The absence of immediate stimulation delays bone healing.

AIM: Secondary bone healing requires an adequate level of mechanical stimulation expressed by the extent of interfragmentary motion in the fracture. However, there is no consensus about when the mechanical stimulation should be initiated to ensure a timely healing response. Therefore, this study aims to compare the effect of the immediate and delayed application of mechanical stimulation in a large animal model. METHODS: Twelve Swiss White Alpine sheep underwent partial osteotomy of a tibia that was stabilised with an active fixator inducing well-controlled mechanical stimulation. Animals were randomly assigned into two groups with different stimulation protocols. The immediate group received daily stimulation (1000 cycles/day) from the first day post-operation, while in the delayed group, stimulation began only on the 22nd day post-operation. Healing progression was evaluated daily by measuring the in vivo stiffness of the repair tissue and by quantifying callus area on weekly radiographs. All animals were euthanised five weeks post-op. Post-mortem callus volume was determined from high-resolution computer tomography (HRCT). RESULTS: Fracture stiffness (p < 0.05) and callus area (p < 0.01) were significantly larger for the immediate group compared to the delayed stimulation group. In addition, the callus volume measured on the post-mortem HRCT showed 319 % greater callus volume for the immediate stimulation group (p < 0.01). CONCLUSIONS: This study demonstrates that a delay in the onset of mechanical stimulation retards fracture callus development and that mechanical stimulation already applied in the early post-op phase promotes bone healing.

Arginine concentration in arterial vs venous blood in a bleomycin-induced lung inflammation model in mice.

Pneumonia, always a major malady, became the main public health and economic disaster of historical proportions with the COVID-19 pandemic. This study was based on a premise that pathology of lung metabolism in inflammation may have features invariant to the nature of the underlying cause. Amino acid uptake by the lungs was measured from plasma samples collected pre-terminally from a carotid artery and vena cava in mice with bleomycin-induced lung inflammation (N = 10) and compared to controls treated with saline instillation (N = 6). In the control group, the difference in concentrations between the arterial and venous blood of the 19 amino acids measured reached the level of statistical significance only for arginine (-10.7%, p = 0.0372) and phenylalanine (+5.5%, p = 0.0266). In the bleomycin group, 11 amino acids had significantly lower concentrations in the arterial blood. Arginine concentration was decreased by 21.1% (p<0.0001) and only that of citrulline was significantly increased (by 20.1%, p = 0.0002). Global Arginine Bioavailability Ratio was decreased in arterial blood by 19.5% (p = 0.0305) in the saline group and by 30.4% (p<0.0001) in the bleomycin group. Production of nitric oxide (NO) and citrulline from arginine by the inducible nitric oxide synthase (iNOS) is greatly increased in the immune system's response to lung injury. Deprived of arginine, the endothelial cells downstream may fail to provide enough NO to prevent the activation of thrombocytes. Thrombotic-related vascular dysfunction is a defining characteristic of pneumonia, including COVID-19. This experiment lends further support to arginine replacement as adjuvant therapy in pneumonia.

Prediction of Treatment Response and Survival with Chemotherapy for Metastatic Penile Cancer by the Modified Glasgow Prognostic Score.

PURPOSE: There is increasing evidence that a persistent systemic inflammatory response predicts lower survival in patients with malignant disease. The modified Glasgow Prognostic Score (mGPS) is defined by a combination of elevated C-reactive protein (CRP) (>10 mg/L) and hypoalbuminemia (<35 g/L). It is considered as an independent prognostic marker in several organ malignancies. The aim of this study was to investigate the value of mGPS in metastatic penile carcinoma in predicting treatment response and survival. METHODS: One hundred and fifty-six patients with penile carcinoma treated with chemotherapy were included in this retrospective study. The mGPS before chemotherapy was classified into 3 groups (mGPS 0 [CRP <10, any albumin], mGPS 1 [CRP >10 mg/L, albumin >35 g/L], and mGPS 2 [CRP >10 mg/L, albumin <35 g/L]). Overall survival and disease-free survival were calculated by Kaplan-Meier analysis and chemotherapy toxicity by CTC criteria. Univariate Cox proportional hazards models were calculated to estimate the effect of each predictor on OS and DFS. RESULTS: Survival was significantly different in the 3 mGPS classes, with mGPS 0 patients showing the best treatment response and survival. Univariate analysis showed that mGPS (p < 0.0001), tumor stage (p = 0.004), and venous and lymphatic invasion (p = 0.011) were factors independently associated with prognosis. The response to chemotherapy differed significantly between mGPS groups (mGPS 0, 36/51 [71%]; mGPS 1, 24/70 [34%]; mGPS 2, 9/35 [26%], p = 0.03 and p = 0.37, respectively). mGPS was significantly associated with chemotherapy-associated toxicity, with treatment adaptation (p < 0.01) and toxicity-related deaths (p = 0.028). CONCLUSIONS: Systemic inflammatory response and nutritional status as expressed by the mGPS are independent predictors of treatment response, chemotherapy-associated toxicity, and survival in metastatic penile carcinoma. In addition to other known pathological markers of tumor aggressiveness, the mGPS can be used as a clinical predictor of prognosis.

Protocol for a systematic review of good surgical practice guidelines for experimental rodent surgery.

Objective: Surgery is an integral part of many experimental studies. Aseptic and minimal invasive surgical technique and optimal perioperative and post-operative care are prerequisites to achieve surgical success and best possible animal welfare outcomes. Good surgical practice cannot only improve the animal's postoperative recovery, but also study outcome and validity. There seems to be a lack of implementation of good surgical practice during rodent surgery. The aim of this systematic review is to identify, critically evaluate and compare the currently recommended standards and underlying guidelines for rodent surgery-and finally to compile a comprehensive guideline of good surgical practice for rodent surgery. Search strategy: PubMed, Embase and Web of Science were searched to identify guidelines published in peer-reviewed journals. To identify grey literature and unpublished guidelines, we will perform a Google search for published guidelines and search laboratory animal sciences books for relevant book chapters. Additionally, we will conduct a survey among animal researchers enquiring about the guidelines they use. Screening and study selection: For publications retrieved by the systematic search, unique references are screened by two reviewers, first for eligibility based on title and abstract and subsequently for final inclusion based on full text. Eligibility of books is based on title and content, final inclusion based on chapter full text. Guidelines are either retrieved by Google searches or a survey. Google searches will be conducted by at least four of the authors. Thereafter, guidelines will be screened by two of the authors. Data extraction and synthesis: We will extract data from publications, book chapters and guidelines. Based on the extracted data, we will perform a descriptive synthesis of the bibliographical details, guideline development and endorsement, and the prevalence of individual recommendations, including subgroup analysis of the guidance per continent or country and differences between peer-reviewed versus non-peer-reviewed guidance.

Conversion of anterograde into retrograde trains is an intrinsic property of intraflagellar transport.

Cilia or eukaryotic flagella are microtubule-based organelles found across the eukaryotic tree of life. Their very high aspect ratio and crowded interior are unfavorable to diffusive transport of most components required for their assembly and maintenance. Instead, a system of intraflagellar transport (IFT) trains moves cargo rapidly up and down the cilium (Figure 1A).1-3 Anterograde IFT, from the cell body to the ciliary tip, is driven by kinesin-II motors, whereas retrograde IFT is powered by cytoplasmic dynein-1b motors.4 Both motors are associated with long chains of IFT protein complexes, known as IFT trains, and their cargoes.5-8 The conversion from anterograde to retrograde motility at the ciliary tip involves (1) the dissociation of kinesin motors from trains,9 (2) a fundamental restructuring of the train from the anterograde to the retrograde architecture,8,10,11 (3) the unloading and reloading of cargo,2 and (4) the activation of the dynein motors.8,12 A prominent hypothesis is that there is dedicated calcium-dependent protein-based machinery at the ciliary tip to mediate these processes.4,13 However, the mechanisms of IFT turnaround have remained elusive. In this study, we use mechanical and chemical methods to block IFT at intermediate positions along the cilia of the green algae Chlamydomonas reinhardtii, in normal and calcium-depleted conditions. We show that IFT turnaround, kinesin dissociation, and dynein-1b activation can consistently be induced at arbitrary distances from the ciliary tip, with no stationary tip machinery being required. Instead, we demonstrate that the anterograde-to-retrograde conversion is a calcium-independent intrinsic ability of IFT.

Bacteriophage Therapy for the Prevention and Treatment of Fracture-Related Infection Caused by Staphylococcus aureus: a Preclinical Study.

Although several studies have shown promising clinical outcomes of phage therapy in patients with orthopedic device-related infections, questions remain regarding the optimal application protocol, systemic effects, and the impact of the immune response. This study provides a proof-of-concept of phage therapy in a clinically relevant rabbit model of fracture-related infection (FRI) caused by Staphylococcus aureus. In a prevention setting, phage in saline (without any biomaterial-based carrier) was highly effective in the prevention of FRI, compared to systemic antibiotic prophylaxis alone. In the subsequent study involving treatment of established infection, daily administration of phage in saline through a subcutaneous access tube was compared to a single intraoperative application of a phage-loaded hydrogel and a control group receiving antibiotics only. In this setting, although a possible trend of bacterial load reduction on the implant was observed with the phage-loaded hydrogel, no superior effect of phage therapy was found compared to antibiotic treatment alone. The application of phage in saline through a subcutaneous access tube was, however, complicated by superinfection and the development of neutralizing antibodies. The latter was not found in the animals that received the phage-loaded hydrogel, which may indicate that encapsulation of phages into a carrier such as a hydrogel limits their exposure to the adaptive immune system. These studies show phage therapy can be useful in targeting orthopedic device-related infection, however, further research and improvements of these application methods are required for this complex clinical setting. IMPORTANCE Because of the growing spread of antimicrobial resistance, the use of alternative prevention and treatment strategies is gaining interest. Although the therapeutic potential of bacteriophages has been demonstrated in a number of case reports and series over the past decade, many unanswered questions remain regarding the optimal application protocol. Furthermore, a major concern during phage therapy is the induction of phage neutralizing antibodies. This study aimed at providing a proof-of-concept of phage therapy in a clinically relevant rabbit model of fracture-related infection caused by Staphylococcus aureus. Phage therapy was applied as prophylaxis in a first phase, and as treatment of an established infection in a second phase. The development of phage neutralizing antibodies was evaluated in the treatment study. This study demonstrates that phage therapy can be useful in targeting orthopedic device-related infection, especially as prophylaxis; however, further research and improvements of these application methods are required.

Is a Block of the Femoral and Sciatic Nerves an Alternative to Epidural Analgesia in Sheep Undergoing Orthopaedic Hind Limb Surgery? A Prospective, Randomized, Double Blinded Experimental Trial.

Peripheral nerve blocks are commonly used in human and veterinary medicine. The aim of the study was to compare the analgesic efficacy of a combined block of the femoral and sciatic nerves with an epidural injection of ropivacaine in experimental sheep undergoing orthopaedic hind limb surgery. Twenty-five sheep were assigned to two groups (peripheral nerve block; sciatic and femoral nerves (P); epidural analgesia (E)). In group P 10 mL ropivacaine 0.5% was injected around the sciatic and the femoral nerves under sonographic guidance and 10 mL NaCl 0.9% into the epidural space while in group E 10 mL ropivacaine 0.5% was injected into the epidural space and 10 mL NaCl 0.9% to the sciatic and the femoral nerves. During surgery, heart rate, respiratory rate and mean blood pressure were used as indicators of nociception. In the postoperative phase, nociception was evaluated every hour by use of a purposefully adapted pain score until the animal showed painful sensation at the surgical site. The mean duration of analgesia at the surgical wound was 6 h in group P and 8 h in group E. Mean time to standing was 4 h in group P and 7 h in group E. In conclusion time to standing was significantly shorter in group P while the duration of nociception was comparable in both groups. The peripheral nerve block can be used as an alternative to epidural analgesia in experimental sheep.

Democratising deep learning for microscopy with ZeroCostDL4Mic.

Deep Learning (DL) methods are powerful analytical tools for microscopy and can outperform conventional image processing pipelines. Despite the enthusiasm and innovations fuelled by DL technology, the need to access powerful and compatible resources to train DL networks leads to an accessibility barrier that novice users often find difficult to overcome. Here, we present ZeroCostDL4Mic, an entry-level platform simplifying DL access by leveraging the free, cloud-based computational resources of Google Colab. ZeroCostDL4Mic allows researchers with no coding expertise to train and apply key DL networks to perform tasks including segmentation (using U-Net and StarDist), object detection (using YOLOv2), denoising (using CARE and Noise2Void), super-resolution microscopy (using Deep-STORM), and image-to-image translation (using Label-free prediction - fnet, pix2pix and CycleGAN). Importantly, we provide suitable quantitative tools for each network to evaluate model performance, allowing model optimisation. We demonstrate the application of the platform to study multiple biological processes.

Transdermal Fentanyl Uptake at Two Different Patch Locations in Swiss White Alpine Sheep.

When using animals in biomedical research, investigators have the responsibility to ensure adequate analgesia. Currently, transdermal fentanyl patches (TFP) are often used to provide postoperative analgesia in large laboratory animals. The aim of this study was to compare the fentanyl uptake resulting from TFP applied at two different locations, namely the foreleg and the thorax, in healthy adult sheep. Twelve sheep received a TFP with an intended dosage of 2 ug/kg/h. Blood samples were taken at different time points over a period of five days and the fentanyl plasma levels were measured. The TFP applied on the foreleg allowed a faster fentanyl uptake with higher peaks and a longer time within or above the target concentration of 0.6-1.5 ng/mL, shown to be analgesic in humans, when compared to the one on the thorax. Assuming that the effective plasma concentration described for humans is providing analgesia in sheep as well, the present findings suggest that it should be sufficient to apply the TFP 3-6 h before the painful insult and that its effect should last at least 48 h. Furthermore, when TFP are used to provide postoperative analgesia in sheep, they should be placed on the foreleg rather than on the thorax.

Content-aware image restoration for electron microscopy.

Multiple approaches to use deep neural networks for image restoration have recently been proposed. Training such networks requires well registered pairs of high and low-quality images. While this is easily achievable for many imaging modalities, e.g., fluorescence light microscopy, for others it is not. Here we summarize on a number of recent developments in the fast-paced field of Content-Aware Image Restoration (CARE), in particular, and the associated area of neural network training, more in general. We then give specific examples how electron microscopy data can benefit from these new technologies.