Potentials of CCL21 and CBS as therapeutic approaches for breast cancer

Objective: The present research set out to ascertain the roles of CCL21 and CBS in breast cancer (BC) cell biological behaviors and the relationship of CCL21 and CBS expression with the clinicopathological features of patients with BC. Methods: Immunohistochemistry of CCL21 or CBS was performed in 18 intraductal cancer tissues, 124 invasive BC tissues, 50 paraneoplastic tissues, 50 lobular hyperplasia tissues, and 30 normal breast tissues. For cell experiments, two human BC cell lines (MDA-MB-231 and MCF-7) and a human breast epithelial cell line (MCF-10A) were utilized to detect the expression of CCL21 and CBS. After loss- and gain-of-function assays for CCL21 or CBS, the expression of CBS and CCL21 was measured by qRT-PCR and Western blot. Additionally, BC cell proliferation was assessed by MTT assay and EdU staining, and BC cell migration was determined by scratch test and Transwell assay. Results: In the clinical data, the positive rate of CCL21 or CBS was significantly higher in invasive BC tissues than in intraductal BC tissues, lobular hyperplasia tissues, paraneoplastic tissues, and normal breast tissues (P < 0.05 or P < 0.01). CBS or CCL21 expression shared close association with the clinicopathological characteristic and the poor prognosis of BC patients. In cell experiments, overexpression of CCL21 or CBS enhanced the proliferative and migratory abilities of BC cells. Conclusion: CCL21 and CBS promoted BC cell migration and proliferation. CCL21 or CBS expression was strongly related to the poor prognosis of BC patients.

miR-27b-3p Improved High Glucose-Induced Spermatogenic Cell Damage via Regulating Gfpt1/HBP Signaling

<b><i>Introduction:</i></b> Diabetes mellitus (DM)-induced testicular damage is characterized by abnormal apoptosis of spermatogenic cells. Here, we clarified the roles and the molecular mechanism of microRNA (miR)-27b-3p in high glucose (HG)-induced spermatogenic cell damage. <b><i>Methods:</i></b> GC-1 spg cells were treated with 30 mmol/L glucose for 24 h. Cell viability was assessed by 2.3 3-(4, 5-dimethylthiazolyl2)-2, 5-diphenyltetrazolium bromide (MTT) assay. And, levels of O-linked N-acetylglucosamine (OGT), apoptosis-related proteins, and autophagy-related proteins were evaluated using Western blot. Levels of tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and UDP-N-acetylglucosamine (UDP-GlcNAc) were assessed by enzyme linked immunosorbent (ELISA) assay. Levels of reactive oxygen species (ROS), malonic dialdehyde (MDA) and activity of superoxide dismutase (SOD) in cells were determined using kits. Cell apoptosis was determined using flow cytometry assay. Besides, dual luciferase reporter assay was employed to verify the binding relationship between miR-27b-3p and glutamine-fructose-6-phosphate transaminase 1 (Gfpt1). <b><i>Results:</i></b> miR-27b-3p was markedly downregulated in HG-treated GC-1 spg cells. HG treatment caused decreased cell viability, increased oxidative stress and inflammation, and induced autophagy and apoptosis, which were abolished by miR-27b-3p overexpression. miR-27b-3p suppressed the activation of hexosamine biosynthetic pathway (HBP) signaling in HG-treated spermatogenic cells. miR-27b-3p directly bound to Gfpt1 and negatively regulated its expression. <b><i>Conclusion:</i></b> miR-27b-3p could improve HG-induced spermatogenic cell damage via regulating Gfpt1/HBP signaling, providing a new treatment strategy for the treatment of DM-induced testicular damage.

Alterations in Toll-Like Receptor 7 and -9 mRNA Levels in Lungs after Ovariohysterectomy in a Pyometra Mouse Model

<b><i>Background:</i></b> Pyometra (P) leads to sepsis and multiple organ dysfunction syndrome. Toll-like receptors (TLRs) recognize pathogens which can cause P. The aim of this study was to investigate TLR-7 and -9 via the MYD88 pathway and the nuclear factor kappa B (NFκB) response in the uterus of a P mouse model before and after ovariohysterectomy (RP) as well as potential lung injury. <b><i>Materials and Methods:</i></b> 200 female C57BL/6J mice were randomly divided into groups (<i>N</i> = 10/subgroup; sham 1, 2, 3, 7; P1, 2, 3, 7; 1RP1, 2, 3, 7; 2RP1, 2, 3, 7; 3RP1, 2, 3, 7) according to the day of euthanasia. Pathogens were administrated in the groups P and RP in order to induce P. <b><i>Results:</i></b> Alterations in blood chemistry, histopathology, and RT-qPCT analysis before (P) and after RP were observed. Significant correlations were also found between MYD88, NFκB, and TLR9 in P and RP groups in the lungs and in RP groups in the uterus, suggesting that the immune system responded via the TLR9-MYD88 pathway. <b><i>Conclusions:</i></b> This is the first report of immunohistochemical TLR-7 and -9 localization and of TLR-7, -9, MYD88, and NFκB mRNA expression in the uterus causing lung injury in a P mouse model.

Mitochondrial dysfunction in trauma-related coagulopathy – Is there causality? – Study protocol for a prospective observational study

Hemorrhage control often poses a great challenge for clinicians due to trauma-induced coagulopathy (TIC). The pathogenesis of TIC is not completely revealed; however, growing evidence attributes a central role to altered platelet biology. The activation of thrombocytes and subsequent clot formation are highly energetic processes being tied to mitochondrial activity, and the inhibition of the electron transport chain (ETC) impedes on thrombogenesis, suggesting the potential role of mitochondria in TIC. Our present study protocol provides a guide to quantitatively characterize the derangements of mitochondrial functions in TIC. One hundred eleven severely injured (Injury Severity Score ≥16), bleeding trauma patients with an age of 18 or greater will be included in this prospective observational study. Patients receiving oral antiplatelet agents including cyclooxygenase-1 or adenosine diphosphate receptor inhibitors (aspirin, clopidogrel, prasugrel, and ticagrelor) will be excluded from the final analysis. Hemorrhage will be confirmed and assessed with computer tomography. Conventional laboratory markers of hemostasis such as prothrombin time and international normalized ratio (INR) will be measured and rotational thromboelastometry (ROTEM) will be performed directly upon patient arrival. Platelets will be isolated from venous blood samples and subjected to high-resolution fluororespirometry (Oxygraph-2k, Oroboros Instruments, Innsbruck, Austria) to evaluate the efficacy of mitochondrial respiration. Oxidative phosphorylation (OxPhos), coupling of the ETC, mitochondrial superoxide formation, mitochondrial membrane potential changes and extramitochondrial Ca2+-movement will be recorded. The association between OxPhos capacity of platelet mitochondria and numerical parameters of ROTEM aggregometry will constitute our primary outcome. The relation between OxPhos capacity and results of viscoelastic assays and conventional markers of hemostasis will serve as secondary outcomes. The association of the OxPhos capacity of platelet mitochondria upon patient arrival to the need for massive blood transfusion (MBT) and 24-hour mortality will constitute our tertiary outcomes. Mitochondrial dysfunction and its importance in TIC in are yet to be assessed for the deeper understanding of this common, life-threatening condition. Disclosure of mitochondria-mediated processes in thrombocytes may reveal new therapeutic targets in the management of hemorrhaging trauma patients, thereby leading to a reduction of potentially preventable mortality. The present protocol was registered to ClinicalTrials.gov on 12 August 2021, under the reference number NCT05004844.

Involvement of cannabinoid type 2 (CB2) receptors in the favorable effects of sumatriptan on the random-pattern skin flap survival in rats: a novel potential target

Introduction: Recent investigations have indicated the potential therapeutic role of cannabinoid type 2 (CB2) receptors in various inflammatory-related disorders. However, the role of these receptors has not been studied in skin flap models previously. In this study, we aimed to evaluate the possible involvement of CB2 receptors in the anti-inflammatory effects of sumatriptan and improvement of the random-pattern skin flap survival in rats. Methods: In a controlled experimental study, 36 male Wistar rats were randomly divided into six study groups (n = 6 per group). Two doses of sumatriptan (0.1 and 0.3 mg/kg) were administered intraperitoneally (i.p) 30 minutes before harvesting the flap tissue. In a separate group, SR144528 (a selective CB2 receptor inverse agonist) was injected before the most effective dose of sumatriptan to determine the possible involvement of CB2 receptors in its action. Histopathological examinations, the expression level of CB2 receptors (by western blot analysis), and IL-1 and TNF-α concentrations (ELISA) were explored in the skin flap samples. Results: Sumatriptan 0.3 mg/kg remarkably enhanced the skin flap survival in all macroscopic and microscopic investigations compared to the control group (P <0.001). IL-1 and TNF-α levels were significantly attenuated (P <0.001), and the expression of CB2 receptors in skin cells was amplified in rats treated with sumatriptan 0.3 mg/kg (p <0.05) compared to the control group. However, the administration of SR144528 (2 mg/kg) nullified all the protective effects of sumatriptan (0.3 mg/kg). Conclusion: We discovered that CB2 receptors play a crucial role in the favorable effects of sumatriptan on skin flap survival as a novel mechanism of action. So, targeting these receptors seems to be a dependable method in skin flap surgeries to ensure its survival and prevent tissue necrosis. Further experimental and clinical investigations are needed to ensure the safe clinical application of this method.

The dot-probe attention bias task as a method to assess psychological wellbeing after anesthesia: A study with adult female long-tailed macaques (Macaca fascicularis)

Understanding the impact routine research and laboratory procedures have on animals is crucial to improving their wellbeing and to the success and reproducibility of the research they are involved in. Cognitive measures of welfare offer insight into animals’ internal psychological state, but require validation. Attention bias - the tendency to attend to one type of information over another – is a cognitive phenomenon documented in humans and animals that is known to be modulated by affective state (i.e., emotions). Hence, changes in attention bias may offer researchers a deeper perspective of their animals’ psychological wellbeing. The dot-probe task is an established method for quantifying attention bias in humans (by measuring reaction time to a dot-probe replacing pairs of stimuli), but has yet to be validated in animals. We developed a dot-probe task for long-tailed macaques (Macaca fascicularis) to determine if the task can detect changes in attention bias following anesthesia, a context known to modulate attention and trigger physiological arousal in macaques. Our task included the following features: stimulus pairs of threatening and neutral facial expressions of conspecifics and their scrambled counterparts, two stimuli durations (100 and 1000 ms), and counterbalancing of the dot-probe’s position on the touchscreen (left, right) and location relative to the threatening stimulus. We tested eight group-housed adult females on different days relative to being anesthetized (baseline and one-, three-, seven-, and 14-days after). At baseline, monkeys were vigilant to threatening content when stimulus pairs were presented for 100 ms, but not 1000 ms. On the day immediately following anesthesia, we found evidence that attention bias changed to an avoidance of threatening content. Attention bias returned to threat vigilance by the third day post-anesthesia and remained so up to the last day of testing (14 days after anesthesia). We also found that attention bias was independent of the type of stimuli pair (i.e., whole face vs. scrambled counterparts), suggesting that the scrambled stimuli retained aspects of the original stimuli. Nevertheless, whole faces were more salient to the monkeys as responses to these trials were generally slower than to scrambled stimulus pairs. Overall, our study suggests it is feasible to detect changes in attention bias following anesthesia using the dot-probe task in non-human primates. Our results also reveal important aspects of stimulus preparation and experimental design.

Smartphone-based DIY home microsurgical training with 3D printed microvascular clamps and Japanese noodles

We have recently incorporated simple modifications of the konjac flour noodle model to enable DIY home microsurgical training by (i) placing a smartphone on a mug to act as a microscope with at least 3.5-5x magnification, and (ii) rather than cannulating with a 22G needle as described by others, we have found that cannulation with a 23G needle followed by a second pass with an 18G needle will create a lumen (approx. 0.83 mm) without an overly thick and unrealistic “vessel” wall. The current set-up however, did not allow realistic evaluation of anastomotic patency as the noodles became macerated after application of standard microvascular clamps, which also did not facilitate practice of back-wall anastomoses. In order to simulate the actual operative environment as much as possible, we introduced the use of 3D printed microvascular clamps. These were modified from its previous iteration (suitable for use in silastic and chicken thigh vessels) and video recordings were submitted for internal validation by senior surgeons. A “wet” operative field where the knojac noodle lumen can be distended or collapsed, unlike other non-living models, was noted by senior surgeons. With the 3D clamps, the noodle could now be flipped over for back-wall anastomosis and allowed patency testing upon completion as it did not become macerated, unlike that from clinical microvascular clamps. The perceived advantages of this model are numerous. Not only does it comply with the 3Rs of simulation-based training, it can also reduce the associated costs of training by up to a hundred-fold or more when compared to a traditional rat course, and potentially, be extended to low-middle income countries (LMICs) without routine access to microsurgical training for capacity development. That it can be utilised remotely also bodes well with the current limitations on face to-face training due to COVID restrictions and lockdowns.

Implementation of the Surgical Apgar Score in Laboratory Animal Science: A Showcase Pilot Study in a Porcine Model and a Review of the Literature

<b><i>Introduction:</i></b> In an attempt to further improve surgical outcomes, a variety of outcome prediction and risk-assessment tools have been developed for the clinical setting. Risk scores such as the surgical Apgar score (SAS) hold promise to facilitate the objective assessment of perioperative risk related to comorbidities of the patients or the individual characteristics of the surgical procedure itself. Despite the large number of scoring models in clinical surgery, only very few of these models have ever been utilized in the setting of laboratory animal science. The SAS has been validated in various clinical surgical procedures and shown to be strongly associated with postoperative morbidity. In the present study, we aimed to review the clinical evidence supporting the use of the SAS system and performed a showcase pilot trial in a large animal model as the first implementation of a porcine-adapted SAS (pSAS) in an in vivo laboratory animal science setting. <b><i>Methods:</i></b> A literature review was performed in the PubMed and Embase databases. Study characteristics and results using the SAS were reported. For the in vivo study, 21 female German landrace pigs have been used either to study bleeding analogy (<i>n</i> = 9) or to apply pSAS after abdominal surgery in a kidney transplant model (<i>n</i> = 12). The SAS was calculated using 3 criteria: (1) estimated blood loss during surgery; (2) lowest mean arterial blood pressure; and (3) lowest heart rate. <b><i>Results:</i></b> The SAS has been verified to be an effective tool in numerous clinical studies of abdominal surgery, regardless of specialization confirming independence on the type of surgical field or the choice of surgery. Thresholds for blood loss assessment were species specifically adjusted to &#x3e;700 mL = score 0; 700–400 mL = score 1; 400–55 mL score 2; and &#x3c;55 mL = score 3 resulting in a species-specific pSAS for a more precise classification. <b><i>Conclusion:</i></b> Our literature review demonstrates the feasibility and excellent performance of the SAS in various clinical settings. Within this pilot study, we could demonstrate the usefulness of the modified SAS (pSAS) in a porcine kidney transplantation model. The SAS has a potential to facilitate early veterinary intervention and drive the perioperative care in large animal models exemplified in a case study using pigs. Further larger studies are warranted to validate our findings.

Role of Different Doses of Ketamine in Postoperative Neurocognitive Function in Aged Mice Undergoing Partial Hepatectomy by Regulating the Bmal1/NMDA/NF-Κb Axis

<b><i>Background:</i></b> The current study set out to probe the function of different doses of ketamine in postoperative neurocognitive disorder (PND) in aged mice undergoing partial hepatectomy (PH) with the involvement of the brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1)/n-methyl-D-aspartate (NMDA)/nuclear factor-kappa B (NF-κB) axis. <b><i>Methods:</i></b> First, aged mice were intraperitoneally injected with different doses of ketamine prior to surgery, followed by hepatic lobectomy. Afterward, mice cognitive function was assessed. In addition, Bmal1 mRNA expression patterns were quantified, while NMDA 2B receptor, NF-κB p65, synapsin 1, and postsynaptic density 95 (PSD95) levels were determined; the release of inflammatory factors was detected, and ionized calcium-binding adapter molecule-1 expression was measured to quantify microglia activation. In addition, Bmal1-knockout (Bmal1-KO) mice were intraperitoneally injected with a subanesthetic dose of ketamine to verify the mechanism of Bmal1 in regulating the NMDA 2B subunit (NR2B)/NF-κB axis to affect PH in aged patients. <b><i>Results:</i></b> After PH, hippocampal-dependent memory was impaired, and synapsin 1 and PSD95 levels were downregulated. On the other hand, PH diminished Bmal1 expression but elevated NR2B and NF-κB p65 levels and anesthetic doses of ketamine further regulated the Bmal1/NMDA/NF-κB axis. In Bmal1-KO mice, the NMDA/NF-κB axis was activated, the release of inflammatory cytokines was promoted, and hippocampus-dependent memory was impaired, which were reversed by a subanesthetic dose of ketamine. <b><i>Conclusion:</i></b> Altogether, findings obtained in our study indicated that a subanesthetic dose of ketamine activated Bmal1, downregulated the NMDA/NF-κB axis, and reduced inflammation and microglia activation to alleviate PND in aged mice undergoing PH.

Ischemic Conditioning in the Right Colon and Terminal Ileum: An Experimental Rat Model

Introduction: Preoperative gastric ischemic conditioning (IC) improves the outcome of esophageal replacement gastroplasty and is associated with low morbidity. However, when the stomach cannot be used for esophageal replacement, a colonic replacement is required. The study aim was to assess the viability of right colon and terminal ileum IC in a rat model, the histological damage/recovery sequence, and determine if neovascularization is a potential adaptive mechanism. Methods: The study was conducted in Rattus norvegicus with ileocolic vascular ligation. Seven groups of animals were established (six rats per group) with groups defined by the date of their post-IC euthanasia (+1, +3, +6, +10, +15, and +21 days). Comparisons were made with a sham group. Viability of the model was defined as <10% of transmural necrosis. The evaluation of histological damage used the Chiu score in hematoxylin and eosin sections of paraffin-embedded specimens with CD31 immunohistochemical assessment of neovascularization by the median of submucosal vessel counts in five high-magnification fields. Results: Transmural colon necrosis occurred in 1/36 animals (2.78%) with no animal demonstrating transmural ileal necrosis. The maximum damage was observed in the colon on +1 day post-IC (average Chiu score 1.67, P = 0.015), whereas in the ileum, it was on days +1, +3, and +6 (average Chiu score 1.5, 1.3, and 1.17; P = 0.015, 0.002, and 0.015, respectively). In the +21-day group, histological recovery was complete in the colon in four (66.7%) of the six animals and in the ileum in five (83.3%) of six animals. There were no significant differences in quantitative neovascularization in any of the groups when compared with the sham group or when comparisons were made between groups. Conclusions: The tested animal model for IC of the colon and terminal ileum appeared to be feasible. Histological damage was maximal between the 1st and 3rd day following IC, but by day 21, recovery was complete in two-thirds of the rats. There was no evidence in this preliminary IC model that would suggest neovascularization as an adaptive mechanism.