Iron deficiency-induced thrombocytosis increases thrombotic tendency in rats.

Iron deficiency (ID) is globally prevalent, and apart from anemia is associated with thrombocytosis. While considered benign, studies linking thrombotic events with prior ID anemia suggest otherwise. Herein we used animal models to assess the influence of ID on thrombotic tendency. Sprague-Dawley rats were fed control or iron deficient diets and ferric carboxymaltose was used to reverse ID. Thrombosis was induced via stenosis of the inferior vena cava or damage to the right carotid artery using ferric chloride. Thrombi were evaluated histologically and via high frequency ultrasound in the venous model. ID consistently induced thrombocytosis alongside anemia. Venous thrombus growth and final dimensions in both arterial and venous thrombi were largest in ID. In both models, platelet numbers correlated with the final thrombus size, with ID thrombi having the largest platelet areas. Platelet function was also evaluated in surgically naive rats. Coagulability on thromboelastography and hemostasis on tail transection were augmented in ID. Platelet and plasma P-selectin expression were both higher in ID. Platelet adhesion and aggregation in ID was impaired under shear flow but was intact on static assays. Iron replacement therapy reversed all ID-related changes in hematological parameters, thrombus dimensions, and platelet assays. In summary, ID alone increases thrombotic tendency. Iron replacement therapy reverses these changes, making it a viable strategy for prevention of ID-related thrombotic disease. This may be of importance in patients with chronic illnesses which may already be at increased risk for thrombosis such as inflammatory bowel disease, chronic kidney disease, or cancer.

Tenascin C promotes valvular remodeling in two large animal models of ischemic mitral regurgitation.

Ischemic mitral regurgitation (MR) is a frequent complication of myocardial infarction (MI) characterized by adverse remodeling both at the myocardial and valvular levels. Persistent activation of valvular endothelial cells leads to leaflet fibrosis through endothelial-to-mesenchymal transition (EMT). Tenascin C (TNC), an extracellular matrix glycoprotein involved in cardiovascular remodeling and fibrosis, was also identified in inducing epithelial-to-mesenchymal transition. In this study, we hypothesized that TNC also plays a role in the valvular remodeling observed in ischemic MR by contributing to valvular excess EMT. Moderate ischemic MR was induced by creating a posterior papillary muscle infarct (7 pigs and 7 sheep). Additional animals (7 pigs and 4 sheep) served as controls. Pigs and sheep were sacrificed after 6 weeks and 6 months, respectively. TNC expression was upregulated in the pig and sheep experiments at 6 weeks and 6 months, respectively, and correlated well with leaflet thickness (R = 0.68; p < 0.001 at 6 weeks, R = 0.84; p < 0.001 at 6 months). To confirm the translational potential of our findings, we obtained mitral valves from patients with ischemic cardiomyopathy presenting MR (n = 5). Indeed, TNC was also expressed in the mitral leaflets of these. Furthermore, TNC induced EMT in isolated porcine mitral valve endothelial cells (MVEC). Interestingly, Toll-like receptor 4 (TLR4) inhibition prevented TNC-mediated EMT in MVEC. We identified here for the first time a new contributor to valvular remodeling in ischemic MR, namely TNC, which induced EMT through TLR4. Our findings might set the path for novel therapeutic targets for preventing or limiting ischemic MR.

Establishment of an optimized guinea pig model of cisplatin-induced ototoxicity.

Background: Cisplatin is among the most effective antineoplastic agents and has revolutionized the treatment of many cancer diseases. However, one of its serious side effects is a progressive and irreversible hearing loss, occurring in both adults and children. For the development of otoprotective therapies that prevent this side effect, cisplatin-induced hearing loss animal models are indispensable. Due to the high toxicity of cisplatin, the establishment of such animal models is a difficult and time-consuming task. Here we introduce the detailed protocol of a sophisticated guinea pig model with a sufficient and permanent hearing loss induced by cisplatin. This manuscript is intended to provide guidance in the development of future cisplatin guinea pig models which may reduce the mortality rate of the animals and help to gain more reproducible results. Methods: Pigmented and unpigmented guineapigs were treated with an intravenous single application of 8 mg/kg cisplatin under general anesthesia. An extensive and long-term intensive care protocol consisting of scheduled application of fluids, antiemetics, analgesics, glucose and supportive feeding among others, was used to ensure wellbeing of the animals. Hearing tests were performed prior to and 5 days after cisplatin application. Animals were then euthanized. Results: The ABR audiometry 5 days after cisplatin application revealed a hearing threshold ranging from 70 dB to 90 dB in the frequencies from 1 kHz to 32 kHz respectively.All animals presented a good health condition despite the treatment with cisplatin. Discussion: The introduced care protocol in this manuscript is intended to serve as a guidance for the establishment of a stable guinea pig model for short- and long-term investigation regarding the inner ear and its protection in the frame work of cisplatin-induced damage.

A novel ex-vivo isolated rabbit heart preparation to explore the cardiac effects of cervical and cardiac vagus nerve stimulation.

The cardiac responses to vagus nerve stimulation (VNS) are still not fully understood, partly due to uncontrollable confounders in the in-vivo experimental condition. Therefore, an ex-vivo Langendorff-perfused rabbit heart with intact vagal innervation is proposed to study VNS in absence of cofounding anesthetic or autonomic influences. The feasibility to evoke chronotropic responses through electrical stimulation ex-vivo was studied in innervated isolated rabbit hearts (n = 6). The general nerve excitability was assessed through the ability to evoke a heart rate (HR) reduction of at least 5 bpm (physiological threshold). The excitability was quantified as the charge needed for a 10-bpm HR reduction. The results were compared to a series of in-vivo experiments rabbits (n = 5). In the ex-vivo isolated heart, the baseline HR was about 20 bpm lower than in-vivo (158 ± 11 bpm vs 181 ± 19 bpm). Overall, the nerve remained excitable for about 5 h ex-vivo. The charges required to reduce HR by 5 bpm were 9 ± 6 µC and 549 ± 370 µC, ex-vivo and in-vivo, respectively. The charges needed for a 10-bpm HR reduction, normalized to the physiological threshold were 1.78 ± 0.8 and 1.22 ± 0.1, in-vivo and ex-vivo, respectively. Overall, the viability of this ex-vivo model to study the acute cardiac effects of VNS was demonstrated.

Feasibility of an Animal Model for Cavopulmonary Support With a Double-Outflow Pump.

Both single- and double-outflow cavopulmonary assist devices (CPADs) were recently proposed for the Fontan population, whereas single-outflow configurations were evaluated in large animal trials and double-outflow concepts are lacking an equivalent in vivo assessment. The aim of this study was to test the hemodynamic properties of a double-outflow CPAD device in an acute sheep model. The two inflow cannulae of a CPAD were anastomosed to the caval veins. Outflow graft connection was performed via end-to-side anastomosis to the right (RPA) and main pulmonary artery (MPA). Speed ramp protocols were conducted, and hemodynamic effects were monitored in terms of caval flows, cardiac output (CO), central venous pressure (CVP), pulmonary artery pressure (PAP), and left atrial pressure (LAP). Six experiments were conducted (53.35 ± 5.1 kg). In three experiments, the animal model was established, the CPAD was examined, and restoration of biventricular equivalency in terms of venous return was achieved. Venous pressures (CVP) declined linearly with increasing pump speed (r > 0.879), whereas caval flow (r > 0.973), CO (r > 0.993), PAP (r > 0.973), and LAP (r > 0.408) increased. Despite the considerable complexity of the sheep model caused by the sheep pulmonary arterial anatomy that requires substantial graft bending, the CPAD was evaluated in three acute experiments and showed the potential to completely substitute a subpulmonary ventricle.

Closed-loop vagus nerve stimulation for heart rate control evaluated in the Langendorff-perfused rabbit heart.

Persistent sinus tachycardia substantially increases the risk of cardiac death. Vagus nerve stimulation (VNS) is known to reduce the heart rate, and hence may be a non-pharmacological alternative for the management of persistent sinus tachycardia. To precisely regulate the heart rate using VNS, closed-loop control strategies are needed. Therefore, in this work, we developed two closed-loop VNS strategies using an in-silico model of the cardiovascular system. Both strategies employ a proportional-integral controller that operates on the current amplitude. While one control strategy continuously delivers stimulation pulses to the vagus nerve, the other applies bursts of stimuli in synchronization with the cardiac cycle. Both were evaluated in Langendorff-perfused rabbit hearts (n = 6) with intact vagal innervation. The controller performance was quantified by rise time (Tr), steady-state error (SSE), and percentual overshoot amplitude (%OS). In the ex-vivo setting, the cardiac-synchronized variant resulted in Tr = 10.7 ± 4.5 s, SSE = 12.7 ± 9.9 bpm and %OS = 5.1 ± 3.6% while continuous stimulation led to Tr = 10.2 ± 5.6 s, SSE = 10 ± 6.7 bpm and %OS = 3.2 ± 1.9%. Overall, both strategies produced a satisfying and reproducible performance, highlighting their potential use in persistent sinus tachycardia.

Normothermic blood polarizing versus depolarizing cardioplegia in a porcine model of cardiopulmonary bypass.

OBJECTIVES: We have previously demonstrated beneficial cardiac protection with hypothermic polarizing cardioplegia compared to a hyperkalemic depolarizing cardioplegia. In this study, a porcine model of cardiopulmonary bypass was used to compare the protective effects of normothermic blood-based polarizing and depolarizing cardioplegia during cardiac arrest. METHODS: Thirteen pigs were randomized to receive either normothermic polarizing (n = 8) or depolarizing (n = 5) blood-based cardioplegia. After initiation of cardiopulmonary bypass, normothermic arrest (34°C, 60 min) was followed by 60 min of on-pump and 90 min of off-pump reperfusion. Primary outcome was myocardial injury measured as arterial myocardial creatine kinase concentration. Secondary outcome was haemodynamic function and the energy state of the hearts. RESULTS: During reperfusion, release of myocardial creatine kinase was comparable between groups (P = 0.36). In addition, most haemodynamic parameters showed comparable results between groups, but stroke volume (P = 0.03) was significantly lower in the polarizing group. Adenosine triphosphate levels were significantly (18.41 ± 3.86 vs 22.97 ± 2.73 nmol/mg; P = 0.03) lower in polarizing hearts, and the requirement for noradrenaline administration (P = 0.002) and temporary pacing (6 vs 0; P = 0.02) during reperfusion were significantly higher in polarizing hearts. CONCLUSIONS: Under normothermic conditions, polarizing blood cardioplegia was associated with similar myocardial injury to depolarizing blood cardioplegia. Reduced haemodynamic and metabolic outcome and a higher need for temporary pacing with polarized arrest may be associated with the blood-based dilution of this solution.

Ethanol reduces the minimum alveolar concentration of sevoflurane in rats.

A high number of trauma patients are under the influence of alcohol. Since many of them need immediate surgical procedures, it is imperative to be aware of the interaction of alcohol with general anesthesia. To counter challenges that arise from clinical studies, we designed an animal experiment in which 48 adult Wistar rats either received 1 g · kg-1 ethanol, 2 g · kg-1 ethanol or placebo via intraperitoneal application. Subsequently, they were anesthetized with an individual concentration of sevoflurane. The minimum alveolar concentration (MAC) of the different groups was assessed using Dixon's up-and-down design and isotonic regression methods. The bootstrap estimate of the MAC of sevoflurane in the placebo group was 2.24 vol% (95% CI 1.97-2.94 vol%). In the low dose ethanol group, the bootstrap estimate was 1.65 vol% (95% CI 1.40-1.98 vol%), and in the high dose ethanol group, it was 1.08 vol% (95% CI 0.73-1.42 vol%). We therefore report that intraperitoneal application of 1 g · kg-1 or 2 g · kg-1 ethanol both resulted in a significant reduction of the MAC of sevoflurane in adult Wistar rats: by 26.3% and 51.8% respectively as compared to placebo.

Single-incision cochlear implantation and hearing evaluation in piglets and minipigs.

OBJECTIVES: Various animal models have been established and applied in hearing research. In the exploration of novel cochlear implant developments, mainly rodents have been used. Despite their important contribution to the understanding of auditory function, translation of experimental observations from rodents to humans is limited due to the size differences and genetic variability. Large animal models with better representation of the human cochlea are sparse. For this reason, we evaluated domestic piglets and Aachen minipigs for the suitability as a cochlear implantation animal model with commercially available cochlear implants. METHODS: Four domestic piglets (two male and two female) and six Aachen minipigs were implanted with either MED-EL Flex24 or Flex20 cochlear implants respectively, after a step-by-step surgical approach was trained with pig cadavers. Electrophysiological measurements were performed before, during and after implantation for as long as 56 days after surgery. Auditory brainstem responses, electrocochleography as well as electrically and acoustically evoked compound action potentials were recorded. Selected cochleae were further analyzed histologically or with micro-CT imaging. RESULTS: A surgical approach was established using a retroauricular single incision. Baseline auditory thresholds were 27 ± 3 dB sound pressure level (SPL; auditory brainstem click responses, mean ± standard error of the mean) and ranged between 30 and 80 dB SPL in frequency-specific responses (0.5 - 32 kHz). Follow-up measurements revealed deafness within the first two weeks after surgery, but some animals partially recovered to a hearing threshold of 80 dB SPL in certain frequencies as well as in click responses. Electrically evoked compound action potential thresholds increased within the first week after surgery, which led to lower stimulation responses or increase of necessary charge input. Immune reactions and consecutive scalar fibrosis following implantation were confirmed with histological analysis of implanted cochleae and may result in increased impedances. A three-dimensional minipig micro-CT segmentation revealed cochlear volumetric data similar to human inner ear dimensions. CONCLUSIONS: This study underlines the feasibility of cochlear implantation with clinically used cochlear implants in a large animal model with representative inner ear dimensions comparable to humans. To bridge the gap between small animal models and humans in translational research and to account for the structural and size differences, we recommend the minipig as a valuable animal model for hearing research. First insights into the induced trauma in minipigs after cochlear implant surgery and a partial hearing recovery present important data of the cochlear health changes in large animal cochleae.

MicroRNA Expression Profile Changes after Cardiopulmonary Bypass and Ischemia/Reperfusion-Injury in a Porcine Model of Cardioplegic Arrest.

Identification of microRNAs (miRNA) associated with cardiopulmonary bypass, cardiac arrest and subsequent myocardial ischemia/reperfusion may unravel novel therapeutic targets and biomarkers. The primary aim of the present study was to investigate the effects of cardiopulmonary bypass and temperature of cardioplegic arrest on myocardial miRNA profile in pigs' left ventricular tissue. We employed next-generation sequencing to analyse miRNA profiles in the following groups: (1) hearts were arrested with antegrade warm St Thomas Hospital No. 2 (STH2) cardioplegia (n = 5; STH2-warm, 37 °C) and (2) cold STH2 (n = 6; STH2-cold, 4 °C) cardioplegia. Sixty min of ischemia was followed by 60 min of on-pump reperfusion with an additional 90 min of off-pump reperfusion. In addition, two groups without cardiac arrest (off-pump and on-pump group; n = 3, respectively) served as additional controls. STH2-warm and STH2-cold cardioplegia revealed no hemodynamic differences. In contrast, coronary venous creatine kinase-myocardial band (CK-MB) levels were significantly lower in pigs receiving STH2-warm cardioplegia (p < 0.05). Principal component analysis revealed that cardiopulmonary bypass and cardioplegic arrest markedly affected miRNAs in left ventricular tissue. Accordingly, ssc-miR-122, ssc-miR-10a-5p, ssc-miR-193a-3p, ssc-miR-499-3p, ssc-miR-374a-5p, ssc-miR-345-5p, ssc-miR-142-3p, ssc-miR-424-5p, ssc-miR-545-3p, ssc-miR-30b-5p, ssc-miR-145-5p, ssc-miR-374b-5p and ssc-miR-139-3p were differently regulated by cardiopulmonary bypass (false discovery rate (FDR) < 0.05 versus off-pump group). However, only ssc-miR-451 was differently expressed between STH2-warm and STH2-cold (FDR < 0.05). These data demonstrate for the first time that cardiopulmonary bypass and temperature of cardioplegic solution affected the expression of miRNAs in left ventricular tissue. In conclusion, specific miRNAs are potential therapeutic targets for limiting ischemia-reperfusion injury in patients undergoing cardiac surgery.

3D Printed Biomimetic Rabbit Airway Simulation Model for Nasotracheal Intubation Training.

Rabbit inhalation anesthesia by endotracheal intubation involves a higher risk among small animals owing to several anatomical and physiological features, which is pathognomonic to this species of lagomorphs. Rabbit-specific airway devices have been designed to prevent misguided intubation attempts. However, it is believed that expert anesthetic training could be a boon in limiting the aftermaths of this procedure. Our research is aimed to develop a novel biomimetic 3D printed rabbit airway model with representative biomechanical material behavior and radiodensity. Imaging data were collected for two sacrificed rabbit heads using micro-computed tomography (µCT) and micro-magnetic resonance imaging for the first head and cone beam computed tomography (CBCT) for the second head. Imaging-based life-size musculoskeletal airway models were printed using polyjet technology with a combination of hard and soft materials in replicates of three. The models were evaluated quantitatively for dimensional accuracy and radiodensity and qualitatively using digital microscopy and endoscopy for technical, tactic, and visual realism. The results displayed that simulation models printed with polyjet technology have an overall surface representation of 93% for µCT-based images and 97% for CBCT-based images within a range of 0.0-2.5 mm, with µCT showing a more detailed reproduction of the nasotracheal anatomy. Dimensional discrepancies can be caused due to inadequate support material removal and due to the limited reconstruction of microstructures from the imaging on the 3D printed model. The model showed a significant difference in radiodensities in hard and soft tissue regions. Endoscopic evaluation provided good visual and tactile feedback, comparable to the real animal. Overall, the model, being a practical low-cost simulator, comprehensively accelerates the learning curve of veterinary nasotracheal intubation and paves the way for 3D simulation-based image-guided interventional procedures.

CA1 Hippocampal Pyramidal Cells in Rats, Resuscitated From 8 Minutes of Ventricular Fibrillation Cardiac Arrest, Recover After 20 Weeks of Survival: A Retrospective Pilot Study.

PURPOSE: The cornu ammonis 1 (CA1) region of the hippocampus is specifically vulnerable to global ischemia. We hypothesized that histopathological outcome in a ventricular fibrillation cardiac arrest (VFCA) rat model depends on the time point of the examination. METHODS: Male Sprague-Dawley rats were put into VFCA for 8 min, received chest compressions for 2 min, and were defibrillated to achieve return of spontaneous circulation. Animals surviving for 80 min, 14 days and 140 days were compared with controls. Viable neurons were counted in a 500 µm sector of the CA1 region and layer thickness measured. Microglia cells and astrocytes were counted in a 250×300 µm aspect. RESULTS: Control and 80 min surviving animals had similar numbers of pyramidal neurons in the CA1 region. In 14 days and 140 days survivors neuron numbers and layer thickness were severely diminished compared with controls (P < 0.001). Two-thirds of the 140 days survivors showed significantly more viable neurons than the last third. Microglia was increased in 14 days survivors compared with controls and 140 days survivors, while astrocytes increased in 14 days and 140 days survivors compared with controls (P < 0.001). 140 days survivors had significantly higher astrocyte counts compared with 14 days survivors. CONCLUSIONS: The amount and type of brain lesions present after global ischemia depend on the survival time. A consistent reduction in pyramidal cells in the CA1 region was present in all animals 14 days after VFCA, but in two-thirds of animals a repopulation of pyramidal cells seems to have taken place after 140 days.

Motor Cortex and Hippocampus Display Decreased Heme Oxygenase Activity 2 Weeks After Ventricular Fibrillation Cardiac Arrest in Rats.

Heme oxygenase (HO) and biliverdin reductase (BVR) activities are important for neuronal function and redox homeostasis. Resuscitation from cardiac arrest (CA) frequently results in neuronal injury and delayed neurodegeneration that typically affect vulnerable brain regions, primarily hippocampus (Hc) and motor cortex (mC), but occasionally also striatum and cerebellum. We questioned whether these delayed effects are associated with changes of the HO/BVR system. We therefore analyzed the activities of HO and BVR in the brain regions Hc, mC, striatum and cerebellum of rats subjected to ventricular fibrillation CA (6 min or 8 min) after 2 weeks following resuscitation, or sham operation. From all investigated regions, only Hc and mC showed significantly decreased HO activities, while BVR activity was not affected. In order to find an explanation for the changed HO activity, we analyzed protein abundance and mRNA expression levels of HO-1, the inducible, and HO-2, the constitutively expressed isoform, in the affected regions. In both regions we found a tendency for a decreased immunoreactivity of HO-2 using immunoblots and immunohistochemistry. Additionally, we investigated the histological appearance and the expression of markers indicative for activation of microglia [tumor necrosis factor receptor type I (TNFR1) mRNA and immunoreactivity for ionized calcium-binding adapter molecule 1 (Iba1])], and activation of astrocytes [immunoreactivity for glial fibrillary acidic protein (GFAP)] in Hc and mC. Morphological changes were detected only in Hc displaying loss of neurons in the cornu ammonis 1 (CA1) region, which was most pronounced in the 8 min CA group. In this region also markers indicating inflammation and activation of pro-death pathways (expression of HO-1 and TNFR1 mRNA, as well as Iba1 and GFAP immunoreactivity) were upregulated. Since HO products are relevant for maintaining neuronal function, our data suggest that neurodegenerative processes following CA may be associated with a decreased capacity to convert heme into HO products in particularly vulnerable brain regions.

Cochlear Implantation in the Guinea Pig.

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception outcomes, candidacy criteria have been expanded over the last few decades. This includes patients with substantial residual hearing that benefit from electrical and acoustical stimulation of the same ear, which makes hearing preservation during cochlear implantation an important issue. Electrode impedances and the related issue of energy consumption is another major research field, as progress in this area could pave the way for fully implantable auditory prostheses. To address these issues in a systematic way, adequate animal models are essential. Therefore, the goal of this protocol is to provide an animal model of cochlear implantation, which can be used to address various research questions. Due to its large tympanic bulla, which allows easy surgical access to the inner ear, as well as its hearing range which is relatively similar to the hearing range of humans, the guinea pig is a commonly used species in auditory research. Cochlear implantation in the guinea pig is performed via a retroauricular approach. Through the bullostomy a cochleostomy is drilled and the cochlear implant electrode is inserted into the scala tympani. This electrode can then be used for electrical stimulation, determination of electrode impedances and the measurement of compound action potentials of the auditory nerve. In addition to these applications, cochlear implant electrodes can also be used as drug delivery devices, if a topical delivery of pharmaceutical agents to the cells or fluids of the inner ear is intended.

Feasibility of profound hypothermia as part of extracorporeal life support in a pig model.

OBJECTIVE: To investigate the feasibility of a refined aortic flush catheter and pump system to induce emergency preservation and resuscitation before extracorporeal cardiopulmonary resuscitation in a normovolemic cardiac arrest swine model simulating near real size/weight conditions of adults. METHODS: In this feasibility study, 8 female Large White breed pigs weighing 70 to 80 kg underwent ventricular fibrillation cardiac arrest for 15 minutes, followed by 4°C aortic flush (150 mL/kg for the brain; 50 mL/kg for the spine) via a new hardware ensued by resuscitation with extracorporeal cardiopulmonary resuscitation. RESULTS: Brain temperature was lowered from 39.9°C (interquartile range [IQR] 39.6-40.3) to 24.0°C (IQR 20.8-28.9) in 12 minutes (IQR 11-16) with a median cooling rate of 1.3°C (IQR 0.7-1.6) per minute. A median of 776 mL (IQR 673-840) per minute with a median pump pressure of 1487 mm Hg (IQR 1324-1545) were pumped to the brain. CONCLUSIONS: With the new hardware, we were able to cool the brain within a few minutes in a large pig cardiac arrest model. The exact position; the design, diameter, and length of the flush catheter; and the brain perfusion pressure seem to be critical to effectively reduce brain temperature. Redistribution of peripheral blood could lead to sterile inflammation again and might be avoided.