Christensen, Trace

ABSTRACT The enteric nervous system (ENS) is a complex peripheral neural network that coordinates gastrointestinal motility through highly organized synaptic communication. Although tissue-resident muscularis macrophages (MMs) closely associate with enteric neurons, whether they regulate enteric synaptic organization remains unknown. In the central nervous system (CNS), microglia sculpt neural circuits through complement-dependent synaptic remodeling, raising the possibility that analogous neuroimmune mechanisms operate in the gut. Here, we identify a previously unrecognized role for C1qa□ MMs in regulating enteric synaptic homeostasis and gastrointestinal motility. Using macrophage-specific constitutive and inducible C1qa deletion models, single-cell RNA sequencing, enteric synaptosome proteomics, physiology, and advanced imaging, we demonstrate that loss of MMs-derived C1qa increases enteric synaptic density without altering neuronal numbers. C1qa deficiency induced broad transcriptional changes in enteric neurons and macrophages, including altered synapse-associated, lysosomal, and endocytic programs. Proteomic analysis revealed that enteric synapses share a conserved molecular architecture with brain synapses while exhibiting distinct gastrointestinal-specific complement-associated synaptic networks enriched for structural and receptor-localization pathways. Functionally, macrophage-specific C1qa deletion altered excitatory and inhibitory enteric neurotransmission, enhanced cholinergic signaling, reduced nitrergic responses, and accelerated gastrointestinal transit, while smooth muscle responsiveness remained preserved. C1qa□ MMs displayed transcriptional and functional features consistent with a phagocytic synapse-remodeling phenotype, including enrichment of complement, lysosomal, and engulfment pathways. Loss of C1qa impaired macrophage phagocytic activity both in vitro and in vivo and was associated with synapse accumulation and altered macrophage morphology. Importantly, inducible deletion of C1qa in adulthood recapitulated the synaptic and motility phenotypes, demonstrating that C1qa□ MMs continuously regulate enteric synaptic organization beyond development. Together, these findings identify a complement-dependent neuroimmune mechanism that regulates enteric circuit organization and gut motility, establishing MMs as active modulators of adult ENS synaptic homeostasis. Abstract Figure

In the aerodigestive system (ventilation, swallow, speech and airway defense manoeuvres), age-related neuromotor declines contribute to the increased risk of aspiration pneumonia, a leading cause of mortality in the elderly. In the Fischer 344 (F344) rat model, we and others have identified that brainstem hypoglossal motor neuron (MN) degenerations and death contribute to motor dyscoordination and tongue muscle weakness, respectively. In F344 rats, both MNs and striated muscle exhibit morphological and functional deficits of mitochondria in aging, consistent with elderly human post mortem results. However, to date the dendritic and axonal compartments have been relatively ignored. In young (6-months old; n=6 rats) and old (24-months-old; n=6 rats) female and male F344 hypoglossal motor axons were assessed using serial block face scanning electron microscopy (SDFEM). Within myelinated hypoglossal motor axons and hypoglossal MN dendrites, mitochondrial volume density and morphology (mitochondrial CSA, form factor and aspect ratios) were assessed. In old age, mitochondrial volume density was reduced in dendrites and, surprisingly, increased in axons. Mitochondrial fragmentation (reduced CSA, form factors and aspect ratios was apparent in dendrites. In axons, old age mitochondria were larger, but more simplistic. We interpret these dendritic findings to be consistent with altered neural network function in old age. In the axons, excessive mitochondria may physically impair the anterograde and retrograde transit along the axons. Our results suggest that a more nuanced compartment-specific evaluation of mitochondrial structure and function is required to fully elucidate the pathophysiology underlying age-related neuromotor dysfunction. Funding for this research was provided by National Institutes of Health (NIH) grant AG086136 (MJF). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Abstract Intravital imaging studies have provided insights into the spatial and temporal variations of platelet activation and thrombin generation that occur during hemostasis; however, these studies are generally limited to small vessels due to the practical limitations of imaging in thicker tissues. Recent advances in cleared tissue fluorescence imaging as well as volume electron microscopy (vEM) coupled with machine learning–based image segmentation provide an opportunity for analysis of the 3-dimensional structure of complex tissues. We utilized these technologies to examine hemostatic plugs from murine jugular veins and carotid arteries to investigate the spatial distribution of platelet activation and biochemical responses in these disparate physiologic contexts. Both venous and arterial hemostatic plugs had a heterogeneous structure with regions of sparsely and densely packed platelets. Despite similar injury sizes, arterial hemostatic plugs were at least an order of magnitude larger than venous plugs. The difference in plug size was primarily due to a 19-fold increase in the population of densely packed platelets in the extravascular compartment. Venous plugs displayed significant platelet aggregation extending into the vessel lumen and developed distinctive fibrin and red blood cell–filled cavities. Complementary fluorescence microscopy revealed that platelet activation was spatially heterogeneous in both contexts, with α-granule secretion and phosphatidylserine exposure confined to specific microenvironments, highlighting tightly regulated thrombin activity. Overall, our findings reveal both conserved and distinct mechanisms of hemostatic thrombus formation in different physiologic contexts. They also demonstrate the power of vEM coupled with machine learning–based image segmentation for the quantitative analysis of large imaging data sets from complex tissues.

Key Points Understanding how heme proteins/heme induce AKI and whether NAD + -boosting ameliorates heme protein–mediated AKI is clinically relevant. Heme proteins/heme directly decrease kidney NAD + content and NAD + -boosting is markedly protective in heme protein–mediated AKI. These findings provide new insights in heme protein–mediated AKI and support interest in NAD + -boosting as a therapeutic strategy in AKI. Background Understanding how heme proteins and heme induce AKI is clinically relevant from numerous perspectives, and, in this regard, a widely used model of heme protein–mediated AKI (HP-AKI) involves the glycerol model in mice. Using this model, we have previously demonstrated that NAD + content is decreased in HP-AKI. Because there is significant current interest in NAD + -boosting as a strategy in clinical AKI, we examined the effect of NAD + -boosting in this model. Methods NAD + -boosting was achieved by the administration of nicotinamide mononucleotide (NMN) in the glycerol model of HP-AKI. The effect of NMN, compared with vehicle, was examined in mice with HP-AKI and with sham AKI at day 1 after HP-AKI was induced. Results The administration of NMN preserved kidney NAD + content in HP-AKI, and compared with the administration of vehicle in HP-AKI, NMN improved glomerular filtration markers; reduced histologic injury as assessed by tubular necrosis, dilation, and cast formation and by assessment of dystrophic calcification; reduced expression of kidney injury marker 1, a sensitive marker of AKI; preserved mitochondrial ultrastructure and increased expression of proteins that promote mitochondrial integrity; reduced apoptosis as assessed by terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling staining and genes and protein expression that contribute to apoptosis, while increasing expression of an anti-apoptotic gene; reduced expression of several renal injury-related genes; and mitigated the severity of the senescence phenotype, as assessed by multiple markers. Finally, the administration of myoglobin or heme in vivo diminished kidney NAD + content in mice with intact kidneys. Conclusions These findings demonstrate the remarkable protective effects of NAD + -boosting by NMN in HP-AKI as revealed by a multitude of markers relevant to AKI. The reduction in NAD + content in the kidney in HP-AKI may reflect the direct effects of heme proteins and/or heme. We suggest that these findings support current interest in NAD + -boosting as a therapeutic strategy in clinical AKI.

Diabetic pregnancy increases the offspring’s risk of neonatal and adult cardiovascular disease (CVD). We previously used a rat model (Sprague–Dawley) to show that diabetic pregnancy impairs mitochondrial bioenergetics, dynamics, mitophagy, and quality control in the offspring’s heart, and we hypothesized that mitochondrial dysfunction during early development influences the adult myocardium structure to confer cardiometabolic disease risk with aging. Here, we used 3D serial block face-scanning electron microscopy (SBF-SEM) to analyze perinuclear (PN) and intrafibrillar (IF) mitochondrial networks in the left ventricular sections from control and pregestational diabetes-exposed newborn (NB) rats that were three-week-old and four-month-old. Diabetes-exposed myocardium had 50% fewer PN and 20% fewer IF mitochondria at birth but counts increased more rapidly, resulting in no difference at three weeks and 35% more PN and 49% more IF mitochondria by four months. Despite rising counts, mitochondria volumes remained significantly lower at every developmental timepoint. This shows that diabetic pregnancy causes maldevelopment of the myocardial mitochondrial reticulum which likely contributes to adult CVD.

In the elderly, age-associated muscle weakness and atrophy (sarcopenia) is prevalent in both limb and respiratory muscles. In the tongue muscles, dyscoordination and sarcopenia predisposes elderly individuals to dysphagia, dysphonia and a greater risk for aspiration pneumonias and obstructive sleep apnea. In humans and in the Fischer 344 rat model of aging, motor neuron (MN) death occurs in old age, and the selective vulnerability of fast-fatigable motor units to loss is consistent with behavioral deficits in airway defense (e.g., cough). However, whether the timing of MN loss is gradual across the lifespan or precipitous from late-middle-age is unknown. Recently, the concept of an age -related increase of systemic inflammatory cytokines has been implicated in the development of sarcopenia, although the mechanisms by which this occurs remain obscure. In other cell types, inflammation triggers an unfolded protein response and mitochondrial fragmentation – processes implicated in neuronal death and neurodegeneration. In this study of female and male Fischer 344 rats at 6-, 18- and 24-months-old, we evaluated serum inflammatory cytokines (TNFα, IL1β & IL6) via ELISA, medullary unfolded protein response (pIRE1a S724 & CDK5) and mitochondrial fragmentation (pDRP1 S616 ) via Western blot, hypoglossal MN mitochondrial volume density and mitochondrial fragmentation via serial blockface electron microscopy (SBFEM), hypoglossal MN survival via Nissl histology and stereological counting and tongue muscle (longitudinal and genioglossus) sarcopenia via uniaxial specific force assessment and cross-sectional muscle fiber areas. All data were analyzed by 1-way ANOVAs or Kruskal-Wallis nonparametric tests, depending on data distributions. Here, we show that a systemic inflammation-induced unfolded protein response in the medulla and hypoglossal MNs mitochondrial fragmentation is present at 18-months-old. This mitochondrial fragmentation is consistent with increased medullary expression of pDRP1 S616 . At this age, we did not observe any evidence of tongue muscle weakness or atrophy, nor any evidence of hypoglossal MN death. At 24-months old, hypoglossal MN mitochondrial deficits remained readily observable using SBFEM, with hypoglossal MN death (specific to larger MNs) and tongue muscle sarcopenia (selective to fast-fatigable type IIx fibers) present at this age. Overall, we conclude that denervation-related sarcopenia is precipitous from late-middle-age to old age in Fischer 344 rats. This MN death is triggered (or at least preceded), by an unfolded protein response and mitochondrial fragmentation. Interventions ameliorating inflammation, the unfolded protein response and/or mitochondrial breakdown may preserve MNs and prevent or reduce age-associated sarcopenia. This work was supported by NIH grants: HL166204 (MJF), AG086136 (MJF), HL157984 (GCS) & AG44615 (GCS) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

The exact physiological role of BACE2 in endothelial function of systemic arteries is unknown. Our study shows that thromboxane A2 and prostaglandin F2α are responsible for impairment of endothelium-dependent relaxations in endothelium-specific BACE2-deficient mice. The data support an important role of BACE2 in the control of endothelial arachidonic acid metabolism and vasomotor function.

Abstract Background Phage therapy is being evaluated to treat bacterial infections. Host-related factors that might abrogate phage efficacy remain incompletely characterized. Phage aggregation has been hypothesized to protect phages in harsh environments. Herein, effects of immunized rabbit sera on phage aggregation were assessed. Methods Three New Zealand rabbits were immunized weekly for 3 weeks by intramuscular injection of phage K. Serum samples were collected weekly at times 0, 7, 14, 21 and 28. Development of an antibody response was confirmed by ELISA. Phage K was exposed to immunized serum with or without heat-inactivated host bacterium (Staphylococcus aureus ATCC 19685) as follows. 45 µl of phage K (1010±1 plaque forming units/mL [pfu/ml]) was mixed with 5 µl immunized serum diluted 1:1000 with or without heat-inactivated bacterial host in phosphate buffered saline (PBS). After incubation, exposed phage was visualized using transmission electron microscopy (TEM, JEOL) at 80kV. Results Phage K immunization elicited a strong IgG response. Exposure to immunized serum led to a decrease in viable phage over time, abrogated by adding heat-inactivated bacteria (Fig 1). TEM imaging showed phage clustering with exposure to immunized serum (Fig 2A); clusters appeared disorganized with concomitant exposure to heat-inactivated host bacteria (Fig 2B). Exposure of phage to heat-inactivated bacteria without serum yielded evenly distributed phage, with minimal clustering (Fig 2C). Conclusion Phage K exposure elicited a neutralizing immune response associated with decreased phage survival in immunized serum, prolonged by adding heat-inactivated host bacteria. TEM imaging showed phage K clustering in different ways depending on the microenvironment encountered. Biological relevance and mechanistic underpinnings of these phenomena need further characterization. Disclosures Robin Patel, MD, a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, a: See above|MicuRx Pharmaceuticals and BIOFIRE: Grant/Research Support|PhAST, Day Zero Diagnostics, Abbott Laboratories, Sysmex, DEEPULL DIAGNOSTICS, S.L., Netflix, Oxford Nanopore Technologies and CARB-X: Advisor/Consultant|Up-to-Date and the Infectious Diseases Board Review Course.: Honoraria