n = 10) via an open-head controlled cortical impact. The mice underwent fecal microbiota transplantation (FMT) or vehicle alone via oral gavage once weekly for 4 weeks after injury. At 59 days after TBI, mice underwent three-dimensional, contrast-enhanced magnetic resonance imaging. Following imaging, mice were killed, brains harvested at 60 DPI, and CD45+ cells isolated via florescence-activated cell sorting. cDNA libraries were prepared using the 10x Genomics Chromium Single Cell 3' Reagent kit followed by sequencing on a HiSeq4000 instrument, and computational analysis was performed. Results: Fecal microbiota transplantation resulted in a >marked reduction of ventriculomegaly (P < 0.002) and preservation of white matter connectivity at 59 days after TBI (P < 0.0001). In addition, microglia from FMT-treated mice significantly reduced inflammatory gene expression and enriched pathways involving the heat-shock response compared with mice treated with vehicle alone. Conclusions: We hypothesized that restoring gut microbial community structure via FMT would attenuate microglial activation and reduce neuropathology after TBI. Our data demonstrated significant preservation of cortical volume and white matter connectivity after an injury compared with mice treated with vehicle alone. This preservation of neuroanatomy after TBI was associated with a marked reduction in inflammatory gene expression within the microglia of FMT-treated mice. Microglia from FMT-treated mice enriched pathways in the heat-shock response, which is known to play a neuroprotective role in TBI and other neurodegenerative disease processes.

PLG nanoparticles target fibroblasts and MARCO+ monocytes to reverse multiorgan fibrosis.

Systemic sclerosis (SSc) is a chronic, multisystem orphan disease with a highly variable clinical course, high mortality rate, and a poorly understood complex pathogenesis. We have identified an important role for a subpopulation of monocytes and macrophages characterized by surface expression of the scavenger receptor macrophage receptor with collagenous structure (MARCO) in chronic inflammation and fibrosis in SSc and in preclinical disease models. We show that MARCO+ monocytes and macrophages accumulate in lesional skin and lung in topographic proximity to activated myofibroblasts in patients with SSc and in the bleomycin-induced mouse model of SSc. Short-term treatment of mice with a potentially novel nanoparticle, poly(lactic-co-glycolic) acid (PLG), which is composed of a carboxylated, FDA-approved, biodegradable polymer and modulates activation and trafficking of MARCO+ inflammatory monocytes, markedly attenuated bleomycin-induced skin and lung inflammation and fibrosis. Mechanistically, in isolated cells in culture, PLG nanoparticles inhibited TGF-dependent fibrotic responses in vitro. Thus, MARCO+ monocytes are potent effector cells of skin and lung fibrosis and can be therapeutically targeted in SSc using PLG nanoparticles.

Preclinical Safety of a 3D-Printed Hydroxyapatite-Demineralized Bone Matrix Scaffold for Spinal Fusion.

STUDY DESIGN: Prospective, randomized, controlled preclinical study. OBJECTIVE: The objective of this study was to compare the host inflammatory response of our previously described hyperelastic, 3D-printed (3DP) hydroxyapatite (HA)-demineralized bone matrix (DBM) composite scaffold to the response elicited with the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) in a preclinical rat posterolateral lumbar fusion model. SUMMARY OF BACKGROUND DATA: Our group previously found that this 3D-printed HA-DBM composite material shows promise as a bone graft substitute in a preclinical rodent model, but its safety profile had yet to be assessed. METHODS: Sixty female Sprague-Dawley rats underwent bilateral posterolateral intertransverse lumbar spinal fusion using with the following implants: 1) type I absorbable collagen sponge (ACS) alone; 2) 10 µg rhBMP-2/ACS; or 3) the 3DP HA-DBM composite scaffold (n = 20). The host inflammatory response was assessed using magnetic resonance imaging, while the local and circulating cytokine expression levels were evaluated by enzyme-linked immunosorbent assays at subsequent postoperative time points (N = 5/time point). RESULTS: At both 2 and 5 days postoperatively, treatment with the HA-DBM scaffold produced significantly less soft tissue edema at the fusion bed site relative to rhBMP-2-treated animals as quantified on magnetic resonance imaging. At every postoperative time point evaluated, the level of soft tissue edema in HA-DBM-treated animals was comparable to that of the ACS control group. At 2 days postoperatively, serum concentrations of tumor necrosis factor-α and macrophage chemoattractant protein-1 were significantly elevated in the rhBMP-2 treatment group relative to ACS controls, whereas these cytokines were not elevated in the HA-DBM-treated animals. CONCLUSION: The 3D-printed HA-DBM composite induces a significantly reduced host inflammatory response in a preclinical spinal fusion model relative to rhBMP-2.Level of Evidence: N/A.

Low-level whole-brain radiation enhances theranostic potential of single-domain antibody fragments for human epidermal growth factor receptor type 2 (HER2)-positive brain metastases.

Background: Single-domain antibody fragments (aka VHH, ~ 13 kDa) are promising delivery systems for brain tumor theranostics; however, achieving efficient delivery of VHH to intracranial lesions remains challenging due to the tumor-brain barrier. Here, we evaluate low-dose whole-brain irradiation as a strategy to increase the delivery of an anti- human epidermal growth factor receptor type 2 (HER2) VHH to breast cancer-derived intracranial tumors in mice. Methods: Mice with intracranial HER2-positive BT474BrM3 tumors received 10-Gy fractionated cranial irradiation and were evaluated by noninvasive imaging. Anti-HER2 VHH 5F7 was labeled with 18F, administered intravenously to irradiated mice and controls, and PET/CT imaging was conducted periodically after irradiation. Tumor uptake of 18F-labeled 5F7 in irradiated and control mice was compared by PET/CT image analysis and correlated with tumor volumes. In addition, longitudinal dynamic contrast-enhanced MRI (DCE-MRI) was conducted to visualize and quantify the potential effects of radiation on tumor perfusion and permeability. Results: Increased 18F-labeled 5F7 intracranial tumor uptake was observed with PET in mice receiving cranial irradiation, with maximum tumor accumulation seen approximately 12 days post initial radiation treatment. No radiation-induced changes in HER2 expression were detected by Western blot, flow cytometry, or on tissue sections. DCE-MRI imaging demonstrated transiently increased tumor perfusion and permeability after irradiation, consistent with the higher tumor uptake of 18F-labeled anti-HER2 5F7 in irradiated mice. Conclusion: Low-level brain irradiation induces dynamic changes in tumor vasculature that increase the intracranial tumor delivery of an anti-HER2 VHH, which could facilitate the use of radiolabeled VHH to detect, monitor, and treat HER2-expressing brain metastases.

Detection of memory- and learning-related brain connectivity changes following trace eyeblink-conditioning using resting-state functional magnetic resonance imaging in the awake rabbit.

Animal imaging studies have the potential to further establish resting-state fMRI (rs-fMRI) and enable its validation for clinical use. The rabbit subjects used in this work are an ideal model system for studying learning and behavior and are also an excellent established test subject for awake scanning given their natural tolerance for restraint. We found that analysis of rs-fMRI conducted on a cohort of rabbits undergoing eyeblink conditioning can reveal functional brain connectivity changes associated with learning, and that rs-fMRI can be used to capture differences between subjects with different levels of cognitive performance. rs-fMRI sessions were conducted on a cohort of rabbits before and after trace eyeblink conditioning. MRI results were analyzed using independent component analysis (ICA) and network analysis. Behavioral data were collected with standard methods using an infrared reflective sensor aimed at the cornea to detect blinks. Behavioral results were analyzed, and a median split was used to create two groups of rabbits based on their performance. The cohort of rabbits undergoing eyeblink conditioning exhibited increased functional connectivity in the cingulate cortex, retrosplenial cortex, and thalamus consistent with brain reorganization associated with increased learning. Differences in the striatum and right cerebellum were also identified between rabbits in the top or bottom halves of the group as measured by the behavioral assay. Thus, rs-fMRI can provide not only a tool to detect and monitor functional brain changes associated with learning, but also to discriminate between different levels of cognitive performance.

Differential neuropathology and functional outcome after equivalent traumatic brain injury in aged versus young adult mice.

The CDC estimate that nearly 3 million Americans sustain a traumatic brain injury (TBI) each year. Even when medical comorbidities are accounted for, age is an independent risk factor for poor outcome after TBI. Nonetheless, few studies have examined the pathophysiology of age-linked biologic outcomes in TBI. We hypothesized that aged mice would demonstrate more severe neuropathology and greater functional deficits as compared to young adult mice after equivalent traumatic brain injuries. Young adult (14-week-old) and aged (80-week-old) C57BL/6 male mice underwent an open-head controlled cortical impact to induce TBI or a sham injury. At 30-days post-injury groups underwent behavioral phenotyping, magnetic resonance imaging, and histologic analyses. Contrary to our hypothesis, young adult TBI mice exhibited more severe neuropathology and greater loss of white matter connectivity as compared to aged mice after TBI. These findings correlated to differential functional outcomes in anxiety response, learning, and memory between young adult and aged mice after TBI. Although the mechanisms underlying this age-effect remain unclear, attenuated signs of secondary brain injury in aged TBI mice point towards different inflammatory and repair processes between age groups. These data suggest that age may need to be an a priori consideration in future clinical trial design.

Use of X-Ray Fluorescence Microscopy for Studies on Research Models of Hepatocellular Carcinoma.

Introduction: TheraSphere® microspheres containing yttrium 90Y are among many radioembolization agents used clinically to reduce liver tumor burden, and their effects on cancer volume reduction are well-established. At the same time, concerns about off target tissue injury often limit their use. Deeper investigation into tissue distribution and long-term impact of these microspheres could inform us about additional ways to use them in practice. Methods: Healthy rat liver and rabbit liver tumor samples from animals treated with TheraSpheres were sectioned and their elemental maps were generated by X-ray fluorescence microscopy (XFM) at the Advanced Photon Source (APS) synchrotron at Argonne National Laboratory (ANL). Results: Elemental imaging allowed us to identify the presence and distribution of TheraSpheres in animal tissues without the need for additional sample manipulation or staining. Ionizing radiation produced by 90Y radioactive contaminants present in these microspheres makes processing TheraSphere treated samples complex. Accumulation of microspheres in macrophages was observed. Conclusions: This is the first study that used XFM to evaluate the location of microspheres and radionuclides in animal liver and tumor samples introduced through radioembolization. XFM has shown promise in expanding our understanding of radioembolization and could be used for investigation of human patient samples in the future.

Activation of the dorsal, but not the ventral, hippocampus relieves neuropathic pain in rodents.

ABSTRACT: Accumulating evidence suggests hippocampal impairment under the chronic pain phenotype. However, it is unknown whether neuropathic behaviors are related to dysfunction of the hippocampal circuitry. Here, we enhanced hippocampal activity by pharmacological, optogenetic, and chemogenetic techniques to determine hippocampal influence on neuropathic pain behaviors. We found that excitation of the dorsal (DH), but not the ventral (VH) hippocampus induces analgesia in 2 rodent models of neuropathic pain (SNI and SNL) and in rats and mice. Optogenetic and pharmacological manipulations of DH neurons demonstrated that DH-induced analgesia was mediated by N-Methyl-D-aspartate and µ-opioid receptors. In addition to analgesia, optogenetic stimulation of the DH in SNI mice also resulted in enhanced real-time conditioned place preference for the chamber where the DH was activated, a finding consistent with pain relief. Similar manipulations in the VH were ineffective. Using chemo-functional magnetic resonance imaging (fMRI), where awake resting-state fMRI was combined with viral vector-mediated chemogenetic activation (PSAM/PSEM89s) of DH neurons, we demonstrated changes of functional connectivity between the DH and thalamus and somatosensory regions that tracked the extent of relief from tactile allodynia. Moreover, we examined hippocampal functional connectivity in humans and observe differential reorganization of its anterior and posterior subdivisions between subacute and chronic back pain. Altogether, these results imply that downregulation of the DH circuitry during chronic neuropathic pain aggravates pain-related behaviors. Conversely, activation of the DH reverses pain-related behaviors through local excitatory and opioidergic mechanisms affecting DH functional connectivity. Thus, this study exhibits a novel causal role for the DH but not the VH in controlling neuropathic pain-related behaviors.