C57BL6 Mice Research Articles

Morphological and functional observations of a novel model of retinal ischemia injury induced by bilateral carotid artery stenosis in mice.

To investigate the features of retinal ischemic injuries in a novel mouse model with bilateral carotid artery stenosis (BCAS). BCAS was induced with microcoil implantation in 6-8-week-old C57BL6 mice. Cerebral blood flow was monitored at 2, 7, and 28d postoperatively. Retinal morphological changes were evaluated by fundus photography and hematoxylin-eosin staining. Fluorescein fundus angiography (FFA) was performed to detect retinal vascular changes and circulation. The levels of apoptosis, activation of neurogliosis, and expression of hypoxia-inducible factor (HIF)-1α in the retina were assessed by Western blotting and immunofluorescence staining, followed by retinal ganglion cell (RGC) density detection. Additionally, electrophysiological examinations including photopic negative response (PhNR) was also performed. The mice demonstrated an initial rapid decrease in cerebral blood flow, followed by a 4-week recovery period after BCAS. The ratio of retinal artery and vein was decreased under fundus photography and FFA. Compared with the sham mice, BCAS mice showed thinner retinal thickness on day 28. Additionally, apoptosis was increased and RGC density was decreased mainly in peripheral retinal region. Neurogliosis was mainly located in the inner retinal layers, with a stable increase in HIF-1α expression. The dark-adapted electroretinogram showed a notable reduction in the a-, b-, and oscillatory potential (OP) wave amplitudes between days 2 and 7; this gradually recovered over the following 4wk. However, the b- and OP-wave amplitudes were still significantly decreased on PhNR examination on day 28. BCAS can result in relatively mild retinal ischemia injuries in mice, mainly in the inner layer and peripheral region. Our study provides a novel animal model for investigating retinal ischemic diseases.

Unique Leishmania mexicana clones secrete populations of extracellular vesicles with unique protein profile and variable infectious capability

The study of extracellular vesicles has become an incredibly important field of study, but the inherent heterogeneity of these vesicles continues to make their study challenging. The genetic variability and well-documented protocols for the growth and vesicle isolation from Leishmania parasites provide a unique opportunity to compare the heterogeneity of different populations secreted by Leishmania clones. Leishmania mexicana was cultured on solid SDM agar plates and 8 clonal colonies were selected. The EVs collected from the liquid cultures of these 8 clones were assessed by NTA, TEM, and proteomic analysis. We found that all 8 clonal L. mexicana cultures were visually indistinguishable from each other and had similar growth rate, and these physical similarities extended to their EVs. However, proteomic analysis reveals that the EVs collected have unique protein profiles compared to each other and EVs isolated from a heterogeneous liquid culture of L. mexicana. We selected 3 clonal EVs for further mouse infection experiments and found that EVs from CL7 L. mexicana consistently caused reduced footpad swelling in C57BL6 mice footpads compared to EVs from CL1, CL8, and heterogenous L. mexicana. This trend was not observed when infecting Balb/C mice and C57BL6 with the parasites alone, with only CL1 L. mexicana causing significantly increased infection in Balb/c mice. Our results together show that EVs isolated from different clonal colonies of L. mexicana have distinct differences in protein cargo which can lead to varying outcomes on Leishmania infection. Further evaluation will be needed to determine the underlying mechanisms behind this and verify that differences observed in infectivity are directly caused by variations between our L. mexicana clones, especially genetic sequencing and immunoblotting to validate our results.

In Vivo Mouse Abdominal Oxygen Imaging And Assessment of Subcutaneously Implanted Beta Cell Replacement Devices.

Type 1 diabetes (T1D) is an autoimmune disease that leads to the loss of insulin-producing pancreatic beta cells. Beta cell replacement devices or bioartificial pancreas (BAP) have shown promise in curing T1D and providing long-term insulin independence without the need for immunosuppressants. Hypoxia in BAP devices damages cells and imposes limitations on device dimensions. Noninvasive in vivo oxygen imaging assessment of implanted BAP devices will provide the necessary feedback and improve the chances of success. Pulse-mode electron paramagnetic resonance (EPR) oxygen imaging (EPROI) using injectable trityl OX071 as the oxygen-sensitive agent is an excellent technique for obtaining partial oxygen pressure (pO2) maps in vitro and in vivo. In this study, our goal was to optimize in vivo mouse abdominal EPROI and demonstrate proof-of-concept pO2 imaging of subcutaneously implanted BAP devices. All EPROI experiments were performed using a 25 mT EPROI instrument, JIVA-25®. For in vivo EPROI experiments, trityl OX071, a whole-body mouse resonator (∅32mm × 35mm), C57BL6 mice, and the inversion recovery electron spin echo (IRESE) pulse sequence were utilized. We investigated the signal amplitude and pO2 in mouse abdomen region for intravenous (i.v.) and intraperitoneal (i.p.) injection methods with either only a single bolus (B) or bolus plus infusion (BI) for 72.2mM OX071 and the effect of OX071 concentrations from 18 to 72.2 mM for the i.p.-B injection method. We also investigated the impact of animal respiratory rate on mouse abdominal pO2. Finally, we performed proof-of-concept pO2 imaging of two subcutaneously implanted BAP devices, OxySite and TheraCyte. At the end of the four-week study, the TheraCyte devices were extracted and analyzed for fibrosis, vascular differentiation, and immune cell infiltration. We established that mouse abdominal pO2 remains stable irrespective of trityl injection methods, concentrations, imaging time, or animal breathing rate. We demonstrate that the i.p.-B and i.p.-BI methods are suitable for EPROI, and i.p.-B method provides higher signal amplitude compared to i.v.-BIand up to 75min of imaging. An injection with a reduced trityl concentration and higher volume provides higher signal amplitude for i.p.-B method at the beginning. We also highlight the advantage of milder anesthesia for consistent, reliable mouse pO2 imaging. Finally, we demonstrate that EPROI could provide longitudinal noninvasive oxygen assessment of subcutaneously implanted BAP devices in vivo. In vivo EPROI is a reliable technique for mouse abdominal oxygen imaging and longitudinal assessment of subcutaneously implanted BAP devices noninvasively. This work reports abdominal oxygen imaging in the mouse model and demonstrates its application for the assessment of BAP devices. Even though the application focus of this work was on cell therapy, the techniques developed will have a much broader use in the biomedical field.

Resolving spatiotemporal electrical signaling within the islet via CMOS microelectrode arrays.

Glucose-stimulated beta-cells exhibit synchronized calcium dynamics across the islet that recruit beta-cells to enhance insulin secretion. Compared to calcium dynamics, the formation and cell-to-cell propagation of electrical signals within the islet are poorly characterized. To determine factors that influence the propagation of electrical activity across the islet underlying calcium oscillations and beta-cell synchronization, we used high-resolution CMOS multielectrode arrays (MEA) to measure voltage changes associated with the membrane potential of individual cells within intact C57BL6 mouse islets. We measured fast (milliseconds, spikes) and slow (seconds, waves) voltage dynamics. Single spike activity and wave signal velocity were both glucose-dependent, but only spike activity was influenced by NMDA receptor activation or inhibition. A repeated glucose stimulus revealed a highly responsive subset of cells in terms of spike activity. When islets were pretreated for 72 hours with glucolipotoxic medium, the wave velocity was significantly reduced. Network analysis confirmed that in response to glucolipotoxicity the synchrony of islet cells was affected due to slower propagating electrical waves and not due to altered spike activity. In summary, this approach provided novel insight regarding the propagation of electrical activity and the disruption of cell-to-cell communication due to excessive stimulation.

Berbamine Promotes the Repair of Lower Limb Muscle Damage in Chronic Limb-Threatening Ischemia by Inhibiting Local Inflammation and NF-κB Nuclear Translocation

Background/Objectives: Chronic Limb-Threatening Ischemia (CLTI) is a chronic limb ischemic disease caused by vascular lesions, characterized by pain, ulcers, and gangrene, which can be life-threatening in severe cases. The objective of this study is to explore whether Berbamine (BBM) can protect against and repair ischemic muscle tissue in the lower limbs; Methods: Using a mouse hindlimb ischemia (HLI) model, 36 C57BL6 mice were divided into sham, HLI, and HLI+BBM treatment groups. Results: Our findings indicate that BBM can restore motor function and muscle tissue pathology in mice, potentially by inhibiting the nuclear translocation of nuclear factor kappa-B (NF-κB), thereby alleviating tissue inflammation caused by chronic ischemia, reducing muscle cell apoptosis, inhibiting M1 macrophage polarization, and promoting angiogenesis. Conclusions: Our research suggests that BBM has the potential to protect against ischemic damage in lower limb muscle tissue, providing a new approach to the treatment of CLTI.

Proanthocyanidins alleviate acute alcohol liver injury by inhibiting pyroptosis via inhibiting the ROS-MLKL-CTSB-NLRP3 pathway

BackgroundAlcoholic Liver Disease (ALD) is a hepatic disorder resulting from prolonged or excessive alcohol intake. The predominant manifestation of ALD is fatty liver, which progresses to alcoholic hepatitis as the disease worsens. Pyroptosis is a novel type of programmed cell death that is intricately linked to the inflammatory cascade, presenting a promising avenue for therapeutic intervention in the management of ALD. Oligomeric proanthocyanidins (OPCs) are polyphenols extracted from grape seeds that have anti-inflammatory and antioxidant properties. However, whether OPCs can treat ALD by suppressing pyroptosis is not completely clarified. PurposeTo explore the role of OPCs in ALD to inhibit pyroptosis and its mechanism. MethodsIn vitro, HepG2 cells were employed to evaluate the beneficial impact of OPCs on alcohol-induced pyroptosis. MTT colorimetric method, enzyme-linked immunosorbent assay (ELISA), western blot (WB), immunofluorescence, acridine orange (AO) staining, and reactive oxygen species (ROS) assay were performed. In vivo, C57BL mice were used and gavaged with alcohol and OPCs. Hematoxylin-eosin staining (HE) staining, alanine aminotransferase (ALT), aspartate aminotransferase (AST) level assay, and WB were performed. ResultsThe findings revealed that OPCs could reduce the alcohol-induced increase in pyroptosis-related proteins, such as pyrin domain-containing 3 protein (NLRP3), cleaved-caspase 1, gasdermin D (GSDMD-N), Interleukin-18 (IL-18), IL-1β (IL-1β). In in vitro mechanistic experiments, We discovered that OPCs ameliorate alcohol-induced pyroptosis by decreasing cathepsin B (CTSB) leakage-mediated NLRP3 activation. More significantly, we discovered that alcohol phosphorylates mixed lineage kinase domain-like protein (MLKL), enabling P-MLKL to translocate to the lysosomal membrane and induce lysosomal membrane permeabilization (LMP). OPCs might counteract the effects of alcohol by reducing the leakage of CTSB and inhibiting the phosphorylation of MLKL through the scavenging of ROS. Conclusions: These results suggested that OPCs might counteract ALD by inhibiting pyroptosis through the ROS-MLKL-CTSB-NLRP3 pathway. Our study offered fresh insight into the ways in which naturally occurring chemicals shield ALD against harm.

Age of Cafeteria Diet Onset Influences Obesity Phenotype in Mice in a Sex-Specific Manner.

We investigated the influence of sex and the age of obesogenic diet initiation on the obesity phenotypes at a later age. C57Bl mice started the Cafeteria Diet (CafD, with increased fat and carbohydrates, ad libitum, from 7 weeks of age (7CafD, pre-puberty) or 17 weeks of age (7CafD, post-puberty) while control C57Bl mice were fed regular chow. At 27 weeks of age, 7CafD males (n = 9) compared to 17CafD males (n = 7) had lower body weight, white adipose tissue (WAT) relative weight, and plasma cholesterol levels, and a higher expression of thermogenic genes in WAT and brown adipose tissue (BAT), and fatty acid oxidation (FAO) and insulin signalling genes in muscles. The 7CafD females (n = 8), compared to 17CafD females (n = 6), had higher plasma triglyceride levels and hepatic glycogen content, but lower insulin sensitivity and hepatic expression of FAO and insulin signalling genes. The 7CafD females, compared to 7CafD males, had more WAT, and a reduced expression of FAO genes in muscles and thermogenic genes in WAT. The 17CafD females, compared to 17CafD males, had lower plasma leptin and insulin levels, and higher insulin sensitivity and expression of insulin signalling genes in the liver and muscles. Thus, the initiation of the obesogenic diet before puberty led to a more adaptive metabolic phenotypes in males, and after puberty, in females.

Abstract C009: MK2 knockout and radiation enhances immune cell influx in a mouse model of HPV+ head and neck squamous cell carcinoma

Abstract Introduction: HPV mediated head and neck squamous cell carcinoma (HNSCC) has an immunosuppressive microenvironment which contributes to local and distant failures. We have previously shown that MAPKAPK2 (MK2) inhibition (MK2i) in HNSCC results in improved response to irradiation (RT). MK2i was synergistic with RT resulting in better control than monotherapy. To understand the mechanism behind the improvement of RT in MK2i treated mice we tested the hypothesis that MK2 ablation can enhance the radiotherapy mediated immunogenic response in a solid tumor. Methods: We used CRISPR gene editing to knockout the MK2 gene in the highly metastatic murine cell line, MLM3 (KO). We implanted tumors into c57bl6 and NSG mice and compared tumor growth compared to wild type (WT) with and without radiation. At end point, tumors were excised, digested and immunophenotyped utilizing labelled antibodies and flow cytometry. Before immunophenotyping, 8 mgs of tumor were cut off and incubated in media for 24 hours to assess cytokine release by cytokine array analysis. Results: We found that the differences in tumor growth between WT and KO cells in c57bl6 mice were significantly greater than those seen in NSG mice. Survival studies with c57bl6 mice implanted with WT or KO tumors with and without RT showed that MLM3 KO implanted mice had significantly greater survival (58.5 days) compared to WT (35.5 days). WT tumors treated with RT improved median overall survival with the WT + RT group surviving longer than WT (56 days) and the MK2 KO + RT had the longest survival overall (65 days). Immunophenotyping WT v KO tumors grown in c57bl6 mice showed an increase in CD45 cells in the KO tumors compared to WT. The cells that were increased in number in the KO tumors were labelled by Ly6G (neutrophils), F4/80 (macrophages), CD14+MD11c (dendritic cells), CD4 (Th), CD8 (Tc), CD3+NK1.1 (NKt) and NK1.1 (NK cells). Irradiating the tumors accentuated the influx of immune cells causing a greater influx of CD45 cells at 3 days post-RT which included increases in F4/80+CD80 (M1 macrophages), CD14 (monocytes), CD11c+MHCII, NK1.1, NK1.1+CD3 and CD4 cells. However, the influx of CD8 cells were reduced at 3 days. At 8 days post RT, many cell types remained elevated in the KO tumors compared to wildtype and there was a partial recovery of CD8 cell influx. However, there was also an increase in immune suppressor cells into the tumors including Ly6C+arginase (M-MDSCs), Ly6C+arginase (PMN-MDSCs) and CD4+T-bet (Th1) cells. The cytokine analysis showed that six cytokines were differentially secreted from tumors: Dkk-1, IL-2, IGFBP-5, IL-3, IL-17a and CD14. All these cytokines were suppressed in the KO with respect to the WT tumors. Conclusion: Here we show evidence that genetic ablation of MK2 in a mouse model of HPV mediated HNSCC results in an increase in immune cells infiltrate into tumors. Irradiation enhances this difference by stimulating a greater influx of immune cells. These data suggest that tumoral MK2 may aid in the immunosuppression of tumors in HNSCC. Citation Format: Deri Morgan, Colby Spiess, Alyssa Schmidt, Dakota D.D. Okuwone, Harmony Saunders, Chris E. Lominska, Mary A. Markiewicz, Yelder Tonia, Devin Shrock, Yuting Lin, Hao Gao, Gregory N Gan. MK2 knockout and radiation enhances immune cell influx in a mouse model of HPV+ head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C009.

Organic vs. inorganic nitrates: Metabolic and vascular outcomes in STZ-induced diabetes in mice

BackgroundDiabetic animals often display dysregulated nitric oxide (NO) metabolism, contributing to vascular dysfunction. This study evaluates the metabolic and vascular effects of organic nitrate isosorbide mononitrate (ISMN) versus inorganic sodium nitrate (NaNO3) in mice with type 1 diabetes mellitus (T1DM) induced by streptozotocin (STZ). Experimental approachT1DM was induced in male C57Bl6 mice with STZ ip and confirmed by fasting glucose. Mice were treated with ISMN (10 mg·kg-1) or NaNO3 (85 mg·L-1) for 14 days. A combination of in vivo, in vitro, and ex vivo studies assessed cardiometabolic benefits. ResultsBoth nitrates reduced blood and urinary hyperglycemia in T1DM mice, with ISMN exhibiting more significant reductions in blood glucose. ISMN and NaNO3 similarly reduced water and food intake, urinary volume, glucose intolerance, and insulin resistance while increasing insulin and nitrite levels in serum and urine. Both nitrates improved endothelium-independent vascular function and attenuated reactive oxygen species (ROS) while increasing NO levels in the aortic rings of T1DM mice. Furthermore, both nitrates similarly reduced mean arterial pressure in T1DM mice. Conclusion and implicationsISMN and NaNO₃ have demonstrated comparable hypotensive and antioxidant effects, offering metabolic and vascular benefits in STZ-TDM1 mice. The more pronounced reduction in blood glucose with ISMN treatment compared to NaNO₃ is particularly promising. The antihyperglycemic effects of both nitrates were linked to increased serum insulin levels and enhanced insulin sensitivity. These results provide a foundation for future clinical studies to evaluate the potential of ISMN or NaNO3 as antidiabetogenic and antihypertensive adjuvant therapies in diabetic patients.

Piperine‐Loaded Sodium Caseinate Nanoglobules Delivered Transdermally Through Microneedle Patch for Improved Bioavailability at Adipose Tissue

ABSTRACTDespite being a well‐known anti‐obesity chemical, piperine has only found limited therapeutic use because of its poor bioavailability at the site of action. In order to test this hypothesis, we placed piperine sodium caseinate nanoglobules (PSN) in microneedle patches (MNPs), which may enhance piperine transdermal delivery and absorption. Clove oil, sodium caseinate to make nanoglobules, and PEG 400 as a stabilizer were used in a nanoglobules process to create the PSN. The polydispersity index (0.256) and particle size (134.4 nm) of the developed PSN were both smaller, while their zeta potential (−44.4 mV) and encapsulation efficiency (90%–95%) were both greater. Compared to native piperine, the PSN increased solubility, bioaccessibility (6.5 times), and bioavailability (2.8 fold). Moreover, the PSN‐loaded MNPs were created using the micro‐molding (poly‐dimethylsiloxane (PDMS) mold) approach. In both PBS buffer and porcine skin, the PSN‐loaded MNPs were rapidly hydrolyzed, with 50% piperine release in less than 5 min. Also, the produced MNPs demonstrated improved bioavailability of piperine in the plasma (180 ng/mL) and adipose (425 ng/mL) tissue of C57BL6 mice. In conclusion, the work offered a theoretical foundation for utilizing new PSN‐loaded MNPs as a viable piperine delivery method for treating obesity.

Abstract 4132968: CCR5 modulation enhances M-MDSCs functionality in early atherosclerosis

Background: The role of immune suppressive microenvironment, promulgated by the presence of Myeloid Derived Suppressor Cells (MDSC) have been the major focus of research delineating the underlying mechanisms of cardiovascular diseases (CVD). CCR5, a chemokine receptor, has been associated with both immunosuppressive and inflammatory phenotypes, however, the possible role of CCR5 pertaining to MDSCs in the development of atherosclerosis has not been elucidated yet. Therefore, we investigated the therapeutic potential of CCR5 modulation in early atherosclerosis progression. Methods: CCR5 receptor-ligand expression was studied with flow cytometry, real time PCR and immunocytochemistry in 65 individuals (25 Healthy controls, 20 young hypercholesterolemic individuals, and 20 stable [Coronary Artery Disease] CAD patients). To induce atherosclerosis, mice were fed high fat diet (HFD) for 6 weeks following partial ligation of left carotid artery in C57BL6 mice. MDSCs phenotype and functionality were assessed using CCR5 inhibitor in vitro (10 -8 M) and in vivo (i.p 3ng/day for 15 days) . M-MDSCs phenotype switch, atherosclerotic lesion development and plaque phenotype was studied in vivo . Results: We observed CCR5 elevation on Monocytic-MDSCs in the early phase of atherosclerosis. M-MDSCs displayed an intermediate phenotype with expression of both pro and anti-inflammatory mediators in early atherosclerosis, a similar trend was observed in inflammatory cytokines stimulated M-MDSCs ( in vitro ). Notably, DAPTA treatment shifted phenotype of M-MDSCs to anti-inflammatory profile with elevated expression of IL-10 and reduced expression of IFNγ, IL4, and IL22. Further, CCR5 modulation expanded IL10+M-MDSC in vivo . Additionally, DAPTA reduced the migratory potential, reduced cholesterol uptake and improved the functionality (suppression of T cell) of M-MDSCs. Blocking CCR5 with DAPTA led to the formation of stable plaque in HFD-fed mice. Conclusion: Increased expression of CCR5 coupled with inflammatory cytokine secretion by regulatory cells lead to dysfunctional M-MDSCs in CVD risk factor population. Possibly this dysfunctionality contributes to the development and progression of CVD including atherosclerosis. Our findings show targeting of CCR5 with DAPTA reduces atherogenesis, increases plaque stabilization and corrects phenotypic alterations in immune suppressive M-MDSCs. The functionality and phenotype of M-MDSCs in atherosclerosis, may be ameliorated by targeting CCR5.

Abstract 4146302: NETosis Attenuating Nanoprobe Improves Survival after Cardiac Arrest

NETosis, the process of neutrophil cell death due to the formation and release of neutrophil extracellular traps (NETs) has been implicated in the pathogenesis of heart disease. NETs are composed of immunogenic DNA fragments and citrullinated histones. The role of NETosis in the context of cardiac arrest and resuscitation (CA/R) is however unknown. We previously reported the development of a Dextran-Thiazole Orange (DTO) nanoprobe, with nanomolar affinity for nucleic acids, that exerts acute anti-inflammatory effects and reduces ischemia-reperfusion injury in mice. We aimed here to determine whether the DTO nanoprobe would also bind to the nucleic acid in NETS, whether DTO could attenuate the intensity of NETosis, and whether this would improve outcomes and survival after cardiac arrest. C57Bl6 mice were arrested for 8 minutes before resuscitation. DTO or unmodified dextran (vehicle control) was injected at the time of resuscitation, and again 4 hours later (n=38 mice). Survival and neurological function were scored daily. Time-domain resolved (lifetime) and conventional fluorescence imaging was performed at 4 hours after resuscitation in 6 additional mice injected with DTO. NETosis was evaluated by protein blot in the hearts of cardiac arrest mice injected with DTO or control. DTO significantly (p<0.05) improved 10-day survival (Fig. A), mean survival (from 4 to 7 days, Fig. B), and neurological function (Fig. C) following CA/R. Lifetime imaging (Fig. D), but not conventional fluorescence imaging (Fig. E) revealed the presence of DTO in the injured heart, liver and kidneys. In the heart, DTO signal was significantly increased in CA/R compared to sham mice injected with DTO or CA/R mice injected with control dextran (Fig. F). Citrullinated H3 and HMGB-1 (High mobility group box 1, a chromatin protein) were both upregulated in CA/R, and were significantly attenuated by DTO injection (Fig. G-I). We show for the first time that NETosis plays a key role in cardiac arrest and resuscitation. DTO is capable of detecting NETosis as well as attenuating it, likely via a reduction in HMGB-1 activity. The modulation of NETosis may provide a new avenue to improve survival after cardiac arrest.

TMIC-12. LITT RESULTS IN ROBUST T CELL INFILTRATION WITHIN THE TUMOR MICROENVIRONMENT IN A BRAIN METASTASIS MURINE MODEL

Abstract INTRODUCTION Laser interstitial thermal therapy (LITT) is a novel minimally invasive neurosurgical technique used to ablate intra-axial brain tumors with real-time thermometry. The impact of LITT on the brain metastatic tumor microenvironment (TME) is unknown in patients due to limitations of tumor tissue collection post-LITT. The primary objective of this study was to describe the kinetics of immunologic infiltration within the TME in a melanoma brain metastasis model. METHOD: C57BL6 mice underwent intra-cranial implantation of B16F10-OVA tumor cells and treated with LITT using a custom-made murine LITT system developed in our laboratory. The tumors were treated to 42°C (near infra-red 1064 nm DPSS laser system) 7 days post-surgery. Tumors were collected 2 days and 10 days post-LITT for immune-phenotyping using flow cytometry. Blood was collected 6 h and 48 h post-LITT to perform the ELISA for IFN-g and TNF-a. RESULTS LITT treatment triggered a cascade of local infiltration of immune cells at different time points. 2-days post-LITT resulted in increased CD8 T cells (p<0.05), macrophage (p<0.05) and microglia infiltration (p<0.01) within the TME compared to control and sham surgery animals. By 10 days after LITT, significant infiltration by CD8 T cells (p<0.0001) and M1 macrophages (p<0.0001) was observed. The CD8 T cells were T central memory (TCM) and T effector memory (TEM). Moreover, comparison between 2 days and 10 days after LITT revealed significant increase of TEM (p<0.0001) and TCM (p<0.001) 10 days post-LITT. In an ELISA assay, non-significant differences were observed in IFN-g and TNF-a level at 48 h. CONCLUSIONS LITT enhances the recruitment of cytotoxic CD8 T cells within TME in a time-dependent manner. The ability of LITT to recruit TCM and TEM to the TME strongly support its potential therapeutic benefit, especially in the context of immunotherapy.

Exosomes derived from Umbilical cord mesenchymal stem cell promote hair regrowth in C57BL6 mice through upregulation of the RAS/ERK signaling pathway.

Androgenetic alopecia is one of the common types of hair loss and has become a medical and social problem due to its increasingly young onset. Existing therapies, although effective, have serious side effects and therefore better treatments need to be sought. The aim of this study was to evaluate the efficacy of umbilical cord mesenchymal stem cell-derived exosomes in the treatment of androgenetic alopecia and to investigate the mechanism of exosome regulation of hair growth. First, we randomly divided 20 C57BL/6J mice into blank group, model group, positive control group and exosomal hydrogel group, and mice were treated with hair removal on the back. The mice were injected intraperitoneally with dihydrotestosterone solution except for the blank group. At the end of the experiment, new hairs were collected and the differences in length, diameter and number of hair follicles were compared among the groups; the histopathological changes of hair follicles were observed by HE staining; the expression of androgen receptor mRNA and protein in skin tissues were compared; and the skin tissues were analyzed by real-time PCR, western blotting, immunofluorescence staining and transcriptome sequencing. Finally, the results of transcriptome sequencing experiments were verified by real-time PCR, western blotting and other techniques for the corresponding genes and proteins. Compared with the blank group, mice in the model group had shorter hair length and reduced hair diameter, and pathological observation showed that the total number of hair follicles was significantly reduced and the hair follicles were miniaturized; compared with the model group, mice in the positive control and exosome groups had longer hair length, larger hair diameter and more hair follicles; the androgen receptor mRNA content and protein expression in the skin tissue of mice in the model group were significantly higher than those in the blank group, and the protein expression in the exosome gel group was lower than that in the model group. Similarly, compared with the model group, the expression of stemness-related proteins K15 and CD200 in the skin tissues of mice in the exosome group increased, and the expression of PCNA, a protein related to cell proliferation, increased. The KEGG data showed that the differential genes were mainly enriched in the RAS/ERK pathway. In this study, we demonstrated the therapeutic effect of umbilical cord MSC-derived exosomes on androgenetic alopecia and verified that exosomes regulate hair follicle stem cell stemness through the RAS/ERK pathway to promote hair proliferation and thus hair growth in mice with androgenetic alopecia, providing a potential therapeutic strategy for androgenetic alopecia.

Does concomitant diazepam and ethanol use modulate age-related cognitive decline in mice?

BackgroundConcomitant use of alcohol and benzodiazepines are described among elderly, raising concerns about their combined impact on memory. We aimed to evaluate the long-term impact of chronic diazepam use associated with ethanol intoxication on memory in aging mice. MethodsTwelve-month-old male C57BL6 mice were assigned into 4 groups: ethanol (OH), diazepam (DIA), diazepam + ethanol (DOH) and control (CTL). For 16 weeks, ethanol was available ad libitum and diazepam was mixed with food. Behavioral testing, performed during and after treatment cessation included working memory and visual recognition memory assessment. The second session was implemented with spatial reference learning and memory assessment in the Barnes maze test. In vivo magnetic resonance spectroscopy (MRS) acquisitions were performed to quantify hippocampal metabolites during and after cessation treatment. ResultsDuring treatment, visual recognition memory was significantly different between groups with the DIA group exhibiting the worst performance. MRS acquisition highlighted higher glutamate and choline levels in OH and DOH groups in comparison to CTL and DIA groups. After treatment wash-out, there was no difference between in the different memories evaluated. Only the learning phase of the spatial reference memory test differed significantly with worst performance in OH groups. Three months after treatment cessation, there was no remanent effect of diazepam + ethanol on hippocampal metabolites changes. ConclusionsWe did not evidence additive effect of ethanol and diazepam on memory and hippocampal metabolite levels. The disturbances observed during treatment were no remanent, highlighting the benefits of discontinuing these substances.

Overexpression of SULT1E1 alleviates salt-processed Psoraleae Fructus-induced cholestatic liver damage

ObjectiveSalt-processed Psoraleae fructus (SPF) is widely used as a phytoestrogen-like agent in the treatment of osteoporosis. However, due to improper clinical use or misuse, resulting in liver damage. In this study, network pharmacology was employed to analyze the mechanism of cholestatic liver damage. An adeno-associated virus overexpressing SULT1E1 (rAAV8-SULT1E1) was constructed and the hepatotoxicity of SPF, psoralen, and isopsoralen was determined. MethodsBy utilizing three databases inclding TCMSP, TCMID, and BATMAN- TCM, the targets of the three databases were summarized, and a total of 45 psoralen compounds were included. Network pharmacology analysis was then performed. The adenoviral vectors were injected into the tail vein of C57BL6 mice to elucidate the role of SULT1E1 in SPF-induced cholestasis-mediated hepatotoxicity in vivo. SPF (10 g/kg), psoralen, and isopsoralen (50 mg/kg each) were intragastrically administered to mice for 30 d. B-ultrasound and samples were collected and examined for follow-up experiments. ResultsA total of 854 targets were predicted for 45 active components, with 151 cholestasis-mediated hepatotoxicity-related disease targets obtained for SPF. A total of 126 pathways were enriched based on KEGG pathway analysis, with the “estrogen signaling pathway” identified as one of the top 20 pathways. In terms of pathological hepatic changes, treated mice had visually swollen hepatocytes, dilated bile ducts, and elevated serum biochemical markers, which were more prominent in mice treated with isopsoralen than in those treated with other compounds. Notably, the overexpression of SULT1E1 could reverse liver damage in each treatment group. B-ultrasound was used to observe the size of the gallbladder in vivo. The size of the gallbladder was found to significantly increase on day 30 after treatment in the SPF-, psoralen-, and isopsoralen-treated groups, especially the SPF group. Compared with the expression levels in the negative control group (rAAV8-empty+con), the expression levels of FXR, Mrp2, Bsep, SULT1E1, SULT2A1, Ntcp, and Nrf2 decreased, whereas those of CYP7a1 and IL-6 increased in the SPF-, psoralen-, and isopsoralen-treated groups. ConclusionThe overexpression of SULT1E1 could alleviate the decreased or increased expression of indicators, indicating that SULT1E1 is an important target gene for SPF-induced liver damage. The severity of liver damage was significantly lower in the rAAV8-SULT1E1 groups than in the rAAV8-empty groups.

Quantitative proteomic profiling reveals sexual dimorphism in the retina and RPE of C57BL6 mice.

Sex as a biological variable is not a common consideration in molecular mechanistic or preclinical studies of retinal diseases. Understanding the sexual dimorphism of adult RPE and retina under physiological conditions is an important first step in improving our understanding of sex-based physio-pathological mechanisms. Isobaric tags for relative and absolute quantitation (iTRAQ) were used for quantitative proteomics of male and female mouse retina and RPE (10 mice of each sex for each tissue type). Differentially expressed proteins were subjected to Gene Ontology (GO) analysis and Ingenuity Pathway Analysis (IPA). Differential expression analysis identified 21 differentially expressed proteins in the retina and 58 differentially expressed proteins in the RPE. Ingenuity pathway analysis identified the top canonical pathways differentially activated in the retina to be calcium transport I, nucleotide excision repair, molecular transport and cell death and survival. In the RPE, the top canonical pathways were calcium signaling, dilated cardiomyopathy signaling, actin cytoskeletal signaling and cellular assembly and organization. These results provide insights into sex differences in the retina and RPE proteome of mice and begin to shed clues into the sexual dimorphism seen in retinal diseases.

Neonatal hypoxia-ischemia alters the events governing the hippocampal critical period of postnatal synaptic plasticity leading to deficits in working memory in mice

Postnatal critical periods of synaptic plasticity (CPsp) are characterized by profound neural network refinement, which is shaped by synaptic activity and sculpted by maturation of the GABAergic network. Even after therapeutic hypothermia (TH), neonatal hypoxia-ischemia (HI) impairs two triggers for the initiation of the CPsp in the hippocampus: i) PSA-NCAM developmental decline and ii) parvalbumin (PV) + interneuron (IN) maturation. Thus, we investigated whether neonatal HI despite TH disturbs other events governing the onset, consolidation and closure of the postnatal CPsp in the hippocampus. We induced cerebral HI in P10 C57BL6 mice with right carotid ligation and 45 m of hypoxia (FiO2 = 0.08), followed by normothermia (36 °C, NT) or TH (31 °C) for 4 h with anesthesia-exposed shams as controls. ELISA, immunoblotting and immunohistochemistry were performed at 24 h (P11), 5 days (P15), 8 days (P18) and 30 days (P40) after HI injury. We specifically assessed: i) BDNF levels and TrkB activation, controlling the CPsp, ii) Otx2 and NPTX2 immunoreactivity (IR), engaging CPsp onset and iii) NogoR1, Lynx1 IR, PNN formation and myelination (MBP) mediating CPsp closure. Pups aged to P40 also received a battery of tests assessing working memory. Here, we documented deficits in hippocampal BDNF levels and TrkB activation at P18 in response to neonatal HI even with TH. Neonatal HI impaired in the CA1 the developmental increase in PV, Otx2, and NPTX2 between P11 and P18, the colocalization of Otx2 and PV at P18 and P40, the accumulation of NPTX2 in PV+ dendrites at P18 and P40, and the expression of NogoR at P40. Furthermore, neonatal HI decreased BDNF and impaired PNN development and myelination (MBP) at P40. Most of these abnormalities were insensitive to TH and correlated with memory deficits. Neonatal HI appears to disrupt many of the molecular and structural events initiating and consolidating the postnatal hippocampal CPsp, perhaps due to the early and delayed deficits in TrkB activation leading to memory deficits.

Unraveling immune checkpoint inhibitors effects on atherosclerosis: mechanisms and preventive approaches

Abstract Background Immune checkpoint inhibitors (ICIs) represent a novel and rapidly evolving field in cancer treatment. Despite increasing reports of accelerated atherosclerosis and ICI-related atherosclerotic cardiovascular (CV) events in clinical studies and case reports, there is a notable lack of data concerning the underlying mechanisms, appropriate monitoring, and potential interventions. Objectives To explore the role of ICIs in triggering and/or accelerating the atherosclerotic process, with a specific focus on the involvement of CD8+ T-cells. Additionally, we aimed to evaluate and compare the potential beneficial effects of statins and Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) inhibitors on atherosclerosis under ICIs treatment. Methods Thirty 8-week-old C57BL mice were divided into six groups as follows: (1) Control, normal diet; (2) Control, atherogenic diet; (3) Atherogenic diet + anti PD-1; (4) Atherogenic diet + statins + anti-PD1; (5) Atherogenic diet + PCSK9-i + anti PD-1; (6) Atherogenic diet + PCSK9 inhibitors. At 16 weeks, the carotid arteries and heart were removed for histological examination, and serum was withdrawn for blood work and further analysis. Results Anti-PD1 treatment resulted in extensive lymphocytic infiltration of the carotid intima, primarily constituted of CD8+ T cells. This infiltration was significantly attenuated when statins or PCSK-9 inhibitors were used in conjunction with anti-PD1 treatment. Serum analysis for CD4+ and CD8+ T cells by ELISA revealed no difference in CD4+ abundance between the groups. However, plasma CD8+ T cells were significantly increased with anti-PD1 treatment, and furthermore, when PCSK9 inhibitors were used in combination; but not when statins were used. Conclusion The induction of CD8+ T cell infiltration in the arterial carotid walls by anti-PD-1 was prevented with the use of either statins or PCSK9 inhibitors. In the plasma, while statins reduced the hyper-expression of CD8+ T cells observed under PD-1 treatment, PCSK9 inhibitors enhanced this expression. This may provide a first indication of the potential benefit of PCSK9 inhibitor treatment for both preventing arterial inflammation and enhancing the desired anti-tumor immune response under ICIs treatment. Figure legend: 1A. A cross-section of the carotid artery after 16 weeks of treatment, stained with H&E. Magnification is X100. 1B. A cross-section of the carotid artery after 16 weeks of treatment, stained with anti-CD8 Ab. Magnification is X100. 1C. quantitative CD8+ T cells staining in the carotid arteries. 1D. quantitative results of ELISA for plasma CD8+ T cells.

Investigating the effects of empagliflozin on myocardial metabolic and lipid profile in healthy and metabolic syndrome murine models following acute myocardial infarction

Abstract Background/Introduction Empagliflozin (EMPA) is a sodium/glucose co-transporter type 2 inhibitor with clinically documented cardioprotective effect. However, the mechanism of cardioprotection remains elusive. We have previously shown that Western diet induces metabolic syndrome (MS) and deteriorates cardiac function in mice, whereas EMPA treatment for 6 weeks prevents this damage. Also, EMPA administration for 6 weeks suppresses ischemia/reperfusion (I/R) injury in mice, independent of the presence of MS. Purpose The aim of the present study is to investigate whether changes in myocardial metabolic and lipid profile, induced by EMPA treatment, are responsible for its cardioprotective effect in the setting of myocardial I/R injury. Methods C57Bl6 mice received chow diet or Western diet for 14 weeks. At week 8, mice from each dietary group were randomized into 3 subgroups: 1. Control - Sham surgery (5% DMSO-SHAM), 2. Control - I/R (5% DMSO-I/R) and 3. EMPA (10mg/kg/day in 5% DMSO) - I/R (EMPA-I/R) and received the corresponding treatment by daily per os administration for 6 weeks. At week 14, mice were subjected to 30 minutes of I, followed by 2 hours of R, or sham surgery. Myocardial tissue was harvested at the end of R, and metabolomic and lipidomic analysis was performed by nuclear magnetic resonance spectroscopy and mass spectrometry, respectively. Body weight, as well as blood glucose and cholesterol levels were assessed at baseline and at the end of the administrations. Results EMPA administration reduced body weight, as well as blood glucose and cholesterol levels in animals with MS. EMPA decreased anaerobic glycolysis products (UDP-glucose, Lactic acid) and ketone bodies (β-hydroxybutyrate, acetoacetic acid) in both healthy animals and mice with MS. Additionally, an increase in ATP, accompanied by a decrease in AMP and ADP were observed in the EMPA-I/R group, compared to the DMSO-I/R group. Regarding the lipidomic analysis, gangliosides GM3 emerged as the major lipid group decreased by EMPA treatment. Lastly, despite significant changes were presented in the metabolic and lipid profile between healthy animals and those with MS, the effects of EMPA on myocardial metabolic and lipid profile were the same, regardless of the presence of MS. Conclusion(s) EMPA administration induces metabolic changes in the heart tissue and improves myocardial energetics upon myocardial I/R injury. Also, EMPA suppresses gangliosides GM3, a lipid group that has been associated with neutrophil activation and recruitment. Importantly, the effects of EMPA on myocardial metabolic and lipid profile following I/R is independent of the presence of MS.