Corrigendum: Co-treatment of chloroquine and trametinib inhibits melanoma cell proliferation and decreases immune cell infiltration.

[This corrects the article DOI: 10.3389/fonc.2022.782877.].

Microglia at sites of atrophy restrict the progression of retinal degeneration via galectin-3 and Trem2.

Outer retinal degenerations, including age-related macular degeneration (AMD), are characterized by photoreceptor and retinal pigment epithelium (RPE) atrophy. In these blinding diseases, macrophages accumulate at atrophic sites, but their ontogeny and niche specialization remain poorly understood, especially in humans. We uncovered a unique profile of microglia, marked by galectin-3 upregulation, at atrophic sites in mouse models of retinal degeneration and human AMD. In disease models, conditional deletion of galectin-3 in microglia led to phagocytosis defects and consequent augmented photoreceptor death, RPE damage, and vision loss, indicating protective roles. Mechanistically, Trem2 signaling orchestrated microglial migration to atrophic sites and induced galectin-3 expression. Moreover, pharmacologic Trem2 agonization led to heightened protection but in a galectin-3-dependent manner. In elderly human subjects, we identified this highly conserved microglial population that expressed galectin-3 and Trem2. This population was significantly enriched in the macular RPE-choroid of AMD subjects. Collectively, our findings reveal a neuroprotective population of microglia and a potential therapeutic target for mitigating retinal degeneration.

Microglia at Sites of Atrophy Restrict the Progression of Retinal Degeneration via Galectin-3 and Trem2 Interactions.

Degenerative diseases of the outer retina, including age-related macular degeneration (AMD), are characterized by atrophy of photoreceptors and retinal pigment epithelium (RPE). In these blinding diseases, macrophages are known to accumulate ectopically at sites of atrophy, but their ontogeny and functional specialization within this atrophic niche remain poorly understood, especially in the human context. Here, we uncovered a transcriptionally unique profile of microglia, marked by galectin-3 upregulation, at atrophic sites in mouse models of retinal degeneration and in human AMD. Using disease models, we found that conditional deletion of galectin-3 in microglia led to defects in phagocytosis and consequent augmented photoreceptor death, RPE damage and vision loss, suggestive of a protective role. Mechanistically, Trem2 signaling orchestrated the migration of microglial cells to sites of atrophy, and there, induced galectin-3 expression. Moreover, pharmacologic Trem2 agonization led to heightened protection, but only in a galectin-3-dependent manner, further signifying the functional interdependence of these two molecules. Likewise in elderly human subjects, we identified a highly conserved population of microglia at the transcriptomic, protein and spatial levels, and this population was enriched in the macular region of postmortem AMD subjects. Collectively, our findings reveal an atrophy-associated specialization of microglia that restricts the progression of retinal degeneration in mice and further suggest that these protective microglia are conserved in AMD.

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice.

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p < 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths <5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

Co-Treatment of Chloroquine and Trametinib Inhibits Melanoma Cell Proliferation and Decreases Immune Cell Infiltration.

Autophagy is characterized as a cytoprotective process and inhibition of autophagy with medicinally active agents, such as chloroquine (CQ) is proposed as a prospective adjuvant therapy for cancer. Here, we examined the preclinical effects of CQ combined with the MEK inhibitor trametinib (TRA) on melanoma. We found that cotreatment of CQ and TRA markedly slowed melanoma growth induced in Tyr-CreER.BrafCa.Ptenfl/fl mice. Immunostaining showed that trametinib decreased Ki-67+ proliferating cells, and increased TUNEL+ apoptotic cells. The combo treatment induced a further decrease of Ki-67+ proliferating cells. Consistent with the in vivo findings, CQ and TRA inhibited melanoma cell proliferation in vitro, which was correlated by decreased cyclin D1 expression. In addition, we found that tissues treated with CQ and TRA had significantly decreased numbers of CD4+ and CD8+ T-lymphocytes and F4/80+ macrophages. Together, these results indicate that cotreatment of CQ and TRA decreases cancer cell proliferation, but also dampens immune cell infiltration. Further study is warranted to understand whether CQ-induced immune suppression inadvertently affects therapeutic benefits.

Computational 3D microscopy with optical coherence refraction tomography.

Optical coherence tomography (OCT) has seen widespread success as an in vivo clinical diagnostic 3D imaging modality, impacting areas including ophthalmology, cardiology, and gastroenterology. Despite its many advantages, such as high sensitivity, speed, and depth penetration, OCT suffers from several shortcomings that ultimately limit its utility as a 3D microscopy tool, such as its pervasive coherent speckle noise and poor lateral resolution required to maintain millimeter-scale imaging depths. Here, we present 3D optical coherence refraction tomography (OCRT), a computational extension of OCT which synthesizes an incoherent contrast mechanism by combining multiple OCT volumes, acquired across two rotation axes, to form a resolution-enhanced, speckle-reduced, refraction-corrected 3D reconstruction. Our label-free computational 3D microscope features a novel optical design incorporating a parabolic mirror to enable the capture of 5D plenoptic datasets, consisting of millimetric 3D fields of view over up to ±75° without moving the sample. We demonstrate that 3D OCRT reveals 3D features unobserved by conventional OCT in fruit fly, zebrafish, and mouse samples.

Potential Utility of Synthetic D-Lactate Polymers in Skin Cancer.

Increased breakdown of glucose through glycolysis in both aerobic and anaerobic conditions is a hallmark feature of mammalian cancer and leads to increased production of L-lactate. The high-level lactate present within the tumor microenvironment is reused as a crucial biofuel to support rapid cancer cell proliferation, survival, and immune evasion. Inhibitors that target the glycolysis process are being developed for cancer therapy. In this study, we report an approach of using synthetic D-lactate dimers to inhibit melanoma and squamous cell carcinoma cell proliferation and survival. We also provide in vivo evidence that intratumoral injection of D-lactate dimers induced an innate immune response and inhibited subcutaneous melanoma xenograft growth in immunodeficient mice. Our findings support a potential utility of D-lactate dimers in skin cancer treatment and therefore warrant further mechanistic studies.

ENTPD1 (CD39) Expression Inhibits UVR-Induced DNA Damage Repair through Purinergic Signaling and Is Associated with Metastasis in Human Cutaneous Squamous Cell Carcinoma.

UVR and immunosuppression are major risk factors for cutaneous squamous cell carcinoma (cSCC). Regulatory T cells promote cSCC carcinogenesis, and in other solid tumors, infiltrating regulatory T cells and CD8+ T cells express ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) (also known as CD39), an ectoenzyme that catalyzes the rate-limiting step in converting extracellular adenosine triphosphate (ATP) to extracellular adenosine (ADO). We previously showed that extracellular purine nucleotides influence DNA damage repair. In this study, we investigate whether DNA damage repair is modulated through purinergic signaling in cSCC. We found increased ENTPD1 expression on T cells within cSCCs when compared with the expression on T cells from blood or nonlesional skin, and accordingly, concentrations of derivative extracellular adenosine diphosphate (ADP), adenosine monophosphate (AMP), and ADO are increased in tumors compared with those in normal skin. Importantly, ENTPD1 expression is significantly higher in human cSCCs that metastasize than in those that are nonmetastatic. We also identify in a mouse model that ENTPD1 expression is induced by UVR in an IL-27-dependent manner. Finally, increased extracellular ADO is shown to downregulate the expression of NAP1L2, a nucleosome assembly protein we show to be important for DNA damage repair secondary to UVR. Together, these data suggest a role for ENTPD1 expression on skin-resident T cells to regulate DNA damage repair through purinergic signaling to promote skin carcinogenesis and metastasis.

CVN-AD Alzheimer's mice show premature reduction in neurovascular coupling in response to spreading depression and anoxia compared to aged controls.

We compared the efficacy of neurovascular coupling and substrate supply in cerebral cortex during severe metabolic challenges in transgenic Alzheimer's [CVN-AD] and control [C57Bl/6] mice, to evaluate the hypothesis that metabolic insufficiency is a critical component of degeneration leading to dementia. We analyzed cerebral blood flow and metabolic responses to spreading depression (induced by K+ applied to the cortex) and anoxia across aging in CVN-AD + C57Bl/6 genotypes. In the CVN-AD genotype progression to histological and cognitive hallmarks of dementia is a stereotyped function of age. We correlated physiology and imaging of the cortex with the blood flow responses measured with laser doppler probes. The results show that spreading depression resulted in a hyperemic blood flow response that was dramatically reduced (24% in amplitude, 70% in area) in both middle-aged and aged CVN-AD mice compared to C57Bl/6 age-matched controls. However, spreading depression amplitude and conduction velocity (≈6 mm/min) did not differ among groups. Anoxia (100% N2 ) showed significantly decreased (by 62%) reactive blood flow and autoregulation in aged AD-CVN mice compared to aged control animals. Significantly reduced neurovascular coupling occurred prematurely with aging in CVN-AD mice. Abbreviated physiological hyperemia and decreased resilience to anoxia may enhance early-onset metabolic deficiency through decreased substrate supply to the brain. Metabolic deficiency may contribute significantly to the degeneration associated with dementia as a function of aging and regions of the brain involved.

Rapid, Dose-Dependent Enhancement of Cerebral Blood Flow by transcranial AC Stimulation in Mouse.

BACKGROUND: Transcranial electrical stimulation at an appropriate dose may demonstrate intracranial effects, including neuronal stimulation and cerebral blood flow responses. OBJECTIVE: We performed in vivo experiments on mouse cortex using transcranial alternating current [AC] stimulation to assess whether cerebral blood flow can be reliably altered by extracranial stimulation. METHODS: We performed transcranial AC electrical stimulation transversely across the closed skull in anesthetized mice, measuring transcranial cerebral blood flow with a laser Doppler probe and intracranial electrical responses as endpoint biomarkers. We calculated a stimulation dose-response function between intracranial electric field and cerebral blood flow. RESULTS: Stimulation at electric field amplitudes of 5-20 mV/mm at 10-20 Hz rapidly increased cerebral blood flow (within 100 ms), which then quickly decreased with no residual effects. The time to peak and blood flow shape varied with stimulation intensity and duration, showing a linear correlation between stimulation dose and peak blood flow increase. Neither afterdischarges nor spreading depression occurred from this level of stimulation. CONCLUSIONS: Extracranial stimulation amplitudes sufficient to evoke reliable blood flow changes require electric field strengths higher than what is tolerable in unanesthetized humans (<1 mV/mm), but less than electroconvulsive therapy levels (>40 mV/mm). However, anesthesia effects, spontaneous blood flow fluctuations, and sampling error may accentuate the apparent field strength needed for enhanced blood flow. The translation to a human dose-response function to augment cerebral blood flow (i.e., in stroke recovery) will require significant modification, potentially to pericranial, focused, multi-electrode application or intracranial stimulation.

Quantitative 129Xe MRI detects early impairment of gas-exchange in a rat model of pulmonary hypertension.

Hyperpolarized 129Xe magnetic resonance imaging (MRI) is capable of regional mapping of pulmonary gas-exchange and has found application in a wide range of pulmonary disorders in humans and animal model analogs. This study is the first application of 129Xe MRI to the monocrotaline rat model of pulmonary hypertension. Such models of preclinical pulmonary hypertension, a disease of the pulmonary vasculature that results in right heart failure and death, are usually assessed with invasive procedures such as right heart catheterization and histopathology. The work here adapted from protocols from clinical 129Xe MRI to enable preclinical imaging of rat models of pulmonary hypertension on a Bruker 7 T scanner. 129Xe spectroscopy and gas-exchange imaging showed reduced 129Xe uptake by red blood cells early in the progression of the disease, and at a later time point was accompanied by increased uptake by barrier tissues, edema, and ventilation defects-all of which are salient characteristics of the monocrotaline model. Imaging results were validated by H&E histology, which showed evidence of remodeling of arterioles. This proof-of-concept study has demonstrated that hyperpolarized 129Xe MRI has strong potential to be used to non-invasively monitor the progression of pulmonary hypertension in preclinical models and potentially to also assess response to therapy.

Direct detection of brown adipose tissue thermogenesis in UCP1-/- mice by hyperpolarized 129Xe MR thermometry.

Brown adipose tissue (BAT) is a type of fat specialized in non-shivering thermogenesis. While non-shivering thermogenesis is mediated primarily by uncoupling protein 1 (UCP1), the development of the UCP1 knockout mouse has enabled the study of possible UCP1-independent non-shivering thermogenic mechanisms, whose existence has been shown so far only indirectly in white adipose tissue and still continues to be a matter of debate in BAT. In this study, by using magnetic resonance thermometry with hyperpolarized xenon, we produce the first direct evidence of UCP1-independent BAT thermogenesis in knockout mice. We found that, following adrenergic stimulation, the BAT temperature of knockout mice increases more and faster than rectal temperature. While with this study we cannot exclude or separate the physiological effect of norepinephrine on core body temperature, the fast increase of iBAT temperature seems to suggest the existence of a possible UCP1-independent thermogenic mechanism responsible for this temperature increase.

Induction of hair follicle neogenesis with cultured mouse dermal papilla cells in de novo regenerated skin tissues.

De novo skin regeneration with human keratinocytes amplified in culture is a life-saving procedure for patients with extensive skin loss and chronic wounds. It also provides a valuable platform for gene function and therapeutic assessments. Nevertheless, tissues generated in this manner lack hair follicles that are important for skin homeostasis, barrier function, and repair. In this study, we generated skin tissues with human keratinocytes combined with dermal papilla (DP) cells isolated from mouse whisker hair. For this, cultured keratinocytes and mouse DP (mDP) cells were mixed at 10:1 ratio and seeded onto devitalized human dermal matrix derived from surgically discarded human abdominoplasty skin. After 1 week in submerged culture, the cell/matrix composites were grafted onto the skin wound beds of immunocompromised NSG.SCID mice. Histological analysis of 6-week-old skin grafts showed that tissues generated with the addition of mDP cells contained Sox2-positive dermal condensates and well-differentiated folliculoid structures that express human keratinocyte markers. These results indicate that cultured mDP cells can induce hair follicle neogenesis in the de novo regenerated skin tissues. Our method offers a new experimental system for mechanistic studies of hair follicle morphogenesis and tissue regeneration and provides insights to solving an important clinical challenge in generation of fully functional skin with a limited source of donor cells.

Unraveling the molecular nature of melanin changes in metastatic cancer.

More people die from melanoma after a stage I diagnosis than after a stage IV diagnosis, because the tools available to clinicians do not readily identify which early-stage cancers will be aggressive. Near-infrared pump-probe microscopy detects fundamental differences in melanin structure between benign human moles and melanoma and also correlates with metastatic potential. However, the biological mechanisms of these changes have been difficult to quantify, as many different mechanisms can contribute to the pump-probe signal. We use model systems (sepia, squid, and synthetic eumelanin), cellular uptake studies, and a range of pump and probe wavelengths to demonstrate that the clinically observed effects come from alterations of the aggregated mode from "thick oligomer stacks" to "thin oligomer stacks" (due to changes in monomer composition) and (predominantly) deaggregation of the assembled melanin structure. This provides the opportunity to use pump-probe microscopy for the detection and study of melanin-associated diseases.

Calibration of methylene-referenced lipid-dissolved xenon frequency for absolute MR temperature measurements.

PURPOSE: Absolute MR temperature measurements are currently difficult because they require precalibration procedures specific for tissue types and conditions. Reference of the lipid-dissolved 129 Xe resonance frequency to temperature-insensitive methylene protons (rLDX) has been proposed to remove the effect of macro- and microscopic susceptibility gradients to obtain absolute temperature information. The scope of this work is to evaluate the rLDX chemical shift (CS) dependence on lipid composition to estimate the precision of absolute temperature measurements in lipids. METHODS: Neat triglycerides, vegetable oils, and samples of freshly excised human and rodent adipose tissue (AT) are prepared under 129 Xe atmosphere and studied using high-resolution NMR. The rLDX CS is measured as a function of temperature. 1 H spectra are also acquired and the consistency of methylene-referenced water proton and rLDX CS values are compared in human AT. RESULTS: Although rLDX CS shows a dependence on lipid composition, in human and rodent AT samples the rLDX shows consistent CS values with a similar temperature dependence (-0.2058 ± 0.0010) ppm/°C × T (°C) + (200.15 ± 0.03) ppm, enabling absolute temperature measurements with an accuracy of 0.3°C. Methylene-referenced water CS values present variations of up to 4°C, even under well-controlled conditions. CONCLUSIONS: The rLDX can be used to obtain accurate absolute temperature measurements in AT, opening new opportunities for hyperpolarized 129 Xe MR to measure tissue absolute temperature.

Optical coherence refraction tomography.

Optical coherence tomography (OCT) is a cross-sectional, micron-scale imaging modality with widespread clinical application. Typical OCT systems sacrifice lateral resolution to achieve long depths of focus for bulk tissue imaging, and hence tend to have better axial than lateral resolution. Such anisotropic resolution can obscure fine ultrastructural features. Furthermore, conventional OCT suffers from refraction-induced image distortions. Here, we introduce optical coherence refraction tomography (OCRT), which extends the superior axial resolution to the lateral dimension, synthesising undistorted cross-sectional image reconstructions from multiple conventional images acquired with angular diversity. In correcting refraction-induced distortions to register the OCT images, OCRT also achieves spatially resolved refractive index imaging. We demonstrate >3-fold improvement in lateral resolution as well as speckle reduction in imaging tissue ultrastructure, consistent with histology. With further optimisation in optical designs to incorporate angular diversity into clinical instruments, OCRT could be widely applied as an enhancement over conventional OCT.

UBE2N Promotes Melanoma Growth via MEK/FRA1/SOX10 Signaling.

UBE2N is a K63-specific ubiquitin conjugase linked to various immune disorders and cancer. Here, we demonstrate that UBE2N and its partners UBE2V1 and UBE2V2 are highly expressed in malignant melanoma. Silencing of UBE2N and its partners significantly decreased melanoma cell proliferation and subcutaneous tumor growth. This was accompanied by increased expression of E-cadherin, p16, and MC1R and decreased expression of melanoma malignancy markers including SOX10, Nestin, and ABCB5. Mass spectrometry-based phosphoproteomic analysis revealed that UBE2N loss resulted in distinct alterations to the signaling landscape: MEK/ERK signaling was impaired, FRA1 and SOX10 gene regulators were downregulated, and p53 and p16 tumor suppressors were upregulated. Similar to inhibition of UBE2N and MEK, silencing FRA1 decreased SOX10 expression and cell proliferation. Conversely, exogenous expression of active FRA1 increased pMEK and SOX10 expression, and restored anchorage-independent cell growth of cells with UBE2N loss. Systemic delivery of NSC697923, a small-molecule inhibitor of UBE2N, significantly decreased melanoma xenograft growth. These data indicate that UBE2N is a novel regulator of the MEK/FRA1/SOX10 signaling cascade and is indispensable for malignant melanoma growth. Our findings establish the basis for targeting UBE2N as a potential treatment strategy for melanoma.Significance: These findings identify ubiquitin conjugase UBE2N and its variant partners as novel regulators of MAPK signaling and potential therapeutic targets in melanoma. Cancer Res; 78(22); 6462-72. ©2018 AACR.

A portable ventilator with integrated physiologic monitoring for hyperpolarized 129Xe MRI in rodents.

Hyperpolarized (HP) 129Xe MRI is emerging as a powerful, non-invasive method to image lung function and is beginning to find clinical application across a range of conditions. As clinical implementation progresses, it becomes important to translate back to well-defined animal models, where novel disease signatures can be characterized longitudinally and validated against histology. To date, preclinical 129Xe MRI has been limited to only a few sites worldwide with 2D imaging that is not generally sufficient to fully capture the heterogeneity of lung disease. To address these limitations and facilitate broader dissemination, we report on a compact and portable HP gas ventilator that integrates all the gas-delivery and physiologic monitoring capabilities required for high-resolution 3D hyperpolarized 129Xe imaging. This ventilator is MR- and HP-gas compatible, driven by inexpensive microcontrollers and open source code, and allows for precise control of the tidal volume and breathing cycle in perorally intubated mice and rats. We use the system to demonstrate data acquisition over multiple breath-holds, during which lung motion is suspended to enable high-resolution 3D imaging of gas-phase and dissolved-phase 129Xe in the lungs. We demonstrate the portability and versatility of the ventilator by imaging a mouse model of lung cancer longitudinally at 2 Tesla, and a healthy rat at 7 Tesla. We also report the detection of subtle spectroscopic fluctuations in phase with the heart rate, superimposed onto larger variations stemming from the respiratory cycle. This ventilator was developed to facilitate duplication and gain broad adoption to accelerate preclinical 129Xe MRI research.

KIND1 Loss Sensitizes Keratinocytes to UV-Induced Inflammatory Response and DNA Damage.

Loss of function of KIND1, a cytoskeletal protein involved in ß1-integrin function, causes Kindler syndrome, a genetic disease characterized by skin fragility, photosensitivity, and increased risk of squamous cell carcinoma. Dysregulation of ß1-integrin underlies Kindler syndrome skin fragility. However, the mechanisms underlying squamous cell carcinoma susceptibility are unclear. Here, we demonstrate that gene silencing of KIND1 decreased keratinocyte proliferation and increased apoptosis in vitro and in skin grafts regenerated on mice, which was correlated with reduced cyclinB1. In addition, KIND1 loss sensitized keratinocytes to cytokine and UV-induced NF-κB and c-Jun N-terminal kinase activation and upregulation of CXCL10 and tumor necrosis factor-α. Moreover, KIND1 loss impaired DNA repair, as indicated by the increased detection of γH2AX and cyclobutane pyrimidine dimers 24 hours after UVB radiation. Genetic or pharmacological c-Jun N-terminal kinase inhibition and NF-κB inhibition markedly reduced cyclobutane pyrimidine dimers-positive cells. Further, we show that KIND1 was regulated by JunB at the transcriptional level and, like JunB, it was downregulated in human squamous cell carcinoma cells. Together, these results indicate that KIND1 is important not only for keratinocyte proliferation but also for the suppression of UV-induced inflammation and DNA damage. These latter findings support a tumor suppressor function for KIND1, and identify c-Jun N-terminal kinase and NF-κB as potential therapeutic targets for prevention of squamous cell carcinoma in patients with Kindler syndrome.

Establishing an accurate gas phase reference frequency to quantify 129 Xe chemical shifts in vivo.

PURPOSE: 129 Xe interacts with biological media to exhibit chemical shifts exceeding 200 ppm that report on physiology and pathology. Extracting this functional information requires shifts to be measured precisely. Historically, shifts have been reported relative to the gas-phase resonance originating from pulmonary airspaces. However, this frequency is not fixed-it is affected by bulk magnetic susceptibility, as well as Xe-N2 , Xe-Xe, and Xe-O2 interactions. In this study, we addressed this by introducing a robust method to determine the 0 ppm 129 Xe reference from in vivo data. METHODS: Respiratory-gated hyperpolarized 129 Xe spectra from the gas- and dissolved-phases were acquired in four mice at 2T from multiple axial slices within the thoracic cavity. Complex spectra were then fitted in the time domain to identify peaks. RESULTS: Gas-phase 129 Xe exhibited two distinct resonances corresponding to 129 Xe in conducting airways (varying from -0.6 ± 0.2 to 1.3 ± 0.3 ppm) and alveoli (relatively stable, at -2.2 ± 0.1 ppm). Dissolved-phase 129 Xe exhibited five reproducible resonances in the thorax at 198.4 ± 0.4, 195.5 ± 0.4, 193.9 ± 0.2, 191.3 ± 0.2, and 190.7 ± 0.3 ppm. CONCLUSION: The alveolar 129 Xe resonance exhibits a stable frequency across all mice. Therefore, it can provide a reliable in vivo reference frequency by which to characterize other spectroscopic shifts. Magn Reson Med 77:1438-1445, 2017. © 2016 International Society for Magnetic Resonance in Medicine.