Large Experimental Animals Research Articles

Endoscopic Balloon Dilatation of the Eustachian Tube via the Soft Palate Approach in Miniature Pigs.

The eustachian tube (ET) is one of the most complex organs in the human body, and its dysfunction may lead to a variety of diseases. In recent years, an increasing number of scholars have opted to conduct ET-related studies using large experimental animals such as miniature pigs or sheep, yielding promising results. Typically, conventional endoscopic procedures are performed through the nasal approach for large experimental animals. However, due to the elongated and narrow nasal cavity in these animals, transnasal surgeries are challenging. To address this issue, we explored an ET surgery approach via the soft palate. The animal was placed in a supine position. After endotracheal intubation under general anesthesia, a mouth opener was used to fully expose the upper palate. Local infiltration with diluted adrenal fluid was performed for anesthesia of the area. A sickle knife was then used to make a longitudinal soft palate incision at the junction of the soft and hard palates. After hemostasis, an endoscope was inserted into the nasopharynx cavity, allowing the visualization of the pharyngeal opening of the ET on the posterior lateral wall of the nasal cavity. Subsequently, a specialized pusher was used to insert a balloon into ET. The balloon was inflated, maintained at 10 bar for 2 min, and then removed. The incision in the soft palate was then sutured to ensure proper alignment. The soft palate healed well after the operation. This surgical approach is suitable for ET-related procedures in large experimental animals (e.g., miniature pigs, sheep, and dogs). The surgical procedure is simple, with a short surgical time, and wound healing is rapid. Under endoscopy, the pharyngeal opening of the ET is visible, and it is thus a good choice for procedures such as balloon dilation of the ET.

Comparative studies of influence of palm oil on activity of xanthine oxidase and free radicals in liver and blood: Diabetes and metabolic syndrom

Diabetes is a chronic metabolic disease, which has affected approximately 10.5% of the global population [1]. The most frequent types of diabetes are type-1 and type-2. The third frequent type is gestational diabetes. The pathogenicity of diabetes is multifactorial and involves various factors such as genetic, food, stress, environmental pollutants, and others, in most of the cases combination of two or more factors are noticed. Comparative studies of the influence of palm oil on the activity of xanthine oxidase (XOD) and free radicals in liver and blood. The research compared the effect of palm oil on carbohydrate metabolism in male rats: in young rats and adult rats. The research compared the effect of palm oil on free radicals in male rats: in adults and juvenile rats. Experimental animal body weight change research, 30 days after palm oil treatment. For this scientific research we utilized experimental animals of the male genus: mice of the Wistar type, small mice (45-60 g) and large mice (160-180 g). Experimental animals were divided into groups of six-eight experimental mice and details are shown in the corresponding part of the results. The results are shown in six figures and two tables. Results indicate an increase in the formation of free radicals in blood plasma, both in experimental juvenile mice and in experimental large mice under the influence of palm oil. Palm oil increases the activity of XOD in blood, both in small animals and in large experimental animals. Palm oil increases the formation of XOD in the black liver in both elderly groups. In all groups during the experiment there was an increase in body weight. Results from studies of the effect of palm oil on carbohydrate metabolism showed changes in glucose tolerance test. Current studies for the first time convincingly demonstrate the risk of using palm oil. Analysis of the results of two studies on XOD and carbohydrate metabolism showed a correlation between increased XOD levels and the development of prediabetes.

Chenopodium murale Juice Shows Anti-Fungal Efficacy in Experimental Oral Candidiasis in Immunosuppressed Rats in Relation to Its Chemical Profile.

Chenopodium murale (Syn. Chenopodiastrum murale) (amaranthaceae) is used in the rural Egypt to treat oral ulcers in newborn children. The current study aimed to discover new natural products suitable for treating candidiasis disease with minimal side effects. Characterization of bioactive compounds by LC-QTOF-HR-MS/MS from Chenopodium murale fresh leaves' juice (CMJ) was carried out in order to elucidate their potential anti-fungal and immunomodulatory effects in oral candidiasis in immunosuppressed rats. An oral ulcer candidiasis model was created in three stages: (i) immunosuppression by drinking dexamethasone (0.5 mg/L) for two weeks; (ii) Candida albicans infection (3.00 × 106 viable cell/mL) for one week; and (iii) treatment with CMJ (0.5 and 1.0 g/kg orally) or nystatin (1,000,000 U/L orally) for one week. Two doses of CMJ exhibited antifungal effects, for example, through a significant reduction in CFU/Petri (236.67 ± 37.86 and 4.33 ± 0.58 CFU/Petri), compared to the Candida control (5.86 × 104 ± 1.21 CFU/Petri), p ≤ 0.001. In addition, CMJ significantly induced neutrophil production (32.92% ± 1.29 and 35.68% ± 1.77) compared to the Candida control level of 26.50% ± 2.44. An immunomodulatory effect of CMJ at two doses appeared, with a considerable elevation in INF-γ (103.88 and 115.91%), IL-2 (143.50, 182.33%), and IL-17 (83.97 and 141.95% Pg/mL) compared with the Candida group. LC-MS/MS analysis operated in negative mode was used for tentative identification of secondary (SM) metabolites based on their retention times and fragment ions. A total of 42 phytoconstituents were tentatively identified. Finally, CMJ exhibited a potent antifungal effect. CMJ fought Candida through four strategies: (i) promotion of classical phagocytosis of neutrophils; (ii) activation of T cells that activate IFN-γ, IL-2, and IL-17; (iii) increasing the production of cytotoxic NO and H2O2 that can kill Candida; and (iv) activation of SOD, which converts superoxide to antimicrobial materials. These activities could be due to its active constituents, which are documented as anti-fungal, or due to its richness in flavonoids, especially the active compounds of kaempferol glycosides and aglycone, which have been documented as antifungal. After repetition on another type of small experimental animal, their offspring, and an experimental large animal, this study may lead to clinical trials.

Cardiac electrophysiological remodeling associated with enhanced arrhythmia susceptibility in a canine model of elite exercise.

The health benefits of regular physical exercise are well known. Even so, there is increasing evidence that the exercise regimes of elite athletes can evoke cardiac arrhythmias including ventricular fibrillation and even sudden cardiac death (SCD). The mechanism of exercise-induced arrhythmia and SCD is poorly understood. Here, we show that chronic training in a canine model (12 sedentary and 12 trained dogs) that mimics the regime of elite athletes induces electrophysiological remodeling (measured by ECG, patch-clamp, and immunocytochemical techniques) resulting in increases of both the trigger and the substrate for ventricular arrhythmias. Thus, 4 months sustained training lengthened ventricular repolarization (QTc: 237.1±3.4 ms vs. 213.6±2.8 ms, n=12; APD90: 472.8±29.6 ms vs. 370.1±32.7 ms, n=29 vs. 25), decreased transient outward potassium current (6.4±0.5 pA/pF vs. 8.8±0.9 pA/pF at 50 mV, n=54 vs. 42), and increased the short-term variability of repolarization (29.5±3.8 ms vs. 17.5±4.0 ms, n=27 vs. 18). Left ventricular fibrosis and HCN4 protein expression were also enhanced. These changes were associated with enhanced ectopic activity (number of escape beats from 0/hr to 29.7±20.3/hr) in vivo and arrhythmia susceptibility (elicited ventricular fibrillation: 3 of 10 sedentary dogs vs. 6 of 10 trained dogs). Our findings provide in vivo, cellular electrophysiological and molecular biological evidence for the enhanced susceptibility to ventricular arrhythmia in an experimental large animal model of endurance training.

Use of a local hemostatic agent based on chitosan and external compression of the abdominal area to control intra-abdominal bleeding

The paper presents the intermediate results of an experimental study of three samples of chitosan-based local hemostatic agent in a model of intense intra-abdominal bleeding in an average-sized experimental animal (a Soviet Chinchilla rabbit) with a standard wound on the parenchymal organ (liver) in combination with external compression of the abdominal area to achieve hemostasis in the acute phase of the experiment. At the first stage, nine experimental groups and one control group, with three biological objects each, were involved (n = 30). The composition and properties of laboratory preparations of hemostatics were different from the concentration of the main component. To select the optimal sample of a local hemostatic agent with the least local irritant effect on the organs and structures of the abdominal cavity, three experimental groups with three animals each were formed at the second stage, without modeling blood loss (n = 9) with follow-up from 24 to 72 h. The studied drugs have high hemostatic activity in the model of intense intra-abdominal bleeding with follow-up periods from several hours to 3 days. Moreover, intraperitoneal administration of hemostatic agent samples does not lead to a pronounced local irritant effect. However, during the first 180 min of the experiment, only one fatal outcome was recorded in a group of animals with 15% chitosan in the studied drug because of a technical error in modeling the source of bleeding. External compression of the abdominal area demonstrated its effectiveness as an auxiliary technique for temporary control of the intra-abdominal source of bleeding in the experimental model. The objectification of the obtained results was achieved through a control sectional study and laboratory screening of peripheral blood indicators of experimental animals at different stages of the experiment. To further evaluate the hemostatic activity of chitosan-based drugs, creating an experimental model using a large experimental animal is necessary.

P29. DEVELOPING A LONG-TERM SURVIVAL HEMI-FACIAL VASCULARIZED COMPOSITE ALLOTRANSPLANTATION SWINE MODEL TO STUDY FACIAL NERVE REGENERATION

PURPOSE: We present a survival large animal model for preclinical facial transplant research to evaluate facial nerve regeneration. METHODS: 19 survival surgeries (16 auto-transplant, 3 allotransplant) were performed to develop and validate a hemi-facial composite flap based on the external carotid artery and vein. Assessment of nerve regeneration using serial compound muscle action potentials (CMAP), electroneurograms (ENG) and histology in a 3-4 month survival model was established. Immunosuppression in allotransplanted pigs started on POD-3 and maintained with weight-based Cyclosporin (CSA) (target levels of 200-300), mycophenolic acid and prednisone. Postoperative course and complications were documented. RESULTS: The buccal branch was selected to assess nerve regeneration using nerve conduction studies. In our allotransplant and auto-transplant models, minor wound breakdown and sialoceles in early postoperative period were managed conservatively or with superficial debridement. There were no flap losses. No systemic infections were experienced in the allotransplanted pigs (n=3); one pig developed an abscess requiring debridements, extended antibiotics usage, but had complete resolution at 8 weeks. At 3 months nerve regeneration reached 75% distance to target muscle with comparable ENG to normal side in auto-transplanted Sinclair mini-pig group (n=5), but a slower rate of regeneration in the allotransplanted group (n=3). CONCLUSION: This study demonstrates proof of concept for an allotransplant large experimental animal model for research to inform post-surgical care to improve facial nerve regeneration and functional outcomes.

Sperm quality and testicular histopathology of Wistar albino male rats treated with hydroethanolic extract of Cordia dichotoma fruits

Context Cordia dichotoma Forst. (Boraginaceae) has potent pharmacological impact. Meanwhile, its effect on fertility is unclear. Objective This study investigates the effect of Cordia fresh fruits hydroethanolic extract on fertility. Materials and methods 120 Wistar albino male rats were divided into four groups (n = 30). The first group was negative control, and the second, third, and fourth groups received 125, 250, and 500 mg extract/kg bodyweight for 56 days. After 56 days, Cordia force-feeding stopped, and all groups were kept under laboratory conditions for another month to study the recovering effect. Results After day 56, extract at 500 mg/kg significantly reduced sperm total count, motility%, and alive%, to 47.60 ± 2.27 × 106 sperm/mL, 43.33% ± 1.49, and 63.67% ± 1.19, respectively, abnormalities% increased considerably (26.67% ± 0.54), compared to the negative control. Also, significant depletion on follicle-stimulating hormone (2.66 ± 0.21 mIU/L), luteinizing hormone (1.07 ± 0.06 mIU/L), and testosterone (2.69 ± 0.13 nmol/L) level was recorded, compared to the negative control. Cordia negative effect showed on histopathological studies of testes, prostate, and seminal vesicles. Fortunately, these adverse effects of Cordia recovered remarkably after stopping administration for one month. Conclusions Cordia antifertility effect may be due to its hypocholesterolemic effect, where cholesterol, the steroid cycle precursor, was significantly reduced. This study can be incorporated in clinical research after being repeated on another small experimental animal, their offspring, and one large experimental animal, then going to a clinical study that we plan to do in the future.

Chronic recording of cortical activity underlying vocalization in awake minipigs

BackgroundInvestigating brain dynamics underlying vocal production in animals is a powerful way to inform on the neural bases of human speech. In particular, brain networks underlying vocal production in non-human primates show striking similarities with the human speech production network. However, despite increasing findings also in birds and more recently in rodents, the extent to which the primate vocal cortical network model generalizes to other non-primate mammals remains unclear. Especially, no domestic species has yet been proposed to investigate vocal brain activity using electrophysiological approaches. New methodIn the present study, we introduce a novel experimental paradigm to identify the cortical dynamics underlying vocal production in behaving minipigs. A key problem to chronically implant cortical probes in pigs is the presence and growth of frontal sinuses extending caudally to the parietal bone and preventing safe access to neural structures with conventional craniotomy in adult animals. ResultsHere we first show that implantations of soft ECoG grids can be done safely using conventional craniotomy in minipigs younger than 5 months, a period when sinuses are not yet well developed. Using wireless recordings in behaving animals, we further show activation of the motor and premotor cortex around the onset of vocal production of grunts, the most common vocalization of pigs. ConclusionThese results suggest that minipigs, which are very loquacious and social animals, can be a good experimental large animal model to study the cortical bases of vocal production.

Feasibility of large experimental animal models in testing novel therapeutic strategies for diabetes.

Diabetes is among the top 10 causes of death in adults and caused approximately four million deaths worldwide in 2017. The incidence and prevalence of diabetes is predicted to increase. To alleviate this potentially severe situation, safer and more effective therapeutics are urgently required. Mice have long been the mainstay as preclinical models for basic research on diabetes, although they are not ideally suited for translating basic knowledge into clinical applications. To validate and optimize novel therapeutics for safe application in humans, an appropriate large animal model is needed. Large animals, especially pigs, are well suited for biomedical research and share many similarities with humans, including body size, anatomical features, physiology, and pathophysiology. Moreover, pigs already play an important role in translational studies, including clinical trials for xenotransplantation. Progress in genetic engineering over the past few decades has facilitated the development of transgenic animals, including porcine models of diabetes. This article discusses features that attest to the attractiveness of genetically modified porcine models of diabetes for testing novel treatment strategies using recent technical advances.

A fast and easy-to-learn technique for liver resection in a porcine model.

ObjectiveDespite the recent advances in surgical techniques and perioperative care, liver resection (especially extended hepatectomy) is still a high-risk procedure with considerable morbidity and mortality. Experimental large animal models are the best option for studies in this regard. The present study was performed to present an easy-to-learn, fast, and multipurpose model of liver resection in a porcine model.MethodStepwise liver resections (resection of segments II/III, IVa/IVb, and VIII/IV) were performed in eight pigs with intraoperative monitoring of hemodynamic parameters. The technical aspects, tips, and tricks of this method are explained in detail.ResultsBased on the specific anatomical characteristics of the porcine liver, all resection types including segmental resection, hemihepatectomy, and extended hepatectomy could be performed in one animal in an easy-to-learn and fast technique. All animals were hemodynamically stable following stepwise liver resection.ConclusionStepwise liver resection using stapler in a porcine model is a fast and easy-to-learn method with which junior staff and research fellows can perform liver resection up to extended hepatectomy under stable conditions.

Inbreeding Alters the Gut Microbiota of the Banna Minipig.

Simple SummaryThe mammalian gut microbiota is an indispensable part of host health. The gut microbiota plays a crucial role in nutrient digestibility, preventing colonization of pathogens and maintaining the host immune system. Host genetics has been conclusively shown to closely related to gut microbiota. Inbreeding can cause a decrease of the host’s genetic diversity, however, remarkably little is understood about the gut microbiota of pigs during inbreeding. The Banna minipig inbred is the world’s first successful large mammalian experimental animal inbred line since 1980 from full and half-siblings of the Diannan small-ear pig. Now, Banna minipig inbred has been inbred for over 37 generations, and the inbreeding coefficient is more than 99%. This study is the first to characterize and compare the composition and function of gut microbiota between the Diannan small-ear pig and Banna minipig inbred, aiming to better understand the influence of inbreeding on the gut microbiota.The gut microbiota coevolve with the host and can be stably transmitted to the offspring. Host genetics plays a crucial role in the composition and abundance of gut microbiota. Inbreeding can cause a decrease of the host’s genetic diversity and the heterozygosity. In this study, we used 16S rRNA gene sequencing to compare the differences of gut microbiota between the Diannan small-ear pig and Banna minipig inbred, aiming to understand the impact of inbreeding on the gut microbiota. Three dominant bacteria (Stenotrophlomonas, Streptococcus, and Lactobacillus) were steadily enriched in both the Diannan small-ear pig and Banna minipig inbred. After inbreeding, the gut microbiota alpha diversity and some potential probiotics (Bifidobacterium, Tricibacter, Ruminocaccae, Christensenellaceae, etc.) were significantly decreased, while the pathogenic Klebsiella bacteria was significantly increased. In addition, the predicted metagenomic analysis (PICRUSt2) indicated that several amino acid metabolisms (‘‘Valine, leucine, and isoleucine metabolism’’, ‘‘Phenylalanine, tyrosine, and tryptophan biosynthesis’’, ‘‘Histidine metabolism’’) were also markedly decreased after the inbreeding. Altogether our data reveal that host inbreeding altered the composition and the predicted function of the gut microbiome, which provides some data for the gut microbiota during inbreeding.

Impact of Fat Graft Thickness and Harvesting Technique on Adipocyte Viability in a New Porcine Experimental Model: An Immunohistochemical Analysis.

Autologous fat grafting (AFG) has been employed in surgical practice as a filling method. However, controversies remain on the specifics of this technique. So far, few relevant experimental large animal studies have objectively assessed factors related to AFG integration. This study utilized an experimental, medium-sized animal model to compare the feasibility of AFG collected employing 2 different techniques with instruments of distinct thicknesses. Twenty minipigs (Sus scropha domesticus) were subjected to AFG harvesting via en bloc resection utilizing 3- (Group I) and 5-mm-diameter (Group II) round punch blades (PBs) and liposuction (LS) with 3- (Group III) and 5-mm-diameter cannulas (Group IV). Both samples were grafted intramuscularly (biceps femoralis). Hematoxylin and eosin staining was employed to identify intact adipocytes, fat necrosis, fibrosis, inflammation, and oil cysts. Immunohistochemical staining (perilipin-A, tumor necrosis factor alfa, and cluster of differentiation number 31) was utilized to quantify the feasibility of adipocytes, tissue necrosis, and neoangiogenesis, respectively. Hematoxylin and eosin analysis showed that fat necrosis and histiocyte presence were significantly lower in the AFG harvested utilizing a PB than in LS. For perilipin-A, a statistical difference was observed between subgroups I and III (P = 0.001) and I and IV (P = 0.004). Instrument diameter had no effect on graft integration in comparisons between groups II and III (P = 0.059) and II and IV (P = 0.132). In this experimental study, fat collected utilizing a PB demonstrated higher adipocyte viability than fat collected with LS. The diameter of the collection instruments, whether PB or LS, had no effect on graft integration.

Associations of Maternal Glycemia in the First Half of Pregnancy With Alterations in Cardiac Structure and Function in Childhood.

Gestational diabetes mellitus has been associated with offspring cardiac congenital malformations, ventricular hypertrophy, and diastolic dysfunction in large observational cohort studies and experimental animal models. We assessed the associations of maternal random glucose concentrations across the full range with childhood cardiac ventricular structure and function. In a population-based prospective cohort among 1,959 women and their offspring, maternal random glucose concentrations were measured at a median 13.1 weeks' gestation (95% range 10.5-16.8 weeks). We obtained offspring cardiac outcomes, relative to body size, through cardiac MRI at 10 years. The mean maternal random glucose concentration was 4.4 mmol/L (SD 0.8). The highest quintile of maternal glucose concentrations, compared with the lowest quintile, was associated with a lower childhood left ventricular mass (-0.19 SD score [SDS]; 95% CI -0.31, -0.07) and left ventricular end-diastolic volume (-0.17 SDS; 95% -0.28, -0.05). Also, higher maternal glucose concentrations across the full range per 1 mmol/L increase were associated with a lower childhood left ventricular mass and left ventricular end-diastolic volume (P values ≤0.05). Adjustment for maternal prepregnancy BMI, gestational age, and weight at birth or childhood BMI and blood pressure did not influence the effect estimates. Maternal glucose concentrations were not significantly associated with childhood right ventricular end-diastolic volume or left and right ventricular ejection fraction. Higher maternal random glucose concentrations in the first half of pregnancy are associated with a lower childhood left ventricular mass and left ventricular end-diastolic volume, with the strongest associations for childhood left ventricular mass. These associations were not explained by maternal, birth, or childhood characteristics. Further studies are needed to replicate these findings using repeated maternal glucose measurements throughout pregnancy and offspring cardiac outcomes throughout childhood and adulthood.

Near real-time bedside detection of spinal cord ischaemia during aortic repair by microdialysis of the cerebrospinal fluid.

Spinal cord ischaemia (SCI) remains the most devastating complication after thoraco-abdominal aortic aneurysm (TAAA) repair. Its early detection is crucial if therapeutic interventions are to be successful. Cerebrospinal fluid (CSF) is readily available and accessible to microdialysis (MD) capable of detecting metabolites involved in SCI [i.e. lactate, pyruvate, the lactate/pyruvate ratio (LPR), glucose and glycerol] in real time. Our aim was to evaluate the feasibility of CSF MD for the real-time detection of SCI metabolites. In a combined experimental and translational approach, CSF MD was evaluated (i) in an established experimental large animal model of SCI with 2 arms: (a) after aortic cross-clamping (AXC, N = 4), simulating open TAAA repair and (b) after total segmental artery sacrifice (Th4-L5, N = 8) simulating thoracic endovascular aortic repair. The CSF was analysed utilizing MD every 15 min. Additionally, CSF was collected hourly from 6 patients undergoing open TAAA repair in a high-volume aortic reference centre and analysed using CSF MD. In the experimental AXC group, CSF lactate increased 3-fold after 10 min and 10-fold after 60 min of SCI. Analogously, the LPR increased 5-fold by the end of the main AXC period. Average glucose levels demonstrated a 1.5-fold increase at the end of the first (preconditioning) AXC period (0.60±0.14 vs 0.97±0.32 mmol/l); however, they decreased below (to 1/3 of) baseline levels (0.60±0.14 vs 0.19±0.13 mmol/l) by the end of the experiment (after simulated distal arrest). In the experimental segmental artery sacrifice group, lactate levels doubled and the LPR increased 3.3-fold within 30 min and continued to increase steadily almost 5-fold 180 min after total segmental artery sacrifice (P < 0.05). In patients undergoing TAAA repair, lactate similarly increased 5-fold during ischaemia, reaching a maximum at 6 h postoperatively. In 2 patients with intraoperative SCI, indicated by a decrease in the motor evoked potential of >50%, the LPR increased by 200%. CSF is widely available during and after TAAA repair, and CSF MD is feasible for detection of early anaerobic metabolites of SCI. CSF MD is a promising new tool combining bedside availability and real-time capacity to potentially enable rapid detection of imminent SCI, thereby maximizing chances to prevent permanent paraplegia in patients with TAAA.

Ventricular Arrhythmias in First Acute Myocardial Infarction: Epidemiology, Mechanisms, and Interventions in Large Animal Models.

Ventricular arrhythmia and subsequent sudden cardiac death (SCD) due to acute myocardial infarction (AMI) is one of the most frequent causes of death in humans. Lethal ventricular arrhythmias like ventricular fibrillation (VF) prior to hospitalization have been reported to occur in more than 10% of all AMI cases and survival in these patients is poor. Identification of risk factors and mechanisms for VF following AMI as well as implementing new risk stratification models and therapeutic approaches is therefore an important step to reduce mortality in people with high cardiovascular risk. Studying spontaneous VF following AMI in humans is challenging as it often occurs unexpectedly in a low risk subgroup. Large animal models of AMI can help to bridge this knowledge gap and are utilized to investigate occurrence of arrhythmias, involved mechanisms and therapeutic options. Comparable anatomy and physiology allow for this translational approach. Through experimental focus, using state-of-the-art technologies, including refined electrical mapping equipment and novel pharmacological investigations, valuable insights into arrhythmia mechanisms and possible interventions for arrhythmia-induced SCD during the early phase of AMI are now beginning to emerge. This review describes large experimental animal models of AMI with focus on first AMI-associated ventricular arrhythmias. In this context, epidemiology of first AMI, arrhythmogenic mechanisms and various potential therapeutic pharmacological targets will be discussed.

Minimally Invasive Surgical Protocol for Adipose Derived Stem Cells Collection and Isolation - Ovine Model

Many methods of stem cells collection and isolation from various tissue types harvested either from small or large experimental animals or from human tissues have been published so far, all evaluating them as a potential source of adult mesenchymal stem cells with applicability in various pathologies or tissue bioengineering. The present study purposed to describe a minimally invasive surgical protocol for adipose tissue collection from sheep�s inter-scapular area. The procedure was carried out on adult sheeps, in aseptic conditions. A light sedation protocol with Detomidine was performed, the recovery from anesthesia being carried out with Atipamezole. Throughout the sedation, the surgical procedure and the recovery from anesthesia, the vital functions of the animal were monitored. The adipose tissue samples collected in sterile tubes with culture medium (Dulbecco�s modified Eagle�s medium - DMEM/10% / FBS10 - fetal bovine serum, 2% antibiotic/antifungal), have been succesfully used by our research team for adipose tissue derived stem cells (ADSCs) isolation for further use in cardiac valves tissue engineering.

Safety of intradiscal delivery of triamcinolone acetonide by a poly(esteramide) microsphere platform in a large animal model of intervertebral disc degeneration

BACKGROUND CONTEXTLocal corticosteroids have been used to relieve symptoms of chronic low back pain, although treatment effects have been shown to wear off relatively fast. Prolonging corticosteroid presence by controlled release from biomaterials may allow for longer pain relief while circumventing adverse effects such as high bolus dosages. PURPOSEThe purpose of this study was to evaluate the safety and efficacy of intradiscal controlled release of triamcinolone acetonide (TAA) by poly(esteramide) microspheres in a canine degenerated intervertebral disc (IVD) model. STUDY DESIGNIn a preclinical experimental large animal model, the effect of prolonged glucocorticoid exposure on disc degeneration was evaluated. METHODSDegeneration was accelerated by nucleotomy of lumbar IVDs of Beagle dogs. After 4 weeks, microspheres loaded with 8.4 µg TAA, and 0.84mg TAA were administered to the degenerated IVDs by intradiscal injection (n=6 per group). Empty microspheres (n=6) and all adjacent non-nucleotomized noninjected IVDs were included as controls (n=24). Immediately prior to TAA administration and after 12 weeks, magnetic resonance imaging was performed. Degenerative changes were evaluated by disc height index, Pfirrmann grading, T1ρ and T2 mapping values, postmortem CT scans, macroscopic and microscopic grading, and biochemical/immunohistochemical analysis of inflammation and extracellular matrix content. In addition, nerve growth factor (NGF) protein expression, a biomarker for pain, was scored in nucleus pulposus (NP) tissues. The study was funded by a research grant from Health Holland (1.3million euros = 1.5million US dollars). RESULTSMacroscopic evaluation and CT images postmortem were consistent with mild disc degeneration. Other abnormalities were not observed. Nucleotomy-induced degeneration and inflammation was mild, reflected by moderate Pfirrmann grades and PGE2 levels. Regardless of TAA dosage, local sustained delivery did not affect disc height index nor Pfirrmann grading, T1ρ and T2 mapping values, PGE2 tissue levels, collagen, GAG, and DNA content. However, the low dosage of TAA microspheres significantly reduced NGF immunopositivity in degenerated NP tissue. CONCLUSIONSThis is the first in vivo application in a preclinical large animal model of a controlled release formulation of corticosteroids in mild IVD degeneration. Sustained release of TAA locally in the IVD appeared safe and reduced NGF expression, suggesting its potential applicability for pain relief, although beneficial effects were absent on tissue degeneration. CLINICAL SIGNIFICANCEThe present platform seems to be promising in extending the local controlled delivery of TAA with the potency to provide long-standing analgesia in the subset of LBP patients suffering from discogenic pain.

Meet the First Authors.

Meet the First Authors.

Possibility of tissue discrimination in the anterior approach of the cervical spine by Electrical impedance

Objective To verify the feasibility of discriminating tissue types in the anterior approach of the cervical spine by the Electrical impedance (EI) and decrease the rate of severe complications. Methods Six New Zealand white rabbits and 6 mini-pigs were performed standard anterior cervical surgery. The esophagus, carotid artery, tracheal cartilage, annular liga-ments of trachea, longus colli muscle and anterior longitudinal ligament are classified as the prevertebral group; The cortical bone, cancellous bone, anulus fibrosus and nucleus pulposus are classified as the vertebral group. Once all the tissues were exposed com-pletely, over the frequency range of 200-3 000 kHz (the frequency points were 200 kHz, 400 kHz, 600 kHz, 800 kHz, 1 000 kHz, 2 000 kHz, 3 000 kHz), the in vivo EI of prevertebral group and vertebral group were measured by a probe and a precision induc-tance-capacitance-resistance (LCR) meter; then the data were analyzed with IBM SPSS Statistics 22 software. At every frequency, Kruskal-Wallis test followed by all pairwise multiple comparisons was applied for all the four groups (the prevertebral group of rab-bits, the vertebral group of rabbits, the prevertebral group of mini-pigs, and the vertebral group of mini-pigs), respectively. P val-ues < 0.05 were considered statistically significant. Results At every frequency, the results of Kruskal-Wallis test for all the four groups were significant, and the results of multiple comparisons were as follows. (1) In both miniature experimental ani-mals and large experimental animals, the EI of the vertebral group tissues was significantly different at every investigated fre-quency (multiple comparisons). (2) In the miniature experimental animals, the EI between longus colli muscle and esophagus, carotid artery and annular ligaments of trachea, and tracheal cartilage and anterior longitudinal ligament had no significant dif-ference at any frequency (multiple comparisons); significant difference could not be detected in the EI between anterior longi-tudinal ligament and annular ligaments of trachea at the frequencies of 2000-3 000 kHz (multiple comparisonsk) and could be detected at the frequencies of 200-1 000 kHz (multiple comparisons); for comparison of all other paired tissues of prevertebral group there were significant differences at all frequencies (multiple comparisonsk). (3) In the large experimental animals, the EI between longus colli muscle and esophagus, carotid artery and annular ligaments of trachea, and tracheal cartilage and ante-rior longitudinal ligament had no significant difference at any frequency (multiple comparisons), the same was true for the EI between annular ligaments of trachea and esophagus, carotid artery and anterior longitudinal ligament, and longus colli muscle and annular ligaments of trachea at the frequencies of 800-3 000 kHz, 3 000 kHz and 3 000 kHz (multiple comparisons), re-spectively; for comparison of all other paired tissues of prevertebral group there were significant differences at all frequencies (multiple comparisons). Conclusion At certain frequencies, the EI among tissues was significantly different and could discrimi-nate tissues in the anterior approach of the cervical spine. Key words: Cervical vertebrae; Spinal fusion; Electric impedance; Animal experimentation

Recent advancements and new perspectives in animal models for Neurocysticercosis immunopathogenesis

Neurocysticercosis (NCC), one of the most common parasitic diseases of the central nervous system, is caused by Taenia solium. This parasite involves two hosts, intermediate hosts (pig and human) and a definitive host (human) and has various stages in its complex life cycle (eggs, oncosphere, cysticerci and adult tapeworm). Hence, developing an animal model for T.solium that mimics its natural course of infection is quite challenging. We have reviewed here the animal models frequently used to study immunopathogenesis of cysticercosis and also discussed their usefulness for NCC studies. We found that researchers have used mice, rats, guinea pigs, dogs, cats and pigs as models for this disease with varying degrees of success. Mice and rats models have been utilized extensively for immunopathogenesis studies due to their relative ease of handling and abundance of commercially available reagents to study these small animal models. These models have provided some very exciting results for in-depth understanding of the disease. Of late, the experimentally/naturally infected swine model is turning out to be the best animal model as the disease progression closely resembles human infection in pigs. However, handling large experimental animals has its own challenges and limitations.