Effect of the snake venom component crotamine on lymphatic endothelial cell responses and lymph transport.

OBJECTIVE: The pathology of snake envenomation is closely tied to the severity of edema in the tissue surrounding the area of the bite. Elucidating the mechanisms that promote the development of such severe edema is critical to a better understanding of how to treat this life-threatening injury. We focused on one of the most abundant venom components in North American viper venom, crotamine, and the effects it has on the cells and function of the lymphatic system. METHODS: We used RT-PCR to identify the location and relative abundance of crotamine's cellular targets (Kvα channels) within the tissues and cells of the lymphatic system. We used calcium flux, nitrate production, and cell morphometry to determine the effects of crotamine on lymphatic endothelial cells. We used tracer transport, node morphometry, and node deposition to determine the effects of crotamine on lymph transport in vivo. RESULTS: We found that genes that encode targets of crotamine are highly present in lymphatic tissues and cells and that there is a differential distribution of those genes that correlates with phasic contractile activity. We found that crotamine potentiates calcium flux in human dermal lymphatic endothelial cells in response to stimulation with histamine and sheer stress (but not alone) and that it alters the production of nitric oxide in response to shear as well as changes the level of F-actin polymerization of those same cells. Crotamine alters lymphatic transport of large molecular weight tracers to local lymph nodes and is deposited within the node mostly in the immediate subcapsular region. CONCLUSION: This evidence suggests that snake venom components may have an impact on the function of the lymphatic system. This needs to be studied in greater detail as there are numerous venom components that may have effects on aspects of the lymphatic system. This would not only provide basic information on the pathobiology of snakebite but also provide targets for improved therapeutics to treat snakebite.

Intracellular calcium dynamics of lymphatic endothelial and muscle cells co-cultured in a Lymphangion-Chip under pulsatile flow.

The lymphatic vascular function is regulated by pulsatile shear stresses through signaling mediated by intracellular calcium [Ca2+]i. Further, the intracellular calcium dynamics mediates signaling between lymphatic endothelial cells (LECs) and muscle cells (LMCs), including the lymphatic tone and contractility. Although calcium signaling has been characterized on LEC monolayers under uniform or step changes in shear stress, these dynamics have not been revealed in LMCs under physiologically-relevant co-culture conditions with LECs or under pulsatile flow. In this study, a cylindrical organ-on-chip platform of the lymphatic vessel (Lymphangion-Chip) consisting of a lumen formed with axially-aligned LECs co-cultured with transversally wrapped layers of LMCs was exposed to step changes or pulsatile shear stress, as often experienced in vivo physiologically or pathologically. Through real-time analysis of intracellular calcium [Ca2+]i release, the device reveals the pulsatile shear-dependent biological coupling between LECs and LMCs. Upon step shear, both cell types undergo a relatively rapid rise in [Ca2+]i followed by a gradual decay. Importantly, under pulsatile flow, analysis of the calcium signal also reveals a secondary sinusoid within the LECs and LMCs that is very close to the flow frequency. Finally, LMCs directly influence the LEC calcium dynamics both under step changes in shear and under pulsatile flow, demonstrating a coupling of LEC-LMC signaling. In conclusion, the Lymphangion-Chip is able to illustrate that intracellular calcium [Ca2+]i in lymphatic vascular cells is dependent on pulsatile shear rate and therefore, serves as an analytical biomarker of mechanotransduction within LECs and LMCs, and functional consequences.

Lymphangion-chip: a microphysiological system which supports co-culture and bidirectional signaling of lymphatic endothelial and muscle cells.

The pathophysiology of several lymphatic diseases, such as lymphedema, depends on the function of lymphangions that drive lymph flow. Even though the signaling between the two main cellular components of a lymphangion, endothelial cells (LECs) and muscle cells (LMCs), is responsible for crucial lymphatic functions, there are no in vitro models that have included both cell types. Here, a fabrication technique (gravitational lumen patterning or GLP) is developed to create a lymphangion-chip. This organ-on-chip consists of co-culture of a monolayer of endothelial lumen surrounded by multiple and uniformly thick layers of muscle cells. The platform allows construction of a wide range of luminal diameters and muscular layer thicknesses, thus providing a toolbox to create variable anatomy. In this device, lymphatic muscle cells align circumferentially while endothelial cells aligned axially under flow, as only observed in vivo in the past. This system successfully characterizes the dynamics of cell size, density, growth, alignment, and intercellular gap due to co-culture and shear. Finally, exposure to pro-inflammatory cytokines reveals that the device could facilitate the regulation of endothelial barrier function through the lymphatic muscle cells. Therefore, this bioengineered platform is suitable for use in preclinical research of lymphatic and blood mechanobiology, inflammation, and translational outcomes.

A multiscale sliding filament model of lymphatic muscle pumping.

The lymphatics maintain fluid balance by returning interstitial fluid to veins via contraction/compression of vessel segments with check valves. Disruption of lymphatic pumping can result in a condition called lymphedema with interstitial fluid accumulation. Lymphedema treatments are often ineffective, which is partially attributable to insufficient understanding of specialized lymphatic muscle lining the vessels. This muscle exhibits cardiac-like phasic contractions and smooth muscle-like tonic contractions to generate and regulate flow. To understand the relationship between this sub-cellular contractile machinery and organ-level pumping, we have developed a multiscale computational model of phasic and tonic contractions in lymphatic muscle and coupled it to a lymphangion pumping model. Our model uses the sliding filament model (Huxley in Prog Biophys Biophys Chem 7:255-318, 1957) and its adaptation for smooth muscle (Mijailovich in Biophys J 79(5):2667-2681, 2000). Multiple structural arrangements of contractile components and viscoelastic elements were trialed but only one provided physiologic results. We then coupled this model with our previous lumped parameter model of the lymphangion to relate results to experiments. We show that the model produces similar pressure, diameter, and flow tracings to experiments on rat mesenteric lymphatics. This model provides the first estimates of lymphatic muscle contraction energetics and the ability to assess the potential effects of sub-cellular level phenomena such as calcium oscillations on lymphangion outflow. The maximum efficiency value predicted (40%) is at the upper end of estimates for other muscle types. Spontaneous calcium oscillations during diastole were found to increase outflow up to approximately 50% in the range of frequencies and amplitudes tested.

Targeting Lymphangiogenesis and Lymph Node Metastasis in Liver Cancer.

Increased lymphangiogenesis and lymph node metastasis, the important prognostic indicators of aggressive hepatobiliary malignancies such as hepatocellular cancer and cholangiocarcinoma, are associated with poor patient outcome. The liver produces 25% to 50% of total lymphatic fluid in the body and has a dense network of lymphatic vessels. The lymphatic system plays critical roles in fluid homeostasis and inflammation and immune response. Yet, lymphatic vessel alterations and function are grossly understudied in the context of liver pathology. Expansion of the lymphatic network has been documented in clinical samples of liver cancer; and although largely overlooked in the liver, tumor-induced lymphangiogenesis is an important player, increasing tumor metastasis in several cancers. This review aims to provide a detailed perspective on the current knowledge of alterations in the hepatic lymphatic system during liver malignancies, as well as various molecular signaling mechanisms and growth factors that may provide future targets for therapeutic intervention. In addition, the review also addresses current mechanisms and bottlenecks for effective therapeutic targeting of tumor-associated lymphangiogenesis.

Isolation of Lymphatic Muscle Cells (LMCs ) from Rat Mesentery.

Lymphatic muscle cells (LMCs), with unique characteristics resembling a combination of both cardiac and smooth muscle cells, play an essential role in the spontaneous contraction of the lymphatic vessels to pump fluid forward. However, our understanding of the more detailed molecular phenotypes of LMCs is limited. Here, we described a method to isolate the LMCs from rat mesentery and then culture the cells in vitro, which will serve a lot more molecular biology study of LMCs and significantly improve our knowledge about the unique characteristics of LMCs.

Analysis of Lymphatic Vessel Formation by Whole-Mount Immunofluorescence Staining.

Pathological alterations of lymphatic structure and function interfere with lymph transport, resulting in a wide range of clinical disorders that include edema, tissue inflammation, and metabolic syndromes. Mesentery contains abundant lymphatic vessels and plays an important role in transporting absorbed lipid from the intestine. In this manuscript, we describe a whole-mount staining method on isolated mouse mesentery with VEGFR3, Prox1, and Lyve1 antibodies to visualize the morphology of lymphatic vessels.

Spaceflight and hind limb unloading induces an arthritic phenotype in knee articular cartilage and menisci of rodents.

Reduced knee weight-bearing from prescription or sedentary lifestyles are associated with cartilage degradation; effects on the meniscus are unclear. Rodents exposed to spaceflight or hind limb unloading (HLU) represent unique opportunities to evaluate this question. This study evaluated arthritic changes in the medial knee compartment that bears the highest loads across the knee after actual and simulated spaceflight, and recovery with subsequent full weight-bearing. Cartilage and meniscal degradation in mice were measured via microCT, histology, and proteomics and/or biochemically after: (1) ~ 35 days on the International Space Station (ISS); (2) 13-days aboard the Space Shuttle Atlantis; or (3) 30 days of HLU, followed by a 49-day weight-bearing readaptation with/without exercise. Cartilage degradation post-ISS and HLU occurred at similar spatial locations, the tibial-femoral cartilage-cartilage contact point, with meniscal volume decline. Cartilage and meniscal glycosaminoglycan content were decreased in unloaded mice, with elevated catabolic enzymes (e.g., matrix metalloproteinases), and elevated oxidative stress and catabolic molecular pathway responses in menisci. After the 13-day Shuttle flight, meniscal degradation was observed. During readaptation, recovery of cartilage volume and thickness occurred with exercise. Reduced weight-bearing from either spaceflight or HLU induced an arthritic phenotype in cartilage and menisci, and exercise promoted recovery.

Dichotomous effects on lymphatic transport with loss of caveolae in mice.

AIM: Fluid and macromolecule transport from the interstitium into and through lymphatic vessels is necessary for tissue homeostasis. While lymphatic capillary structure suggests that passive, paracellular transport would be the predominant route of macromolecule entry, active caveolae-mediated transcellular transport has been identified in lymphatic endothelial cells (LECs) in vitro. Caveolae also mediate a wide array of endothelial cell processes, including nitric oxide regulation. Thus, how does the lack of caveolae impact "lymphatic function"? METHODS: Various aspects of lymphatic transport were measured in mice constitutively lacking caveolin-1 ("CavKO"), the protein required for caveolae formation in endothelial cells, and in mice with a LEC-specific Cav1 gene deletion (Lyve1-Cre x Cav1flox/flox ; "LyCav") and ex vivo in their vessels and cells. RESULTS: In each model, lymphatic architecture was largely unchanged. The lymphatic conductance, or initial tissue uptake, was significantly higher in both CavKO mice and LyCav mice by quantitative microlymphangiography and the permeability to 70 kDa dextran was significantly increased in monolayers of LECs isolated from CavKO mice. Conversely, transport within the lymphatic system to the sentinel node was significantly reduced in anaesthetized CavKO and LyCav mice. Isolated, cannulated collecting vessel studies identified significantly reduced phasic contractility when lymphatic endothelium lacks caveolae. Inhibition of nitric oxide synthase was able to partially restore ex vivo vessel contractility. CONCLUSION: Macromolecule transport across lymphatics is increased with loss of caveolae, yet phasic contractility reduced, resulting in reduced overall lymphatic transport function. These studies identify lymphatic caveolar biology as a key regulator of active lymphatic transport functions.

Cartiotonic steroids affect monolayer permeability in lymphatic endothelial cells.

Edema is common in preeclampsia (preE), a hypertensive disorder of pregnancy. Cardiotonic steroids (CTSs) such as marinobufagenin (MBG) are involved in the pathogenesis of preE. To assess whether CTSs are involved in the leakage of lymphatic endothelial cell (LEC), we evaluated their effect on monolayer permeability of LECs (MPLEC) in culture. A rat mesenteric LECs were treated with DMSO (vehicle), and CTSs (MBG, CINO, OUB) at concentrations of 1, 10, and 100 nM. Some LECs were pretreated with 1 µM L-NAME (N-Nitro-L-Arginine Methyl Ester) before adding 100 nM MBG or cinobufotalin (CINO). Expression of ß-catenin and vascular endothelial (VE)-cadherin in CTS-treated LECs was measured by immunofluorescence and MPLEC was quantified using a fluorescence plate reader. Western blot was performed to measure ß-catenin and VE-cadherin protein levels and myosin light chain 20 (MLC20) phosphorylation. MBG (≥ 1 nM) and CINO (≥ 10 nM) caused an increase (p < 0.05) in the MPLEC compared to DMSO while ouabain (OUB) had no effect. Pretreatment of LECs with 1 µM L-NAME attenuated (p < 0.05) the MPLEC. The ß-catenin expression in LECs was downregulated (p < 0.05) by MBG and CINO. However, there was no effect on the LECs tight junctions for the CINO group. VE-cadherin expression was downregulated (p < 0.05) by CINO, and MLC20 phosphorylation was upregulated (p < 0.05) by MBG. We demonstrated that MBG and CINO caused an increase in the MPLEC, which were attenuated by L-NAME pretreatment. The data suggest that CTSs exert their effect via nitric-oxide-dependent signaling pathway and may be involved in vascular leak syndrome of LEC lining in preE.

The Role of Lymphatics in Cholestasis: A Comprehensive Review.

Cholestatic liver disease affects millions of people worldwide and stems from a plethora of causes such as immune dysfunction, genetics, cancerous growths, and lifestyle choices. While not considered a classical lymphatic organ, the liver plays a vital role in the lymph system producing up to half of the body's lymph per day. The lymphatic system is critical to the health of an organism with its networks of vessels that provide drainage for lymphatic fluid and routes for surveilling immune cells. Cholestasis results in an increase of inflammatory cytokines, growth factors, and inflammatory infiltrate. Left unchecked, further disease progression will include collagen deposition which impedes both the hepatic and lymphatic ducts, eventually resulting in an increase in hepatic decompensation, increasing portal pressures, and accumulation of fluid within the abdominal cavity (ascites). Despite the documented interplay between these vital systems, little is known about the effect of liver disease on the lymph system and its biological response. This review looks at the current cholestatic literature from the perspective of the lymphatic system and summarizes what is known about the role of the lymph system in liver pathogenesis during hepatic injury and remodeling, immune-modulating events, or variations in interstitial pressures.

Inflammatory state of lymphatic vessels and miRNA profiles associated with relapse in ovarian cancer patients.

Lymphogenic spread is associated with poor prognosis in epithelial ovarian cancer (EOC), yet little is known regarding roles of non-peri-tumoural lymphatic vessels (LVs) outside the tumour microenvironment that may impact relapse. The aim of this feasibility study was to assess whether inflammatory status of the LVs and/or changes in the miRNA profile of the LVs have potential prognostic and predictive value for overall outcome and risk of relapse. Samples of macroscopically normal human lymph LVs (n = 10) were isolated from the external iliac vessels draining the pelvic region of patients undergoing debulking surgery. This was followed by quantification of the inflammatory state (low, medium and high) and presence of cancer-infiltration of each LV using immunohistochemistry. LV miRNA expression profiling was also performed, and analysed in the context of high versus low inflammation, and cancer-infiltrated versus non-cancer-infiltrated. Results were correlated with clinical outcome data including relapse with an average follow-up time of 13.3 months. The presence of a high degree of inflammation correlated significantly with patient relapse (p = 0.033). Cancer-infiltrated LVs showed a moderate but non-significant association with relapse (p = 0.07). Differential miRNA profiles were identified in cancer-infiltrated LVs and those with high versus low inflammation. In particular, several members of the let-7 family were consistently down-regulated in highly inflamed LVs (>1.8-fold, p<0.05) compared to the less inflamed ones. Down-regulation of the let-7 family appears to be associated with inflammation, but whether inflammation contributes to or is an effect of cancer-infiltration requires further investigation.

Ca2+ release-activated Ca2+ channels are responsible for histamine-induced Ca2+ entry, permeability increase, and interleukin synthesis in lymphatic endothelial cells.

The lymphatic functions in maintaining lymph transport, and immune surveillance can be impaired by infections and inflammation, thereby causing debilitating disorders, such as lymphedema and inflammatory bowel disease. Histamine is a key inflammatory mediator known to trigger vasodilation and vessel hyperpermeability upon binding to its receptors and evoking intracellular Ca2+ ([Ca2+]i) dynamics for downstream signal transductions. However, the exact molecular mechanisms beneath the [Ca2+]i dynamics and the downstream cellular effects have not been elucidated in the lymphatic system. Here, we show that Ca2+ release-activated Ca2+ (CRAC) channels, formed by Orai1 and stromal interaction molecule 1 (STIM1) proteins, are required for the histamine-elicited Ca2+ signaling in human dermal lymphatic endothelial cells (HDLECs). Blockers or antagonists against CRAC channels, phospholipase C, and H1R receptors can all significantly diminish the histamine-evoked [Ca2+]i dynamics in lymphatic endothelial cells (LECs), while short interfering RNA-mediated knockdown of endogenous Orai1 or STIM1 also abolished the Ca2+ entry upon histamine stimulation in LECs. Furthermore, we find that histamine compromises the lymphatic endothelial barrier function by increasing the intercellular permeability and disrupting vascular endothelial-cadherin integrity, which is remarkably attenuated by CRAC channel blockers. Additionally, the upregulated expression of inflammatory cytokines, IL-6 and IL-8, after histamine stimulation was abolished by silencing Orai1 or STIM1 with RNAi in LECs. Taken together, our data demonstrated the essential role of CRAC channels in mediating the [Ca2+]i signaling and downstream endothelial barrier and inflammatory functions induced by histamine in the LECs, suggesting a promising potential to relieve histamine-triggered vascular leakage and inflammatory disorders in the lymphatics by targeting CRAC channel functions.

Altered rodent gait characteristics after ~35 days in orbit aboard the International Space Station.

The long-term adaptations to microgravity and other spaceflight challenges within the confines of a spacecraft, and readaptations to weight-bearing upon reaching a destination, are unclear. While post-flight gait change in astronauts have been well documented and reflect multi-system deficits, no data from rodents have been collected. Thus, the purpose of this study was to evaluate gait changes in response to spaceflight. A prospective collection of gait data was collected on 3 groups of mice: those who spent~35 days in orbit (FLIGHT) aboard the International Space Station (ISS); a ground-based control with the same habitat conditions as ISS (Ground Control; GC); and a vivarium control with typical rodent housing conditions (VIV). Pre-flight and post-flight gait measurements were conducted utilizing an optimized and portable gait analysis system (DigiGait, Mouse Specifics, Inc). The total data acquisition time for gait patterns of FLIGHT and control mice was 1.5-5 min/mouse, allowing all 20 mice per group to be assessed in less than an hour. Patterns of longitudinal gait changes were observed in the hind limbs and the forelimbs of the FLIGHT mice after ~35 days in orbit; few differences were observed in gait characteristics within the GC and VIV controls from the initial to the final gait assessment, and between groups. For FLIGHT mice, 12 out of 18 of the evaluated gait characteristics in the hind limbs were significantly changed, including: stride width variability; stride length and variance; stride, swing, and stance duration; paw angle and area at peak stance; and step angle, among others. Gait characteristics that decreased included stride frequency, and others. Moreover, numerous forelimb gait characteristics in the FLIGHT mice were changed at post-flight measures relative to pre-flight. This rapid DigiGait gait measurement tool and customized spaceflight protocol is useful for providing preliminary insight into how spaceflight could affect multiple systems in rodents in which deficits are reflected by altered gait characteristics.

Histamine-mediated autocrine signaling in mesenteric perilymphatic mast cells.

Lymphatic vessels play a critical role in mounting a proper immune response by trafficking peripheral immune cells to draining lymph nodes. Mast cells (MCs) are well known for their roles in type I hypersensitivity reactions, but little is known about their secretory regulation in the lymphatic niche. MCs, as innate sensor and effector cells, reside close to mesenteric lymphatic vessels (MLVs), and their activation and ability to release histamine influences the lymphatic microenvironment in a histamine-NF-κB-dependent manner. Using an established experimental protocol involving surgical isolation of rat mesenteric tissue segments, including MLVs and surrounding perilymphatic tissues, we tested the hypothesis that perilymphatic mesenteric MCs possess histamine receptors (HRs) that bind and respond to the histamine released from these same MCs. Under various experimental conditions, including inflammatory stimulation by LPS, we measured histamine in mesenteric perilymphatic tissues, evaluated expression of histidine decarboxylase in MCs along with the degree of MC degranulation, assessed the functional status of HRs in MCs, and evaluated the ability of histamine itself to induce MC activation. Finally, we evaluated the importance of MCs and HR1 and -2 for MLV-directed trafficking of CD11b/c-positive cells during acute tissue inflammation. Our data indicate the existence of a functionally potent MC-histamine autocrine regulatory loop, the elements of which are crucially important for acute inflammation-induced trafficking of the CD11b/c-positive cells toward MLVs. This MC-histamine loop serves as a first-line cellular servo control system, playing a key role in the innate and adaptive immune response as well as NF-κB-mediated maintenance of body homeostasis.

Modulation of the Tryptophan Hydroxylase 1/Monoamine Oxidase-A/5-Hydroxytryptamine/5-Hydroxytryptamine Receptor 2A/2B/2C Axis Regulates Biliary Proliferation and Liver Fibrosis During Cholestasis.

BACKGROUND AND AIMS: Serotonin (5HT) is a neuroendocrine hormone synthetized in the central nervous system (CNS) as well as enterochromaffin cells of the gastrointestinal tract. Tryptophan hydroxylase (TPH1) and monoamine oxidase (MAO-A) are the key enzymes for the synthesis and catabolism of 5HT, respectively. Previous studies demonstrated that 5-hydroxytryptamine receptor (5HTR)1A/1B receptor agonists inhibit biliary hyperplasia in bile-duct ligated (BDL) rats, whereas 5HTR2B receptor antagonists attenuate liver fibrosis (LF) in mice. Our aim was to evaluate the role of 5HTR2A/2B/2C agonists/antagonists in cholestatic models. APPROACH AND RESULTS: While in vivo studies were performed in BDL rats and the multidrug resistance gene 2 knockout (Mdr2-/- ) mouse model of PSC, in vitro studies were performed in cell lines of cholangiocytes and hepatic stellate cells (HSCs). 5HTR2A/2B/2C and MAO-A/TPH1 are expressed in cholangiocytes and HSCs from BDL rats and Mdr2-/- - mice. Ductular reaction, LF, as well as the mRNA expression of proinflammatory genes increased in normal, BDL rats, and Mdr2-/- - mice following treatment 5HTR2A/2B/2C agonists, but decreased when BDL rats and Mdr2-/- mice were treated with 5HTR2A/2B/2C antagonists compared to BDL rats and Mdr2-/- mice, respectively. 5HT levels increase in Mdr2-/- mice and in PSC human patients compared to their controls and decrease in serum of Mdr2-/- mice treated with 5HTR2A/2B/2C antagonists compared to untreated Mdr2-/- mice. In vitro, cell lines of murine cholangiocytes and human HSCs express 5HTR2A/2B/2C and MAO-A/TPH1; treatment of these cell lines with 5HTR2A/2B/2C antagonists or TPH1 inhibitor decreased 5HT levels as well as expression of fibrosis and inflammation genes compared to controls. CONCLUSIONS: Modulation of the TPH1/MAO-A/5HT/5HTR2A/2B/2C axis may represent a therapeutic approach for management of cholangiopathies, including PSC.

DSS-induced colitis produces inflammation-induced bone loss while irisin treatment mitigates the inflammatory state in both gut and bone.

Chronic pediatric inflammatory bowel disease (IBD) leads to lack of bone accrual, bone loss, and increased fractures. Presently there is no cure, and many IBD treatments incur negative side effects. We previously discovered treatment with exogenous irisin resolved inflammatory changes in the colon, gut lymphatics, and bone in a mild IBD rodent model. Here we assess irisin treatment in severe IBD induced via dextran sodium sulfate (DSS). Male Sprague Dawley rats (2-mo-old) were untreated (Con) or given 2% DSS in drinking water. In week two, half of each group (Con + Ir and DSS + Ir) received injections of recombinant irisin (i.p., 2x/wk). After 4 weeks, gut inflammation was associated with declines in bone mineral density and cancellous bone volume. Furthermore, elevated osteocyte TNF-α, interleukin-6, RANKL, OPG, and sclerostin corresponded with higher osteoclast surfaces and lower bone formation rate in DSS animals as well as lower ultimate load. While irisin treatment improved colon inflammation, there were no improvements in bone density or bone mechanical properties; however, irisin elevated bone formation rate, decreased osteoclast surfaces, and reduced osteocyte pro-inflammatory factors. These data highlight the negative impact of chronic gut inflammation on bone as well as the therapeutic potential of irisin as an anti-inflammatory treatment.

Spaceflight influences gene expression, photoreceptor integrity, and oxidative stress-related damage in the murine retina.

Extended spaceflight has been shown to adversely affect astronaut visual acuity. The purpose of this study was to determine whether spaceflight alters gene expression profiles and induces oxidative damage in the retina. Ten week old adult C57BL/6 male mice were flown aboard the ISS for 35 days and returned to Earth alive. Ground control mice were maintained on Earth under identical environmental conditions. Within 38 (+/-4) hours after splashdown, mice ocular tissues were collected for analysis. RNA sequencing detected 600 differentially expressed genes (DEGs) in murine spaceflight retinas, which were enriched for genes related to visual perception, the phototransduction pathway, and numerous retina and photoreceptor phenotype categories. Twelve DEGs were associated with retinitis pigmentosa, characterized by dystrophy of the photoreceptor layer rods and cones. Differentially expressed transcription factors indicated changes in chromatin structure, offering clues to the observed phenotypic changes. Immunofluorescence assays showed degradation of cone photoreceptors and increased retinal oxidative stress. Total retinal, retinal pigment epithelium, and choroid layer thickness were significantly lower after spaceflight. These results indicate that retinal performance may decrease over extended periods of spaceflight and cause visual impairment.

Lymphatic Cannulation for Lymph Sampling and Molecular Delivery.

Unlike the blood, the interstitial fluid and the deriving lymph are directly bathing the cellular layer of each organ. As such, composition analysis of the lymphatic fluid can provide more precise biochemical and cellular information on an organ's health and be a valuable resource for biomarker discovery. In this study, we describe a protocol for cannulation of mouse and rat lymphatic collectors that is suitable for the following: the "omic" sampling of pre- and postnodal lymph, collected from different anatomical districts; the phenotyping of immune cells circulating between parenchymal organs and draining lymph nodes; injection of known amounts of molecules for quantitative immunological studies of nodal trafficking and/or clearance; and monitoring an organ's biochemical omic changes in pathological conditions. Our data indicate that probing the lymphatic fluid can provide an accurate snapshot of an organ's physiology/pathology, making it an ideal target for liquid biopsy.

Characterization of mouse ocular response to a 35-day spaceflight mission: Evidence of blood-retinal barrier disruption and ocular adaptations.

The health risks associated with spaceflight-induced ocular structural and functional damage has become a recent concern for NASA. The goal of the present study was to characterize the effects of spaceflight and reentry to 1 g on the structure and integrity of the retina and blood-retinal barrier (BRB) in the eye. To investigate possible mechanisms, changes in protein expression profiles were examined in mouse ocular tissue after spaceflight. Ten week old male C57BL/6 mice were launched to the International Space Station (ISS) on Space-X 12 at the Kennedy Space Center (KSC) on August, 2017. After a 35-day mission, mice were returned to Earth alive. Within 38 +/- 4 hours of splashdown, mice were euthanized and ocular tissues were collected for analysis. Ground control (GC) and vivarium control mice were maintained on Earth in flight hardware or normal vivarium cages respectively. Repeated intraocular pressure (IOP) measurements were performed before the flight launch and re-measured before the mice were euthanized after splashdown. IOP was significantly lower in post-flight measurements compared to that of pre-flight (14.4-19.3 mmHg vs 16.3-20.3 mmHg) (p < 0.05) for the left eye. Flight group had significant apoptosis in the retina and retinal vascular endothelial cells compared to control groups (p < 0.05). Immunohistochemical analysis of the retina revealed that an increased expression of aquaporin-4 (AQP-4) in the flight mice compared to controls gave strong indication of disturbance of BRB integrity. There were also a significant increase in the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and a decrease in the expression of the BRB-related tight junction protein, Zonula occludens-1 (ZO-1). Proteomic analysis showed that many key proteins and pathways responsible for cell death, cell cycle, immune response, mitochondrial function and metabolic stress were significantly altered in the flight mice compared to ground control animals. These data indicate a complex cellular response that may alter retina structure and BRB integrity following long-term spaceflight.