Resolution Confocal Microscopy Research Articles

Anti-slit diaphragm antibodies on kidney biopsy identify pediatric patients with steroid-resistant nephrotic syndrome responsive to second-line immunosuppressants

Podocytopathies represent a group of glomerular disorders associated with minimal changes (MC) or focal segmental glomerulosclerosis (FSGS) lesion patterns at biopsy and heterogeneous responses to steroids. Anti-nephrin antibodies were previously found in such patients, suggesting an autoimmune form of podocytopathy. High resolution confocal microscopy on kidney biopsies of a cohort of 128 pediatric patients revealed localization of IgG along the slit diaphragm in 30% of patients with MC and 25% of those with FSGS, but not in other lesion patterns. Anti-nephrin IgG ELISA assay in the serum and stimulated emission depletion microscopy of kidney biopsies showed IgG-nephrin co-localization only in 77.8% of cases. Similar observations were obtained in a cohort of 48 adult patients with MC or FSGS at kidney biopsy, where IgG-nephrin colocalization was only 44.4%, suggesting the existence of autoantibodies binding to other slit proteins. Patients with anti-slit antibodies showed nephrotic syndrome at onset in 94.4% of cases. Patients with primary steroid-resistance had anti-slit antibodies in 27%, while those with secondary steroid-resistance in 87.5% of cases, irrespective of the histopathological lesion pattern. Steroid-resistant patients with anti-slit antibodies responded to second-line immunosuppressants in 92.3% vs. only 20% of patients that were anti-slit negative. No patient with anti-slit antibodies developed kidney failure vs. 51.7% of those negative for antibodies (66.7% with a genetic cause and 41.2% with a non-genetic cause). Thus, the detection of anti-slit antibodies can identify patients with an autoimmune podocytopathy responsive to treatment with second-line immunosuppressants, irrespective of the histopathological lesion pattern at biopsy.

Phase-only mask for superoscillatory enhanced resolution in confocal microscopy

In this work, we present a simple phase mask that generates a superoscillatory spot suitable for confocal scanning microscopy enabling lateral sub-diffraction resolution. When considering linearly polarized light, the mask enhances the resolving power along the axis perpendicular to the polarization direction. The symmetry of the enhancement is recovered if the mask is combined with radial polarization.

Optimizing miRNA transfection for screening in precision cut lung slices.

Precision cut lung slices(PCLS) are complex 3D lung tissue models, which preserve the native microenvironment, including cell diversity and cell-matrix interactions. They are an innovative ex vivo platform that allows studying disease as well as the effects of therapeutic agents or regulatory molecules(e.g. miRNA). The aim of our study was to develop a protocol to transfect PCLS with miRNA using lipid nanoparticles (LNPs) to enable higher throughput screening of miRNA, obviating the need for custom stabilization and internalization approaches. 4mm diameter PCLS were generated using agarose-filled rodent lungs and a vibratome. TYE665 labelled scrambled miRNA was used to evaluate transfection efficacy of six different commercially available LNPs. Transfection efficacy was visualised using live high content fluorescence microscopy, followed by higher resolution confocal fluorescence microscopy in fixed PCLS. Metabolic activity and cellular damage were assessed using WST-1 and lactate dehydrogenase(LDH) release. Using a live staining kit containing a cell membrane impermeant nuclear dye, RedDot2TM, we established that cellular membranes in PCLS are permeable in the initial 24 hours of slicing but diminished thereafter. Therefore, all transfection experiments occurred at least 24 hours after slicing. All six commercially available LNPs enabled transfection without inducing significant cytotoxicity or impaired metabolic function. However, RNAiMAX and INTERFERin led to increases in transfection efficacy as compared to other LNPs, with detection possible as low as 25nM. Therefore, LNP-based transfection of miRNA is possible and can be visualized in live or fixed PCLS, enabling future higher throughput studies using diverse miRNAs.

Internalisation of immunoglobulin light chains by cardiomyocytes in AL amyloidosis: what can biopsies tell us?

Background Cardiac involvement in systemic light chain amyloidosis (AL) leads to chronic heart failure and is a major prognosis factor. Severe cellular defects are provoked in cardiac cells by tissue-deposited amyloid fibrils of misfolded free immunoglobulin light chains (LCs) and their prefibrillar oligomeric precursors. Objective Understanding the molecular mechanisms behind cardiac cell cytotoxicity is necessary to progress in therapy and to improve patient management. One key question is how extracellularly deposited molecules exert their toxic action inside cardiac cells. Here we searched for direct evidence of amyloid LC uptake by cardiomyocytes in patient biopsies. Methods We immunolocalized LCs in cardiac biopsies from four AL cardiac amyloidosis patients and analysed histopathological images by high resolution confocal microscopy and 3D image reconstruction. Results We show, for the first time directly in patient tissue, the presence of LCs inside cardiomyocytes, and report their proximity to nuclei and to caveolin-3-rich areas. Our observations point to macropinocytosis as a probable mechanism of LC uptake. Conclusions Internalisation of LCs occurs in patient cardiomyocytes. This event could have important consequences for the pathogenesis of the cardiac disease by enabling interactions between amyloid molecules and cellular organelles inducing specific signalling pathways, and might bring new insight regarding treatment.

Senescent endothelial cells promote pathogenic neutrophil trafficking in inflamed tissues

Cellular senescence is a hallmark of advanced age and a major instigator of numerous inflammatory pathologies. While endothelial cell (EC) senescence is aligned with defective vascular functionality, its impact on fundamental inflammatory responses in vivo at single-cell level remain unclear. To directly investigate the role of EC senescence on dynamics of neutrophil-venular wall interactions, we applied high resolution confocal intravital microscopy to inflamed tissues of an EC-specific progeroid mouse model, characterized by profound indicators of EC senescence. Progerin-expressing ECs supported prolonged neutrophil adhesion and crawling in a cell autonomous manner that additionally mediated neutrophil-dependent microvascular leakage. Transcriptomic and immunofluorescence analysis of inflamed tissues identified elevated levels of EC CXCL1 on progerin-expressing ECs and functional blockade of CXCL1 suppressed the dysregulated neutrophil responses elicited by senescent ECs. Similarly, cultured progerin-expressing human ECs exhibited a senescent phenotype, were pro-inflammatory and prompted increased neutrophil attachment and activation. Collectively, our findings support the concept that senescent ECs drive excessive inflammation and provide new insights into the mode, dynamics, and mechanisms of this response at single-cell level.

Central Amygdala Astrocyte Plasticity Underlies GABAergic Dysregulation in Ethanol Dependence.

Dependence is a hallmark of alcohol use disorder characterized by excessive alcohol intake and withdrawal symptoms. The central nucleus of the amygdala (CeA) is a key brain structure underlying the synaptic and behavioral consequences of ethanol dependence. While accumulating evidence suggests that astrocytes regulate synaptic transmission and behavior, there is a limited understanding of the role astrocytes play in ethanol dependence. The present study used a combination of viral labeling, super resolution confocal microscopy, 3D image analysis, and slice electrophysiology to determine the effects of chronic intermittent ethanol (CIE) exposure on astrocyte plasticity in the CeA. During withdrawal from CIE exposure, we observed increased GABA transmission, an upregulation in astrocytic GAT3 levels, and an increased proximity of astrocyte processes near CeA synapses. Furthermore, GAT3 levels and synaptic proximity were positively associated with voluntary ethanol drinking in dependent rats. Slice electrophysiology confirmed that the upregulation in astrocytic GAT3 levels was functional, as CIE exposure unmasked a GAT3-sensitive tonic GABA current in the CeA. A causal role for astrocytic GAT3 in ethanol dependence was assessed using viral-mediated GAT3 overexpression and knockdown approaches. However, GAT3 knockdown or overexpression had no effect on somatic withdrawal symptoms, dependence-escalated ethanol intake, aversion-resistant drinking, or post-dependent ethanol drinking in male or female rats. Moreover, intra-CeA pharmacological inhibition of GAT3 also did not alter dependent ethanol drinking. Together, these findings indicate that ethanol dependence induces GABAergic dysregulation and astrocyte plasticity in the CeA. However, astrocytic GAT3 does not appear necessary for the drinking related phenotypes associated with dependence.

Saliva exposure reduces gingival keratinocyte growth on TiO2-coated titanium

Bioactive, nanoporous TiO2-coating has been shown to enhance cell attachment on titanium implant surface. The aim of this study was to evaluate, whether the saliva proteins affect the epithelial cell adhesion on TiO2-coated and non-coated titanium. Grade V titanium discs were polished. Half of the discs were provided with TiO2-coating produced in sol with polycondensation method. Half of the TiO2-coated and non-coated discs were treated with pasteurized saliva for 30 min. After saliva treatment, the total protein amounts on surfaces were measured. Next, the hydrophilicity of discs were measured with water contact angle measurements. Further, the gingival keratinocyte adhesion strength was measured after 2 and 6 h of cultivation using serial trypsinization. In addition, cell growth and proliferation were measured after 1, 3, and 7 days of cell culture. Finally, cell morphology, spreading and adhesion protein signals were detected with high resolution confocal microscopy. As a result, in sol coated TiO2-surface had significantly higher hydrophilicity when compared to non-coated titanium, meanwhile both non-coated and TiO2-coated surfaces with saliva treatment had a significant increase in hydrophilicity. Importantly, the amounts of adhered saliva proteins were equal between TiO2-coated and non-coated surfaces. Adhesion strength against enzymatic detachment was weakest on non-coated titanium after saliva exposure. Cell proliferation and cell spreading were highest on TiO2-coated titanium, but saliva exposure significantly decreased cell proliferation and spreading on TiO2-coated surface. To conclude, even though saliva exposure makes titanium surfaces more hydrophilic, it seems to neutralize the bioactive TiO2-coating and decrease cell attachment to TiO2-coated surface.Graphical

Mitochondrial translocation of TFEB regulates complex I and inflammation.

TFEB is a master regulator of autophagy, lysosome biogenesis, mitochondrial metabolism, and immunity that works primarily through transcription controlled by cytosol-to-nuclear translocation. Emerging data indicate additional regulatory interactions at the surface of organelles such as lysosomes. Here we show that TFEB has a non-transcriptional role in mitochondria, regulating the electron transport chain complex I to down-modulate inflammation. Proteomics analysis reveals extensive TFEB co-immunoprecipitation with several mitochondrial proteins, whose interactions are disrupted upon infection with S. Typhimurium. High resolution confocal microscopy and biochemistry confirms TFEB localization in the mitochondrial matrix. TFEB translocation depends on a conserved N-terminal TOMM20-binding motif and is enhanced by mTOR inhibition. Within the mitochondria, TFEB and protease LONP1 antagonistically co-regulate complex I, reactive oxygen species and the inflammatory response. Consequently, during infection, lack of TFEB specifically in the mitochondria exacerbates the expression of pro-inflammatory cytokines, contributing to innate immune pathogenesis.

DCB Bonding Energy Measurement Using Confocal IR Microscopy

Direct bonding is a widely used technique in the semiconductor industry. Many materials can be assembled and stacked together with this technique, enabling the fabrication of many useful structures. The direct bonding technique is, for instance, a major step of the Smart Cut ™ process flow enabling the industrial-scale production of Silicon-On-Insulator (SOI) substrates [1][2]. Material diversification in the microelectronics industry (i.e. “More than Moore” approach) imposes new technological constraints. The substrate bonding process control has to be improved calling for more and more accurate bonding energy measurement protocols.In this work, a new technique is assessed in order to measure, at the wafer scale, direct bonding energies. It is derived from the standard Double Cantilever Beam (DCB) method [3] and uses interferometry in confocal IR laser source microscopy to measure crack openings (Fig. 1A) [4][5][6]. Such a bonding energy measurement protocol shows a better accuracy compared to other techniques (Fig. 1B & 1D). This is due to a better confocal microscopy resolution and the high intensity of the laser source. The elastic energy stored in bent wafers is obtained by measuring the beam curvature [7][8]. DCB deformation models are discussed from short-range crack opening to long distance beam-bending theories. Comparison is done between results from analytic models, Finite Element Model (FEM) and experiments (Fig. 1C).Near-bonding edge zone modeled with FEM simulation accurately matches experimental data. A new mechanical and universal bending model, valid at both small and large scales, is proposed. With this model, bonding energy extraction is then reduced to a simple scaling parameter extraction directly linked to the stored elastic strain energy (Fig 1C & 1D) [9]. This work opens prospects for deformation and bonding energy measurements in various configurations including bonded heterostructures, polymer, and metal bonding. [1] Bruel, Electron. Lett. 31, 1201 (1995).[2] K. Celler, A.-J. Auberton-Hervé, B. Aspar, C. Lagahe-Blanchard, and C. Maleville, in Wafer Bonding Applications and Technology, Springer Series in Materials Science, edited by M. Alexe and U. Gosele, Springer, Berlin (2004).[3] Maszara, G. Goetz, A. Caviglia, and J. B. Mckitterick, J. Appl. Phys. 64, 4943 (1988).[4] Wu, S. Gowrishankar, R. Huang et al., Int. J. Fract. 202, 1–19 (2016).[5] Gowrishankar, H. Mei, K. M. Liechti et al., Int. J. Fract. 177, 109–128 (2012).[6] Pallares, L. Ponson, A. Grimaldi, M. George, G. Prevot, and M. Ciccotti, Int. J. Fract. 156, 11–20 (2009).[7] Bertholet, “Measurement, optimization and multiscale modeling of silicon wafer bonding interface fracture resistance,” Ph.D. thesis (Catholique de Louvain, 2006).[8] Olbrechts, B. Lejeune, Y. Bertholet, T. Pardoen, and J.-P. Raskin, ECS Trans. 3(6), 279–289 (2006).[9] Colonel, A. Calvez, F. Fournel et al., J. Appl. Phys. 132, 215106 (2022). Fig. 1: A – (Top) Four pasted IR confocal microscope images acquired during the DCB testing (the vertical red dashed line shows the closing point’s fitted position). (Bottom) Air gap height measured as a function of pixel position.B – Simulated reflected intensity (left y-axis) and experimental reflected intensity (right y-axis), both as functions of the estimated distance from the closure point x. The vertical dashed grey line shows the real experimental closing point.C – Air gap height as a function of the position/thickness ratio (in log/log scales) for different analytical and numerical models and experimental data.D – Bonding energies including errors obtained with the standard length measurement method, the curvature interpolation method, the scaling method, or J-integral calculation from FEM as functions of the annealing temperature. Figure 1

Abstract 15932: Lactobacillus Suppresses Glycoprotein-130-Mediated Microtubule Remodeling to Support Right Ventricular Function in Pulmonary Arterial Hypertension

Introduction: Microtubules (MTs) are crucial for cardiomyocyte function as they regulate t-tubule structure, nuclear morphology, and gap junction protein localization. Glycoprotein-130 (GP130) signaling promotes pathological MT remodeling and right ventricular (RV) dysfunction in pulmonary arterial hypertension (PAH), but the trigger for the inflammation-MT connection is unknown. Microbiome dysbiosis activates systemic inflammation, and levels of the anti-inflammatory bacteria Lactobacillus are associated with RV function in rodents. However, the direct effects of Lactobacillus on GP130 signaling, MT remodeling, and RV function in PAH are unknown. Methods: Rats were randomly allocated into 3 groups: control, MCT-water, and MCT rats given Lactobacillus (MCT- Lactobacillus ). Echocardiography and pressure-volume loops defined RV function. Next-generation sequencing evaluated the composition of the bacterial components of the fecal microbiome. SomaScan proteomics quantified the serum proteome. RV and jejunal morphology were assessed with light and super resolution confocal microscopy. The relationship between Lactobacillus abundance and RV response to afterload was assessed in 65 PAH patients. Results: Lactobacillus augmented RV function (A) and reduced RV hypertrophy and fibrosis without altering PAH severity. Lactobacillus shifted the serum proteomic signature towards control (B) and reduced circulating GP130 ligands, normalized RV t-tubule architecture (C), MT density (D), and nucleus size (E), and prevented connexin-43 lateralization in RV cardiomyocytes (F). Lactobacillus restructured the microbiome, and concomitantly improved jejunal epithelial cell morphology with increased villus/microvillus length and glycocalyx thickness. PAH patients with fecal Lactobacillus had superior RV adaptation as afterload increased (G). Conclusion: Lactobacillus combats GP130-mediated MT remodeling to support RV function.

Three-dimensional configuration of crystal plasticity in stainless steel assessed by high resolution digital image correlation and confocal microscopy

In polycrystalline austenitic stainless steels, a comprehensive three-dimensional configuration of crystal plasticity in relation to microstructure is required to elucidate the deformation behaviors during mechanical loading. In the present work, a combination of backscattered electron image (BSE) based high-resolution digital image correlation (HR-DIC) and laser scanning confocal microscopy (LSCM) was employed to simultaneously investigate in-plane and out-of-plane deformations at the sub-grain scale in 316 stainless steel. The high spatial resolution and precise alignment provided by the three-dimensional deformation assessment enable the identification of specific slip and twinning systems. This is achieved through the minimization of the residual L2 norm between the in-plane components of experimental and theoretical strain tensors. Dominant slip and few mechanical twinning activities are initiated at the early-stage of yielding, and their frequency and intensity increase with the applied load. The hierarchical networks formed by nanotwins, along with the presence of grain and annealing twin boundaries, serve as obstacles for slip glide, leading to substantial twin–slip interaction and grain boundary strengthening. As a consequence of wide mechanical twins formed by parallel nanotwins coalescence and strain localization at the twin boundary, slip transmission occurs, resulting in an effective accommodation of external stress. Furthermore, the consistent observation of out-of-plane roughness, in alignment with twinning Burgers vector predictions, offers valuable insights into the significant deformation gradient occurring in the vicinity of boundaries. This deformation gradient is influenced by the neighboring mechanical twinning activities, indicating strong obstacle to twin transmission and high back-stresses resulting from the highly misoriented grain boundary.

Abstract P1027: Confocal And Mass Spectrometry-based Investigation Of REEP5 Depletion By AAV9 In The Mouse Heart

The sarco(endo)plasmic reticulum (SR/ER) is an essential regulator of many key cellular processes, especially those that play a role in the development and progression of cardiac disease. However, many aspects of its structural organization remain poorly defined. Receptor Expression Enhancing Protein 5 (REEP5) is a cardiac enriched SR/ER membrane protein, which regulates organization of the highly differentiated SR/ER network and responses to stress. In zebrafish models, genetic knock-out of reep5 results in cardiac functional defects and reduced heart rate. Within the cardiomyocyte, depletion of Reep5 in vitro results in decreased muscle cell contraction, disrupted Ca 2+ signaling and SR/ER luminal vacuolization. For these studies, in vivo cardiac knock-down of Reep5 in the mouse was achieved using recombinant adeno-associated virus serotype 9 (rAAV9)-mediated gene delivery. Cardiac tissues or isolated cardiomyocytes were harvested at 7 days through to 4 weeks following knock-down, for biochemical and functional assessments. We observed that the largest significant change in REEP5 expression occurred 4 weeks post-rAAV9 injection, correlating to a 78% knock-down, observed by immunoblotting (unpaired t-test, p<0.0001, n=6-8). 5 weeks following knock-down, mice developed lethal cardiac dysfunction. To assess the biochemical changes induced by Reep5 knock-down, we have established an organelle-specific cardiac proteomic profile of the microsome, mitochondria and cytosol, using subcellular fractionation and mass spectrometry (nLC-ESI-MS-HCD-MS). Coupled with high resolution confocal microscopy and 3D mapping techniques, we have examined localization and expression patterns of key SR/ER and mitochondrial proteins, that have altered expression following knock-down of Reep5 at the myocyte-level. These findings provide a detailed understanding of the role that REEP5 plays in maintaining ER homeostasis, SR/ER structure, and general organelle integrity. By identifying the mechanistic significance of REEP5 expression in the heart, we can work to delineate underappreciated pathways in cardiac muscle development.

Non-alcoholic fatty liver disease combined with rheumatoid arthritis exacerbates liver fibrosis by stimulating co-localization of PTRF and TLR4 in rats.

Rheumatoid arthritis (RA) has a high prevalence in patients with non-alcoholic fatty liver disease (NAFLD); however, the underlying mechanism is unclear. To address this, our study established a rat model with both NAFLD and RA by feeding a high-fat diet (HFD) and administering intradermal injection of Freund's complete adjuvant (FCA) with bovine type II collagen. Collagen-induced RA (CIA) was confirmed by hind paw swelling and histological examination. The histomorphological characteristics of NAFLD were evaluated by Masson's trichrome and hematoxylin-eosin staining. The development of NAFLD was further evaluated by measuring serum concentrations of triglyceride (TG), total cholesterol (T-CHO), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lipopolysaccharide (LPS). The results showed that HFD feeding exacerbated secondary inflammation in CIA rats, whereas FCA/bovine type II collagen injection increased serum levels of ALT, AST, TG, T-CHO, and LPS and exacerbated hepatic fibrosis in both normal and NAFLD rats. Interestingly, NAFLD + CIA significantly promoted the expression of PTRF, a caveolae structure protein involved in hepatic lipid metabolism and affecting downstream signaling of Toll-like receptor 4 (TLR4) and PI3K/Akt activation. High resolution confocal microscopy revealed increased PTRF and TLR4 co-localization in hepatic small vessels of NAFLD + CIA rats. AAV9-mediated PTRF knockdown inhibited TLR4 signaling and alleviated hepatic fibrosis in NAFLD + CIA rats. Together, these findings indicate that NAFLD combined with CIA causes synovial injury and enhances non-alcoholic fatty liver fibrosis in rats. PTRF could attenuate the symptoms of NAFLD + CIA likely by affecting TLR4/PTRF co-expression and downstream signaling.

BS-454195-1 ADRENERGIC MODULATION OF CONDUCTION DURING CARDIAC STRESS IS MEDIATED BY CALCIUM CHANNEL AUTOREGULATION OF INTERCALATED DISC NANODOMAINS

During stress, adrenergic signaling enhances cardiac conduction, although the mechanisms are not fully understood. Reducing extracellular calcium in the heart widens an intercalated disc (ID) nanodomain adjacent to gap junctions known as the perinexus. Under acute cardiac stress, the perinexus adaptively remodels; preventing this response by experimentally changing calcium levels slows conduction and increases arrhythmia risk. This raises the question of whether myocytes autoregulate calcium-mediated ID adhesion to stabilize conduction during cardiac stress. The cardiac voltage gated calcium channel (Cav1.2) is a primary source of calcium influx and a key target during adrenergic signaling. Expression of Cav1.2 within the ID has not been described. To test the hypothesis that Cav1.2 is expressed in the ID and autoregulates myocyte adhesion and conduction in the adrenergic response to cardiac stress. Ex vivo guinea pig hearts were Langendorff-perfused and ventricular tissue harvested for microscopy. Cav1.2 cellular localization was quantified by spinning disk confocal microscopy (n=5), conduction velocity (CV) was measured by optical mapping (n=9), and ID ultrastructure was quantified from transmission electron micrographs (n=17). Cav1.2 function was modulated by perfusion with the β-adrenergic agonist isoproterenol (Iso, 0.6μM), the Cav1.2 agonist BayK8644 (BayK, 0.5μM), or the Cav1.2 antagonist verapamil (Ver, 2μM). Super resolution confocal microscopy demonstrated that 2.5% of total Cav1.2 signal co-occurred with connexin43 and N-cadherin. At baseline, CV slowed at shorter CLs (150 vs 300ms = 4% slower), consistent with CV restitution. Treatment with Iso increased both Wp (20.2 vs 17.0 nm at baseline) and CV (300ms: 113% of baseline). Iso reversed CV restitution such that CV increased by 4%. Activation of Cav1.2 by BayK widened Wp to the same degree as Iso (20.1 vs 20.2 nm). Verapamil prevented both the Iso-induced increase in Wp (17.0 nm) and CV (93.9% of baseline), and exacerbated CV restitution. A subpopulation of Cav1.2 was identified within the ID. Adrenergic stimulation widened perinexal width, increased conduction, and reversed CV restitution. Blocking Cav1.2 prevented the adrenergic-mediated change in perinexal structure and conduction. Thus, Cav1.2 function regulates ID ultrastructure and may serve as an adaptive response to enhance conduction during conditions of acute cardiac stress.

Substance P expression and neurochemical characterization in the enteric nervous system of the porcine colon

The pig is a good model for studying intestinal functions and disorders for its homologies with humans such as microbiome composition, size, nutrition being both omnivores and colon fermenters. The enteric nervous system (ENS) of pigs and humans has a multilayered submucosal plexus with an inner submucous plexus (ISP) near the mucosa and an outer plexus (OSP) near the circular muscle in addition to the myenteric plexus (MP) between the muscle layers. We have shown differences in the density and distribution of functionally distinct neurons in different regions and plexuses of the porcine colon. Aim: This study focused on Substance P, a peptide that modulates many functions in the gastrointestinal (GI) tract and plays an important role in neurogenic inflammation. We tested whether there were differences in the density and neurochemical profile of SP neurons in the ISP, OSP and MP of the ascending (AC) and descending (DC) colon of 15 Yucatan minipigs (12M, 3F, 7-months-old, body weight 25-30 kg). We processed colonic wholemounts for multiple labeling immunofluorescence using the HuCD, choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS) as neuronal markers with high resolution confocal microscopy and Imaris software to quantify the number of neurons/mm2 and the % of total neurons identified by the pan-neuronal marker HuCD. Results: HuCD/SP-immunoreactive (IR) neurons were most abundant in the ISP vs. OSP and MP in both AC and DC (p<0.01-p<0.0001) with highest density in ISP of AC (p<0.0001 vs. DC). SP-IR neurons represent 27-28% of HuCD-IR neurons in ISP followed by OSP (19-22%) and MP (13-17%, p<0.05- p<0.0001) in AC and DC. The highest proportion of SP-IR neurons contains ChAT-IR (62-67% in the MP, 74-76% in the ISP, 70-85% in OSP) with no significant differences in the AC and DC. By contrast, SP-IR neurons containing nNOS-IR were much less abundant in both AC and DC with the highest peak in the OSP 36% vs. 16% in the ISP, p<0.01, and 20% in the MP in AC, whereas in the DC the highest peak was in the ISP (31%) followed by OSP and MP (25% and 18%) without significant differences. There was a small population of SP-/ChAT-/nNOS-IR enteric neurons in all plexuses in AC and DC. Varicose SP-IR fibers were abundant in the ganglia and distributed to the muscle layers and mucosa. Conclusions: SP-IR neurons include excitatory, inhibitory and interneurons based on the co-expression of ChAT, nNOS or both. The different density and neurochemical profile likely reflect different functions of SP-IR neurons such as immune modulation, regulation of mucosal function and control of vascular, myogenic and neurogenic activities. Mapping neuromodulators expression in a pre-clinical model provides the basis for elucidating neuronal circuits underlying GI functions and advance our understanding of human diseases. Supported by NIH SPARC OT2OD24899 & NIH-P30DK41301. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Postnatal Inhibition of TrkB Kinase Reduces the Number of Phrenic Motor Neurons

During embryonic development, the number of phrenic motor neurons (PhMNs) declines precipitously during the final trimester and continues into the early postnatal period. When BDNF/TrkB signaling is absent in utero, animals do not survive after birth, most likely due to excessive loss of vital motor neurons (e.g., PhMNs). Far less is known about the early postnatal effect of BDNF/TrkB signaling. We have shown that BDNF/TrkB signaling is important in promoting PhMN neuroplasticity in mature rodents. Taken together, we hypothesized that inhibition of BDNF/TrkB signaling during early postnatal development results in fewer PhMNs. Chemo-genetic TrkBF616A mutant mice were used to induce inhibition of TrkB kinase activity in response to oral administration (via drinking water) of a phosphoprotein phosphatase 1 inhibitor derivative (1NMPP1). 1NMPP1-induced TrkB kinase inhibition was initiated at P0 and continued until P21 and compared to untreated control mice. At P28, PhMNs were retrogradely labeled via rhodamine phrenic nerve dip. One day later, mice were euthanized, perfused with 4% paraformaldehyde, and the cervical spinal cord was excised and processed for longitudinal cryosectioning (70 μm). Labeled PhMNs were imaged by 3D high resolution confocal microscopy. The number of PhMNs were counted. PhMN somal surface area and number of primary dendrites were also determined. 1NMPP1-induced inhibition of TrkB kinase resulted in 24% fewer PhMNs (P=0.01) and 21% more primary dendrites (P=0.0002) than control mice. The distribution of PhMN somal surface areas was not affected. We conclude that BDNF/TrkB signaling is essential in promoting survival of PhMNs in the early postnatal period. BDNF/TrkB signaling during postnatal development also plays a role in paring primary dendrites. While inhibition of TrkB kinase did not significantly alter the distribution of PhMN somal surface areas at P28, it is possible that further growth of PhMNs into maturity might have been altered as larger PhMNs emerge as a result of disproportionate growth beyond this age in mice. Supported by National Institutes of Health grants R01-AG044615 (GCS), R01-HL96750 (GCS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Beyond the Limits of Light: An Application of Super-Resolution Confocal Microscopy (sCLSM) to Investigate Eocene Amber Microfossils.

Amber is known as one of the best sources of fossil organisms preserved with exceptional fidelity. Historically, different methods of imaging have been applied to amber, including optical microscopy and microtomography. These methods are sufficient to resolve millimeter-scaled fossils. However, microfossils, such as microarthropods, require another resolution. Here, we describe a non-destructive method of super resolution confocal microscopy (sCLSM) to study amber-preserved microfossils, using a novel astigmatid mite species (genus Histiogaster, Acaridae) from Eocene Rovno amber as a model. We show that the resolution obtained with sCLSM is comparable to that of scanning electron microscopy (SEM) routinely used to study modern mites. We compare sCLSM imaging to other methods that are used to study amber inclusions and emphasize its advantages in examination of unique fossil specimens. Furthermore, we show that the deterioration of amber, which manifests in its darkening, positively correlates with its increased fluorescence. Our results demonstrate a great potential of the sCLSM method for imaging of the tiniest organisms preserved in amber.

Osmotic pressure enables facile high yield assembly of cell-like giant unilamellar vesicles in solutions of physiological ionic strength.

Giant unilamellar vesicles (GUVs) are single-walled, closed phospholipid bilayers that are useful as cellular mimics for synthetic biology and drug delivery. The assembly of GUVs in solutions of ionic concentrations >150 mM Na/KCl (salty solutions) is challenging. Here, we investigate the effects of temperature, chemical identity of small molecule and macromolecular polymers on the molar yield of free-floating GUVs in salty solutions using high resolution confocal microscopy and large dataset image analysis. We find that low gelling temperature agarose is the singular compound that produces free-floating GUVs at molar yields >10 % in salty solutions. All other macromolecular polymers, such as polyvinyl alcohol (PVA), fructose, and ultralow gelling temperature agarose, produced yields of GUVs of <7 %. Surface images show that for these other compounds, the formation of large numbers of lipid-polymer pseudobuds due to polymer dewetting reduces yields of GUVs. We propose a free-energy model of GUV assembly to explain the effects of polymers on the assembly of GUVs. Modulating the adhesion potential by varying ion concentration and valency yielded experimental data that supports our model's prediction - the free-energy for bud formation can be reduced by a contribution to the osmotic pressure of dissolving polymers. These results provide a quantitative comparison between established and novel methodology, as well as experimental and theoretical framework to guide studies for obtaining GUVs in physiological salt solutions.

Double cantilever beam bonding energy measurement using confocal IR microscopy

A new technique is assessed in order to measure, at the wafer scale, direct bonding energies. It is derived from the standard Double Cantilever Beam (DCB) method and uses interferometry in confocal IR laser source microscopy to measure crack openings. Such a bonding energy measurement protocol has better accuracy compared to other techniques. This is due to a better confocal microscopy resolution and the high intensity of the laser source. The elastic energy stored in bent wafers is obtained by measuring the beam curvature. DCB deformation models are discussed from the short-range crack opening theory to long distance beam-bending theories. Comparison is made between models, experimental results, and FEM simulations. Finally, the bonding energy error during standard measurements is estimated.

Inhibition of Hyper-ActivatedLysosomes Restores Youthful Properties and Functions in Old Hematopoietic Stem Cells

Aging is the primary risk factor for many late-onset diseases including myeloid malignancies and immune deficiencies of the elderly. Aging of hematopoietic stem cells (HSCs) is thought to be in part responsible for these blood disorders. As quiescence maintains HSCs' health and function throughout life, loss of quiescence with age is likely implicated in defects of old HSCs including their myeloid-biased lineage commitment. Despite major progress, mechanisms that promote aging of HSCs remain to be elucidated. We have identified dynamic lysosomal modulations as a switch in the precise control of HSC quiescence vs. priming/activation in young HSCs. Here we find that lysosomal properties and functions are compromised in old HSCs. We find that lysosomes are relatively depleted in old HSCs, as evaluated by high resolution confocal microscopy analyses of immunofluorescence (IF) staining of lysosomal-associated membrane protein (LAMP)-1 and the more specific LAMP-2 markers. Notably, lysosomal properties are altered in old HSCs beyond what is known of their function in autophagy. We find increased recruitment of damage-associated proteins Galectin 1 and 2 to lysosomes in old relative to young highly purified LSKCD150+CD48- HSCs indicating increased lysosomal damage in old HSCs. In addition, lysosomal LC3 flux that is an indication of lysosomal activity was increased by two-fold in highly purified old relative to young HSCs. This was in line with old HSCs' alteration of expression of lysosomal enzymes Cytochalasin C and D and the vacuolar H+ -adenosine triphosphatase ATPase (v-ATPase). The increased lysosomal activity is supported by greatly acidified lysosomes as well as enhanced lysosomal degradation capacity of cargo in old relative to young HSCs. An 18-hour inhibition of lysosomal acidification by repressing v-ATPase' activity using specific and high affinity v-ATPase inhibitor, ex vivo, led to a dramatic enrichment of old HSCs in large lysosomes, restoration of their properties and reducing their Galectin protein burden similar to what was observed in young quiescent and healthy HSCs. This was specific to lysosomes in the endo-lysosomal pathway as the effect on the Golgi and endoplasmic reticulum compartments in HSCs was relatively negligeable. The v-ATPase inhibitory treatment increased substantially the fraction of non-dividing old HSCs in culture, and the frequency of LTC-ICs recovered in limiting dilution analysis of old HSCs. Importantly, a 4-day ex vivo inhibition of lysosomal activity increased old HSCs' in vivo competitive repopulation ability by over 8-fold over 21 weeks and balanced the production of lineages downstream of old HSCs. Remarkably, only v-ATPase inhibitor- but not vehicle-treated old HSCs sustained survival in lethally irradiated secondarily transplanted mice and maintained self-renewal. v-ATPase inhibition normalized the expression of TFEB, master regulator of lysosomal biogenesis, suppressed CDK6 nuclear expression, normalized metabolic properties of old HSCs, including mTOR activity and glucose uptake, normalized expression of histone marks H4K16ac, H3K27ac and H3K56ac in old HSCs, as well as signs of intrinsic inflammation. Overall, our work indicates that inhibiting lysosomal hyper-activation restores optimum mitochondrial sequestration in old HSCs' lysosomes, leading to reduced inflammation and restitution of stem cell function in old HSCs. These findings may be exploited for improving HSC function in the elderly.