Respiratory gating improves correlation between pulse wave transit time and pulmonary artery pressure in experimental pulmonary hypertension.

Objective. Since pulse wave transit time (PWTT) shortens as pulmonary artery pressure (PAP) increases it was suggested as a potential non-invasive surrogate for PAP. The state of tidal lung filling is also known to affect PWTT independently of PAP. The aim of this retrospective analysis was to test whether respiratory gating improved the correlation coefficient between PWTT and PAP.Approach. In each one of five anesthetized and mechanically ventilated pigs two high-fidelity pressure catheters were placed, one directly behind the pulmonary valve, and the second one in a distal branch of the pulmonary artery. PAP was raised using the thromboxane A2 analogue U46619 and animals were ventilated in a pressure controlled mode (I:E ratio 1:2, respiratory rate 12/min, tidal volume of 6 ml kg-1). All signals were recorded using the multi-channel platform PowerLab®. The arrival of the pulse wave at each catheter tip was determined using a MATLAB-based modified hyperbolic tangent algorithm and PWTT calculated as the time interval between these arrivals.Main results. Correlation coefficient for PWTT and mean PAP wasr= 0.932 for thromboxane. This correlation coefficient increased considerably when heart beats either at end-inspiration (r= 0.978) or at end-expiration (r= 0.985) were selected (=respiratory gating).Significance. The estimation of mean PAP from PWTT improved significantly when taking the respiratory cycle into account. Respiratory gating is suggested to improve for the estimation of PAP by PWTT.

7 Tesla MRI Liver Fat Quantification in Mice: Data Quality Assessment.

PURPOSE: The objective of this study was to evaluate the robustness of proton density fat fraction (PDFF) data determined by magnetic resonance imaging (MRI) and spectroscopy (MRS) via spatially resolved error estimation. MATERIALS AND METHODS: Using standard T2* relaxation time measurement protocols, in-vivo and ex-vivo MRI data with water and fat nominally in phase or out of phase relative to each other were acquired on a 7 T small animal scanner. Based on a total of 24 different echo times, PDFF maps were calculated in a magnitude-based approach. After identification of the decisive error-prone variables, pixel-wise error estimation was performed by simple propagation of uncertainty. The method was then used to evaluate PDFF data acquired for an explanted mouse liver and an in vivo mouse liver measurement. RESULTS: The determined error maps helped excluding measurement errors as cause of unexpected local PDFF variations in the explanted liver. For in vivo measurements, severe error maps gave rise to doubts in the acquired PDFF maps and triggered an in-depth analysis of possible causes, yielding abdominal movement or bladder filling as in vivo occurring reasons for the increased errors. CONCLUSION: The combination of pixel-wise acquisition of PDFF data and the corresponding error maps allows for a more specific, spatially resolved evaluation of the PDFF value reliability.

Multi-modal assessment of a cardiac stem cell therapy reveals distinct modulation of regional scar properties.

BACKGROUND: The initial idea of functional tissue replacement has shifted to the concept that injected cells positively modulate myocardial healing by a non-specific immune response of the transplanted cells within the target tissue. This alleged local modification of the scar requires assessment of regional properties of the left ventricular wall in addition to commonly applied measures of global morphological and functional parameters. Hence, we aimed at investigating the effect of cardiac cell therapy with cardiovascular progenitor cells, so-called cardiac induced cells, on both global and regional properties of the left ventricle by a multimodal imaging approach in a mouse model. METHODS: Myocardial infarction was induced in mice by ligation of the left anterior descending artery, the therapy group received an intramyocardial injection of 1 × 106 cardiac induced cells suspended in matrigel, the control group received matrigel only. [18F]FDG positron emission tomography imaging was performed after 17 days, to assess regional glucose metabolism. Three weeks after myocardial infarction, cardiac magnetic resonance imaging was performed for morphological and functional assessment of the left ventricle. Following these measurements, hearts were excised for histological examinations. RESULTS: Cell therapy had no significant effect on global morphological parameters. Similarly, there was no difference in scar size and capillary density between therapy and control group. However, there was a significant improvement in contractile function of the left ventricle - left ventricular ejection fraction, stroke volume and cardiac output. Regional analysis of the left ventricle identified changes of wall properties in the scar area as the putative mechanism. Cell therapy reduced the thinning of the scar and significantly improved its radial contractility. Furthermore, the metabolic defect, assessed by [18F]FDG, was significantly reduced by the cell therapy. CONCLUSION: Our data support the relevance of extending the assessment of global left ventricular parameters by a structured regional wall analysis for the evaluation of therapies targeting at modulation of healing myocardium. This approach will enable a deeper understanding of mechanisms underlying the effect of experimental regenerative therapies, thus paving the way for a successful translation into clinical application.

Synergistic effect of cold gas plasma and experimental drug exposure exhibits skin cancer toxicity in vitro and in vivo.

INTRODUCTION: Skin cancer is often fatal, which motivates new therapy avenues. Recent advances in cancer treatment are indicative of the importance of combination treatments in oncology. Previous studies have identified small molecule-based therapies and redox-based technologies, including photodynamic therapy or medical gas plasma, as promising candidates to target skin cancer. OBJECTIVE: We aimed to identify effective combinations of experimental small molecules with cold gas plasma for therapy in dermato-oncology. METHODS: Promising drug candidates were identified after screening an in-house 155-compound library using 3D skin cancer spheroids and high content imaging. Combination effects of selected drugs and cold gas plasma were investigated with respect to oxidative stress, invasion, and viability. Drugs that had combined well with cold gas plasma were further investigated in vascularized tumor organoids in ovo and a xenograft mouse melanoma model in vivo. RESULTS: The two chromone derivatives Sm837 and IS112 enhanced cold gas plasma-induced oxidative stress, including histone 2A.X phosphorylation, and further reduced proliferation and skin cancer cell viability. Combination treatments of tumor organoids grown in ovo confirmed the principal anti-cancer effect of the selected drugs. While one of the two compounds exerted severe toxicity in vivo, the other (Sm837) resulted in a significant synergistic anti-tumor toxicity at good tolerability. Principal component analysis of protein phosphorylation profiles confirmed profound combination treatment effects in contrast to the monotherapies. CONCLUSION: We identified a novel compound that, combined with topical cold gas plasma-induced oxidative stress, represents a novel and promising treatment approach to target skin cancer.

Establishing safe high hydrostatic pressure devitalization thresholds for autologous head and neck cancer vaccination and reconstruction.

High hydrostatic pressure specifically devitalizes cells and tissues without major changes in their molecular structure. Hence, high hydrostatic pressure may enhance the development of whole-cell anti-tumor vaccines, representing tumor heterogeneity and thus (neo-) antigen diversity. Moreover, safe devitalization of tumor-infiltrated supporting tissue may facilitate reimplantation for functional reconstruction. However, precise high hydrostatic pressure thresholds for safe cancer cell killing are unknown. Here, we show that high hydrostatic pressure of at least 450 MPa is necessary to safely devitalize head and neck squamous cell cancer. A pressure of 300 MPa, which has been used frequently in cancer vaccine preparation, resulted in partial devitalization with 27% live cells in flow cytometry and 4% remaining autofluorescence in cell culture after one week. The remaining cells could form vital tumors in the chorioallantoic membrane assay. In contrast, 450 MPa killed all cells in vitro and prevented tumor outgrowth in ovo. The effectiveness of 450 MPa was attributed to the induction of DNA double-strand breaks, independent of apoptosis, autophagy, or methuosis. Furthermore, 450 MPa continued to induce immunogenic cell death. Our results demonstrate that 450 MPa of high hydrostatic pressure induces safe and sustained devitalization of head and neck cancer cells and tissues. Because of the heterogeneity in pressure resistance, we propose our approach as a starting point for determining the precise thresholds for other cancer entities. Further studies on head and neck cancer should focus on immunological co-cultures, combinations of immune checkpoint inhibition, and accurate anatomical reconstruction with pressure-treated autografts.

Towards substitution of invasive telemetry: An integrated home cage concept for unobtrusive monitoring of objective physiological parameters in rodents.

This study presents a novel concept for a smart home cage design, tools, and software used to monitor the physiological parameters of mice and rats in animal-based experiments. The proposed system focuses on monitoring key clinical parameters, including heart rate, respiratory rate, and body temperature, and can also assess activity and circadian rhythm. As the basis of the smart home cage system, an in-depth analysis of the requirements was performed, including camera positioning, imaging system types, resolution, frame rates, external illumination, video acquisition, data storage, and synchronization. Two different camera perspectives were considered, and specific camera models, including two near-infrared and two thermal cameras, were selected to meet the requirements. The developed specifications, hardware models, and software are freely available via GitHub. During the first testing phase, the system demonstrated the potential of extracting vital parameters such as respiratory and heart rate. This technology has the potential to reduce the need for implantable sensors while providing reliable and accurate physiological data, leading to refinement and improvement in laboratory animal care.

Pressure- vs. volume-controlled ventilation and their respective impact on dynamic parameters of fluid responsiveness: a cross-over animal study.

BACKGROUND AND GOAL OF STUDY: Pulse pressure variation (PPV) and stroke volume variation (SVV), which are based on the forces caused by controlled mechanical ventilation, are commonly used to predict fluid responsiveness. When PPV and SVV were introduced into clinical practice, volume-controlled ventilation (VCV) with tidal volumes (VT) ≥ 10 ml kg- 1 was most commonly used. Nowadays, lower VT and the use of pressure-controlled ventilation (PCV) has widely become the preferred type of ventilation. Due to their specific flow characteristics, VCV and PCV result in different airway pressures at comparable tidal volumes. We hypothesised that higher inspiratory pressures would result in higher PPVs and aimed to determine the impact of VCV and PCV on PPV and SVV. METHODS: In this self-controlled animal study, sixteen anaesthetised, paralysed, and mechanically ventilated (goal: VT 8 ml kg- 1) pigs were instrumented with catheters for continuous arterial blood pressure measurement and transpulmonary thermodilution. At four different intravascular fluid states (IVFS; baseline, hypovolaemia, resuscitation I and II), ventilatory and hemodynamic data including PPV and SVV were assessed during VCV and PCV. Statistical analysis was performed using U-test and RM ANOVA on ranks as well as descriptive LDA and GEE analysis. RESULTS: Complete data sets were available of eight pigs. VT and respiratory rates were similar in both forms. Heart rate, central venous, systolic, diastolic, and mean arterial pressures were not different between VCV and PCV at any IVFS. Peak inspiratory pressure was significantly higher in VCV, while plateau, airway and transpulmonary driving pressures were significantly higher in PCV. However, these higher pressures did not result in different PPVs nor SVVs at any IVFS. CONCLUSION: VCV and PCV at similar tidal volumes and respiratory rates produced PPVs and SVVs without clinically meaningful differences in this experimental setting. Further research is needed to transfer these results to humans.

Effect of Calcitriol and Vitamin D Receptor Modulator 2 on Recovery of Injured Skeletal Muscle in Wistar Rats.

Muscle injuries often result in functional limitations due to insufficient healing. This study assessed the influence of calcitriol and vitamin D Receptor Modulator 2 (VDRM2) on muscle regeneration in male Wistar rats following open blunt muscle injury. The injured left soleus muscle of the rats was treated for the first four days after trauma with local injections of either calcitriol, VDRM2, or a 10% ethanol solution (control). Although muscle strength significantly decreased post-injury, all groups showed gradual improvement but did not achieve full recovery. By the 14th day, calcitriol-treated rats significantly outperformed the control group in the incomplete tetanic force, with VDRM2-treated rats showing muscle strength values that fell between the control and calcitriol groups. Similar trends were observed in complete tetanic contractions and were confirmed histologically via muscle cell width quantification. Additionally, histological analysis showed increased cellular turnover on the fourth postoperative day in the calcitriol group, as indicated by elevated cell proliferation rates and fewer apoptotic cells. VDRM2-treated animals showed only an increased proliferative activity on day 4 after injury. No noticeable differences between the groups for CAE-positive cells or visible muscle tissue area were found. In conclusion, predominantly calcitriol positively influenced post-trauma muscle recovery, where VDRM2 had substantially lower biological activity.

Sarcomeric network analysis of ex vivo cultivated human atrial appendage tissue using super-resolution microscopy.

Investigating native human cardiac tissue with preserved 3D macro- and microarchitecture is fundamental for clinical and basic research. Unfortunately, the low accessibility of the human myocardium continues to limit scientific progress. To overcome this issue, utilizing atrial appendages of the human heart may become highly beneficial. Atrial appendages are often removed during open-heart surgery and can be preserved ex vivo as living tissue with varying durability depending on the culture method. In this study, we prepared living thin myocardial slices from left atrial appendages that were cultured using an air-liquid interface system for overall 10 days. Metabolic activity of the cultured slices was assessed using a conventional methyl thiazolyl tetrazolium (MTT) assay. To monitor the structural integrity of cardiomyocytes within the tissue, we implemented our recently described super-resolution microscopy approach that allows both qualitative and quantitative in-depth evaluation of sarcomere network based on parameters such as overall sarcomere content, filament size and orientation. Additionally, expression of mRNAs coding for key structural and functional proteins was analyzed by real-time reverse transcription polymerase chain reaction (qRT-PCR). Our findings demonstrate highly significant disassembly of contractile apparatus represented by degradation of [Formula: see text]-actinin filaments detected after three days in culture, while metabolic activity was constantly rising and remained high for up to seven days. However, gene expression of crucial cardiac markers strongly decreased after the first day in culture indicating an early destructive response to ex vivo conditions. Therefore, we suggest static cultivation of living myocardial slices derived from left atrial appendage and prepared according to our protocol only for short-termed experiments (e.g. medicinal drug testing), while introduction of electro-mechanical stimulation protocols may offer the possibility for long-term integrity of such constructs.

CCR2 macrophage response determines the functional outcome following cardiomyocyte transplantation.

BACKGROUND: The immune response is a crucial factor for mediating the benefit of cardiac cell therapies. Our previous research showed that cardiomyocyte transplantation alters the cardiac immune response and, when combined with short-term pharmacological CCR2 inhibition, resulted in diminished functional benefit. However, the specific role of innate immune cells, especially CCR2 macrophages on the outcome of cardiomyocyte transplantation, is unclear. METHODS: We compared the cellular, molecular, and functional outcome following cardiomyocyte transplantation in wildtype and T cell- and B cell-deficient Rag2del mice. The cardiac inflammatory response was assessed using flow cytometry. Gene expression profile was assessed using single-cell and bulk RNA sequencing. Cardiac function and morphology were determined using magnetic resonance tomography and immunohistochemistry respectively. RESULTS: Compared to wildtype mice, Rag2del mice show an increased innate immune response at steady state and disparate macrophage response after MI. Subsequent single-cell analyses after MI showed differences in macrophage development and a lower prevalence of CCR2 expressing macrophages. Cardiomyocyte transplantation increased NK cells and monocytes, while reducing CCR2-MHC-IIlo macrophages. Consequently, it led to increased mRNA levels of genes involved in extracellular remodelling, poor graft survival, and no functional improvement. Using machine learning-based feature selection, Mfge8 and Ccl7 were identified as the primary targets underlying these effects in the heart. CONCLUSIONS: Our results demonstrate that the improved functional outcome following cardiomyocyte transplantation is dependent on a specific CCR2 macrophage response. This work highlights the need to study the role of the immune response for cardiomyocyte cell therapy for successful clinical translation.

Chronic oxidative stress adaptation in head and neck cancer cells generates slow-cyclers with decreased tumour growth in vivo.

BACKGROUND: Reactive oxygen species (ROS) are implicated in cancer therapy and as drivers of microenvironmental tumour cell adaptations. Medical gas plasma is a multi-ROS generating technology that has been shown effective for palliative tumour control in head and neck cancer (HNC) patients before tumour cells adapted to the oxidative stress and growth regressed fatally. METHODS: In a bedside-to-bench approach, we sought to explore the oxidative stress adaptation in two human squamous cell carcinoma cell lines. Gas plasma was utilised as a putative therapeutic agent and chronic oxidative stress inducer. RESULTS: Cellular responses of single and multiple treated cells were compared regarding sensitivity, cellular senescence, redox state and cytokine release. Whole transcriptome analysis revealed a strong correlation of cancer cell adaption with increased interleukin 1 receptor type 2 (IL1R2) expression. Using magnetic resonance imaging, tumour growth and gas plasma treatment responses of wild-type (WT) and repeatedly exposed (RE) A431 cells were further investigated in a xenograft model in vivo. RE cells generated significantly smaller tumours with suppressed inflammatory secretion profiles and increased epidermal growth factor receptor (EGFR) activity showing significantly lower gas plasma sensitivity until day 8. CONCLUSIONS: Clinically, combination treatments together with cetuximab, an EGFR inhibitor, may overcome acquired oxidative stress resistance in HNC.

ALM Therapy Promotes Functional and Histologic Regeneration of Traumatized Peripheral Skeletal Muscle.

Skeletal muscle trauma is a common injury with a range of severity. Adenosine, lidocaine and Mg2+ (ALM) is a protective solution and improves tissue perfusion and coagulopathy. Male Wistar rats were anesthetized and subjected to standardized skeletal muscle trauma of the left soleus muscle with the protection of the neurovascular structures. Seventy animals were randomly assigned to saline control or ALM. Immediately after trauma, a bolus of ALM solution was applied intravenously, followed by a one-hour infusion. After 1, 4, 7, 14 and 42 days, the biomechanical regenerative capacity was examined using incomplete tetanic force and tetany, and immunohistochemistry was used to examine for proliferation and apoptosis characteristics. Biomechanical force development showed a significant increase following ALM therapy for incomplete tetanic force and tetany on days 4 and 7. In addition, the histological evaluation showed a significant increase in proliferative BrdU-positive cells with ALM therapy on days 1 and 14. Ki67 histology also detected significantly more proliferative cells on days 1, 4, 7, 14 and 42 in ALM-treated animals. Furthermore, a simultaneous decrease in the number of apoptotic cells was observed using the TUNEL method. ALM solution showed significant superiority in biomechanical force development and also a significant positive effect on cell proliferation in traumatized skeletal muscle tissue and reduced apoptosis.

[18F]GE-180-PET and Post Mortem Marker Characteristics of Long-Term High-Fat-Diet-Induced Chronic Neuroinflammation in Mice.

Obesity is characterized by immoderate fat accumulation leading to an elevated risk of neurodegenerative disorders, along with a host of metabolic disturbances. Chronic neuroinflammation is a main factor linking obesity and the propensity for neurodegenerative disorders. To determine the cerebrometabolic effects of diet-induced obesity (DIO) in female mice fed a long-term (24 weeks) high-fat diet (HFD, 60% fat) compared to a group on a control diet (CD, 20% fat), we used in vivo PET imaging with the radiotracer [18F]FDG as a marker for brain glucose metabolism. In addition, we determined the effects of DIO on cerebral neuroinflammation using translocator protein 18 kDa (TSPO)-sensitive PET imaging with [18F]GE-180. Finally, we performed complementary post mortem histological and biochemical analyses of TSPO and further microglial (Iba1, TMEM119) and astroglial (GFAP) markers as well as cerebral expression analyses of cytokines (e.g., Interleukin (IL)-1ß). We showed the development of a peripheral DIO phenotype, characterized by increased body weight, visceral fat, free triglycerides and leptin in plasma, as well as increased fasted blood glucose levels. Furthermore, we found obesity-associated hypermetabolic changes in brain glucose metabolism in the HFD group. Our main findings with respect to neuroinflammation were that neither [18F]GE-180 PET nor histological analyses of brain samples seem fit to detect the predicted cerebral inflammation response, despite clear evidence of perturbed brain metabolism along with elevated IL-1ß expression. These results could be interpreted as a metabolically activated state in brain-resident immune cells due to a long-term HFD.

Effects on Bone and Muscle upon Treadmill Interval Training in Hypogonadal Male Rats.

Testosterone deficiency in males is linked to various pathological conditions, including muscle and bone loss. This study evaluated the potential of different training modalities to counteract these losses in hypogonadal male rats. A total of 54 male Wistar rats underwent either castration (ORX, n = 18) or sham castration (n = 18), with 18 castrated rats engaging in uphill, level, or downhill interval treadmill training. Analyses were conducted at 4, 8, and 12 weeks postsurgery. Muscle force of the soleus muscle, muscle tissue samples, and bone characteristics were analyzed. No significant differences were observed in cortical bone characteristics. Castrated rats experienced decreased trabecular bone mineral density compared to sham-operated rats. However, 12 weeks of training increased trabecular bone mineral density, with no significant differences among groups. Muscle force measurements revealed decreased tetanic force in castrated rats at week 12, while uphill and downhill interval training restored force to sham group levels and led to muscle hypertrophy compared to ORX animals. Linear regression analyses showed a positive correlation between bone biomechanical characteristics and muscle force. The findings suggest that running exercise can prevent bone loss in osteoporosis, with similar bone restoration effects observed across different training modalities.

Leptin deficiency-caused behavioral change - A comparative analysis using EthoVision and DeepLabCut.

Introduction: Obese rodents e.g., the leptin-deficient (ob/ob) mouse exhibit remarkable behavioral changes and are therefore ideal models for evaluating mental disorders resulting from obesity. In doing so, female as well as male ob/ob mice at 8, 24, and 40 weeks of age underwent two common behavioral tests, namely the Open Field test and Elevated Plus Maze, to investigate behavioral alteration in a sex- and age dependent manner. The accuracy of these tests is often dependent on the observer that can subjectively influence the data. Methods: To avoid this bias, mice were tracked with a video system. Video files were further analyzed by the compared use of two software, namely EthoVision (EV) and DeepLabCut (DLC). In DLC a Deep Learning application forms the basis for using artificial intelligence in behavioral research in the future, also with regard to the reduction of animal numbers. Results: After no sex and partly also no age-related differences were found, comparison revealed that both software lead to almost identical results and are therefore similar in their basic outcomes, especially in the determination of velocity and total distance movement. Moreover, we observed additional benefits of DLC compared to EV as it enabled the interpretation of more complex behavior, such as rearing and leaning, in an automated manner. Discussion: Based on the comparable results from both software, our study can serve as a starting point for investigating behavioral alterations in preclinical studies of obesity by using DLC to optimize and probably to predict behavioral observations in the future.

The Effect of Early Application of Synthetic Peptides 19-2.5 and 19-4LF to Improve Survival and Neurological Outcome in a Mouse Model of Cardiac Arrest and Resuscitation.

The synthetic antimicrobial peptides (sAMPs) Pep19-2.5 and Pep19-4LF have been shown in vitro and in vivo to reduce the release of pro-inflammatory cytokines, leading to the suppression of inflammation and immunomodulation. We hypothesized that intervention with Pep19-2.5 and Pep19-4LF immediately after cardiac arrest and resuscitation (CA-CPR) might attenuate immediate systemic inflammation, survival, and long-term outcomes in a standardized mouse model of CA-CPR. Long-term outcomes up to 28 days were assessed between a control group (saline) and two peptide intervention groups. Primarily, survival as well as neurological and cognitive parameters were assessed. In addition, systemic inflammatory molecules and specific biomarkers were analyzed in plasma as well as in brain tissue. Treatment with sAMPs did not provide any short- or long-term benefits for either survival or neurological outcomes, and no significant benefit on inflammation in the CA-CPR animal model. While no difference was found in the plasma analysis of early cytokines between the intervention groups four hours after resuscitation, a significant increase in UCH-L1, a biomarker of neuronal damage and blood-brain barrier rupture, was measured in the Pep19-4LF-treated group. The theoretical benefit of both sAMPs tested here for the treatment of post-cardiac arrest syndrome could not be proven.

Robustness of a multivariate composite score when evaluating distress of animal models for gastrointestinal diseases.

The fundament of an evidence-based severity assessment in laboratory animal science is reliable distress parameters. Many readouts are used to evaluate and determine animal distress and the severity of experimental procedures. Therefore, we analyzed four distinct parameters like the body weight, burrowing behavior, nesting, and distress score in the four gastrointestinal animal models (pancreatic ductal adenocarcinoma (PDA), pancreatitis, CCl4 intoxication, and bile duct ligation (BDL)). Further, we determined the parameters' robustness in various experimental subgroups due to slight variations like drug treatment or telemeter implantations. We used non-parametric bootstrapping to get robust estimates and 95% confidence intervals for the experimental groups. It was found that the performance of the readout parameters is model-dependent and that the distress score is prone to experimental variation. On the other hand, we also found that burrowing and nesting can be more robust than, e.g., the body weight when evaluating PDA. However, the body weight still was highly robust in BDL, pancreatitis, and CCl4 intoxication. To address the complex nature of the multi-dimensional severity space, we used the Relative Severity Assessment (RELSA) procedure to combine multiple distress parameters into a score and mapped the subgroups and models against a defined reference set obtained by telemeter implantation. This approach allowed us to compare the severity of individual animals in the experimental subgroups using the maximum achieved severity (RELSAmax). With this, the following order of severity was found for the animal models: CCl4 < PDA ≈ Pancreatitis < BDL. Furthermore, the robustness of the RELSA procedure and outcome was externally validated with a reference set from another laboratory also obtained from telemeter implantation. Since the RELSA procedure reflects the multi-dimensional severity information and is highly robust in estimating the quantitative severity within and between models, it can be deemed a valuable tool for laboratory animal severity assessment.

Ventilation Induces Changes in Pulse Wave Transit Time in the Pulmonary Artery.

Pulse wave transit time (PWTT) shortens as pulmonary artery pressure (PAP) increases and was therefore suggested as a surrogate parameter for PAP. The aim of this analysis was to reveal patterns and potential mechanisms of ventilation-induced periodic changes in PWTT under resting conditions. To measure both PWTT and PAP in five healthy pigs, two pulmonary artery Mikro-Tip™ catheters were inserted into the pulmonary vasculature: one with the tip placed in the pulmonary artery trunk, and a second one placed in a distal segment of the pulmonary artery. Animals received pressure-controlled mechanical ventilation. Ventilation-dependent changes were seen in both variables, PWTT and mean PAP; however, changes in PWTT were not synchronous with changes in PAP. Thus, plotting the value of PWTT for each heartbeat over the respective PAP revealed a characteristic hysteresis. At the beginning of inspiration, PAP rose while PWTT remained constant. During further inspiration, PWTT started to decrease rapidly as mPAP was about to reach its plateau. The same time course was observed during expiration: while mPAP approached its minimum, PWTT increased rapidly. During apnea this hysteresis disappeared. Thus, non-synchronous ventilation-induced changes in PWTT and PAP were found with inspiration causing a significant shortening of PWTT. Therefore, it is suggested that the respiratory cycle should be considered when using PWTT as a surrogate for PAP.

The Dorsal Skinfold Chamber as a New Tympanic Membrane Wound Healing Model: Intravital Insights into the Pathophysiology of Epithelialized Wounds.

BACKGROUND: Tympanic membrane perforations (TMPs) are a common complication of trauma and infection. Persisting perforations result from the unique location of the tympanic membrane. The wound is surrounded by air of the middle ear and the external auditory canal. The inadequate wound bed, growth factor, and blood supply lead to circular epithelialization of the perforation's edge and premature interruption of defect closure. Orthotopic animal models use mechanical or chemical tympanic membrane laceration to identify bioactive wound dressings and overcome premature epithelialization. However, all orthotopic models essentially lack repetitive visualization of the biomaterial-wound interface. Therefore, recent progress in 3D printing of customized wound dressings has not yet been transferred to the unique wound setup of the TMP. Here, we present a novel application for the mice dorsal skinfold chamber (DSC) with an epithelialized full-thickness defect as TMP model. METHODS: A circular 2-mm defect was cut into the extended dorsal skinfold using a biopsy punch. The skinfold was either perforated through both skin layers without prior preparation or perforated on 1 side, following resection of the opposing skin layer. In both groups, the wound was sealed with a coverslip or left unclosed (n = 4). All animals were examined for epithelialization of the edge (histology), size of the perforation (planimetry), neovascularization (repetitive intravital fluorescence microscopy), and inflammation (immunohistology). RESULTS: The edge of the perforation was overgrown by the cornified squamous epithelium in all pre-parations. Reduction in the perforation's size was enhanced by application of a coverslip. Microsurgical preparation before biopsy punch perforation and sealing with a coverslip enabled repetitive high-quality intravital fluorescence microscopy. However, spontaneous reduction of the perforation occurred frequently. Therefore, the direct biopsy punch perforation without microsurgical preparation was favorable: spontaneous reduction did not occur throughout 21 days. Moreover, the visualization of the neovascularization was sufficient in intravital microscopy. CONCLUSIONS: The DSC full-thickness defect is a valuable supplement to orthotopic TMP models. Repetitive intravital microscopy of the epithelialized edge enables investigation of the underlying pathophysiology during the transition from the inflammation to the proliferation phase of wound healing. Using established analysis procedures, the present model provides an effective platform for the screening of bioactive materials and transferring progress in tissue engineering to the special conditions of tympanic membrane wound healing.