Identification of predictive MRI and functional biomarkers in a pediatric piglet traumatic brain injury model

Traumatic brain injury(TBI) at a young age can lead to the development of long-term functional impairments. Severity of injury is well demonstrated to have a strong influence on the extent of functional impairments;however, identification of specific magnetic resonance imaging(MRI) biomarkers that are most reflective of injury severity and functional prognosis remain elusive. Therefore, the objective of this study was to utilize advanced statistical approaches to identify clinically relevant MRI biomarkers and predict functional outcomes using MRI metrics in a translational large animal piglet TBI model. TBI was induced via controlled cortical impact and multiparametric MRI was performed at 24 hours and 12 weeks post-TBI using T1-weighted, T2-weighted, T2-weighted fluid attenuated inversion recovery, diffusion-weighted imaging, and diffusion tensor imaging. Changes in spatiotemporal gait parameters were also assessed using an automated gait mat at 24 hours and 12 weeks post-TBI. Principal component analysis was performed to determine the MRI metrics and spatiotemporal gait parameters that explain the largest sources of variation within the datasets. We found that linear combinations of lesion size and midline shift acquired using T2-weighted imaging explained most of the variability of the data at both 24 hours and 12 weeks post-TBI. In addition, linear combinations of velocity, cadence, and stride length were found to explain most of the gait data variability at 24 hours and 12 weeks post-TBI. Linear regression analysis was performed to determine if MRI metrics are predictive of changes in gait. We found that both lesion size and midline shift are significantly correlated with decreases in stride and step length. These results from this study provide an important first step at identifying relevant MRI and functional biomarkers that are predictive of functional outcomes in a clinically relevant piglet TBI model. This study was approved by the University of Georgia Institutional Animal Care and Use Committee(AUP: A2015 11-001) on December 22, 2015.

A new approach to endocochlear potential and potassium ion concentration measures in mini pig models

Mini pig models are large mammals and their ears are more similar with human beings in structure and development than other animals.However,the study on porcine ears is still in the initial stage and there is no description of an ideal operation approach to endocochlear potential and potassium ion concentration measurements.In this article,we describe a pre-auricular surgical approach to access the middle and inner ear for endocochlear potential and potassium ion concentration measures in mini pig models.Ten one-week old normal mini pigs were used in the study.The bulla of the temporal bone was accessed via a pre-auricular approach for endocochlear potential and potassium ion concentration measurements.The condition of the animals during the first post—experiment 24 h was observed.One animal died during surgery.The preauricular approach improved protection and preservation of relevant nervous and vascular elements including the facial nerve and carotid artery.So,the pre-auricular approach can be used for endocochlear potential and potassium ion concentration measurements with improved nerve and artery preservation mini pigs.

Establishment of a male fertility prediction model with sperm RNA markers in pigs as a translational animal model

Background:Male infertility is an important issue that causes low production in the animal industry.To solve the male fertility crisis in the animal industry,the prediction of sperm quality is the most important step.Sperm RNA is the potential marker for male fertility prediction.We hypothesized that the expression of functional genes related to fertilization will be the best target for male fertility prediction markers.To investigate optimum male fertility prediction marker,we compared target genes expression level and a wide range of field data acquired from artificial insemination of boar semen.Results:Among the genes related to acrosomal vesicle exocytosis and sperm–oocyte fusion,equatorin(EQTN),zona pellucida sperm-binding protein 4(ZP4),and sperm acrosome membrane-associated protein 3 exhibited high accuracy(70%,90%,and 70%,respectively)as markers to evaluate male fertility.Combinations of EQTN-ZP4,ZP4-protein unc-13 homolog B,and ZP4-regulating synaptic membrane exocytosis protein 1(RIMS1)showed the highest prediction value,and all these markers are involved in the acrosome reaction.Conclusion:The EQTN-ZP4 model was efficient in clustering the high-fertility group and may be useful for selection of animal that has superior fertility in the livestock industry.Compared to the EQTN-ZP4 model,the ZP4-RIMS1 model was more efficient in clustering the low-fertility group and may be useful in the diagnosis of male infertility in humans and other animals.The appointed translational animal model and established biomarker combination can be widely used in various scientific fields such as biomedical science.

An integrative multivariate approach for predicting functional recovery using magnetic resonance imaging parameters in a translational pig ischemic stroke model

Magnetic resonance imaging(MRI)is a clinically relevant,real-time imaging modality that is frequently utilized to assess stroke type and severity.However,specific MRI biomarkers that can be used to predict long-term functional recovery are still a critical need.Consequently,the present study sought to examine the prognostic value of commonly utilized MRI parameters to predict functional outcomes in a porcine model of ischemic stroke.Stroke was induced via permanent middle cerebral artery occlusion.At 24 hours post-stroke,MRI analysis revealed focal ischemic lesions,decreased diffusivity,hemispheric swelling,and white matter degradation.Functional deficits including behavioral abnormalities in open field and novel object exploration as well as spatiotemporal gait impairments were observed at 4 weeks post-stroke.Gaussian graphical models identified specific MRI outputs and functional recovery variables,including white matter integrity and gait performance,that exhibited strong conditional dependencies.Canonical correlation analysis revealed a prognostic relationship between lesion volume and white matter integrity and novel object exploration and gait performance.Consequently,these analyses may also have the potential of predicting patient recovery at chronic time points as pigs and humans share many anatomical similarities(e.g.,white matter composition)that have proven to be critical in ischemic stroke pathophysiology.The study was approved by the University of Georgia(UGA)Institutional Animal Care and Use Committee(IACUC;Protocol Number:A2014-07-021-Y3-A11 and 2018-01-029-Y1-A5)on November 22,2017.

Characteristics of glucose and lipid metabolism and the interaction between gut microbiota and colonic mucosal immunity in pigs during cold exposure

Background Cold regions have long autumn and winter seasons and low ambient temperatures.When pigs are unable to adjust to the cold,oxidative damage and inflammation may develop.However,the differences between cold and non-cold adaptation regarding glucose and lipid metabolism,gut microbiota and colonic mucosal immunological features in pigs are unknown.This study revealed the glucose and lipid metabolic responses and the dual role of gut microbiota in pigs during cold and non-cold adaptation.Moreover,the regulatory effects of dietary glucose supplements on glucose and lipid metabolism and the colonic mucosal barrier were evaluated in cold-exposed pigs.Results Cold and non-cold-adapted models were established by Min and Yorkshire pigs.Our results exhibited that cold exposure induced glucose overconsumption in non-cold-adapted pig models(Yorkshire pigs),decreasing plasma glucose concentrations.In this case,cold exposure enhanced the ATGL and CPT-1αexpression to promote liver lipolysis and fatty acid oxidation.Meanwhile,the two probiotics(Collinsella and Bifidobacterium)depletion and the enrichment of two pathogens(Sutterella and Escherichia-Shigella)in colonic microbiota are not conducive to colonic mucosal immunity.However,glucagon-mediated hepatic glycogenolysis in cold-adapted pig models(Min pigs)maintained the stability of glucose homeostasis during cold exposure.It contributed to the gut microbiota(including the enrichment of the Rikenellaceae RC9 gut group,[Eubacterium]coprostanoligenes group and WCHB1-41)that favored cold-adapted metabolism.Conclusions The results of both models indicate that the gut microbiota during cold adaptation contributes to the protection of the colonic mucosa.During non-cold adaptation,cold-induced glucose overconsumption promotes thermogenesis through lipolysis,but interferes with the gut microbiome and colonic mucosal immunity.Furthermore,glucagon-mediated hepatic glycogenolysis contributes to glucose homeostasis during cold exposure.

Short-chain fatty acids can improve lipid and glucose metabolism independently of the pig gut microbiota

Background:Previous studies have shown that exogenous short-chain fatty acids(SCFAs)introduction attenuated the body fat deposition in conventional mice and pigs.However,limited studies have evaluated the effects of exogenously introduced SCFAs on the lipid and glucose metabolism independently of the gut microbiota.This study was to investigate the effects of exogenous introduction of SCFAs on the lipid and glucose metabolism in a germ-free(GF)pig model.Methods:Twelve hysterectomy-derived newborn pigs were reared in six sterile isolators.All pigs were hand-fed with sterile milk powder for 21 d,then the sterile feed was introduced to pigs for another 21 d.In the second 21-d period,six pigs were orally administrated with 25 mL/kg sterile saline per day and considered as the GF group,while the other six pigs were orally administrated with 25 mL/kg SCFAs mixture(acetic,propionic,and butyric acids,45,15,and 11 mmol/L,respectively)per day and regarded as FA group.Results:Orally administrated with SCFAs tended to increase the adiponectin concentration in serum,enhance the CPT-1 activity in longissimus dorsi,and upregulate the ANGPTL4 mRNA expression level in colon(P<0.10).Meanwhile,the mRNA abundances of ACC,FAS,and SREBP-1C in liver and CD36 in longissimus dorsi of the FA group were decreased(P<0.05)compared with those in the GF group.Besides,the mRNA expression of PGC-1αin liver and LPL in longissimus dorsi tended to(P<0.10)upregulate and downregulate respectively in the FA group.Moreover,oral administration of SCFAs tended to increase the protein level of GPR43(P<0.10)and decrease the protein level of ACC(P<0.10)in liver.Also,oral administration of SCFAs upregulated the p-AMPK/AMPK ratio and the mRNA expressions of GLUT-2 and GYS2 in liver(P<0.05).In addition,the metabolic pathway associated with the biosynthesis of unsaturated fatty acids was most significantly promoted(P<0.05)by oral administration of SCFAs.Conclusions:Exogenous introduction of SCFAs might attenuate the fat deposition and to some extent improve the glucose control in the pig model,which occurred independently of the gut microbiota.

Electrophysiological changes of autonomic cells in left ventricular outflow tract in guinea pigs with iron deficiency anemia complicated with chronic heart failure

Objective: To investigate the electrophysiological changes of autonomic cells in left ventricular outflow tract in guinea pigs with iron deficiency anemia complicated with chronic heart failure.Methods: Guinea pigs model of iron deficiency anemia complicated with chronic heart failure in 10 guinea pigs of the experimental group was made by feeding a low iron diet,pure water and subcutaneous injection of isoproterenol. The control group consisting of 11 guinea pigs was given normal food, normal water and injected with normal saline. The left ventricular outflow tract model specimen was also prepared. The standard microelectrode technique was used to observe electrophysiological changes of autonomic cells in the outflow tract of left ventricular heart failure complicated with iron deficiency anemia in guinea pig model. The indicators of observation were maximal diastolic potential, action potential amplitude, 0 phase maximal depolarization velocity, 4 phase automatic depolarization velocity, repolarization 50% and 90%, and spontaneous discharge frequency.Results: Compared with the control group, 4 phase automatic depolarization velocity,spontaneous discharge frequency and 0 phase maximal depolarization velocity decreased significantly(P < 0.01) and action potential amplitude reduced(P < 0.01) in model group. Moreover, repolarization 50% and 90% increased(P < 0.01).Conclusions: There are electrophysiological abnormalities of the left ventricular outflow tract in guinea pigs with iron deficiency anemia complicated with heart failure.

Effect of green flickering light on myopia development and expression of M1 muscarinic acetylcholine receptor in guinea pigs

AIM： To investigate the effects of green flickering light on refractive development and expression of muscarinic acetylcholine receptor（mAChR） M1 in the eyes of guinea pigs.METHODS： Thirty guinea pigs（15-20 days old） were randomly divided into three groups（n=10/group）. Animals in group I were raised in a completely closed carton with green flickering light illumination. Those in group II were kept in the open top closed carton under normal natural light. Guinea pigs were raised in a sight-widen cage under normal natural light in group III. The refractive status and axial length were measured before and after 8 weeks＇ illumination. Moreover, total RNA extracted from retinal, choroidal, and scleral tissues were determined by real-time reverse transcription polymerase chain reaction（RT-PCR）. The expressions of the receptor M1 were also explored in the retina, choroid, and sclera using immunohistochemistry.RESULTS： There was a remarkable reduction in refractive error and increase in axial length after 8-weeks＇ green flickering light stimulation（P〈0.001）. The expression of M1 receptor mRNA in sclera and retina in myopia group were remarkably lower than that in group II and III（P〈0.01）. Significant reduced expression of M1 receptor stimulated by green flickering light in retina and sclera tissues were also observed（P〈0.05）. However, there was no M1 receptor expression in choroid in 3 groups.CONCLUSION： Myopia can be induced by 8 weeks＇ green flickering light exposure in the animal model. M1 receptor may be involved causally or protectively in myopia development.

Rapid Establishment of Tracheal Stenosis in Pigs Using Endotracheal Tube Cuff Overpressure and Electrocautery

To apply a new airway treatment to humans, preclinical studies in an appropriate animalmodel is needed. Canine, porcine and leporine tracheas have been employed as animal airwaystenosis models using various methods such as chemical caustic agents, laser, and electrocautery.However, existing models take a long time to develop (3- 8 weeks) and the mechanism of stenosisis different from that in humans. The aim of the present study was to establish a new and fasttracheal stenosis model in pigs using a combination of cuff overpressure intubation (COI) andelectrocautery. Fourteen pigs were divided into three groups: tracheal cautery (TC) group (n=3),COI group (n=3), and COI-TC combination group (n=8). Cuff overpressure (200/400/500 mmHg)was applied using a 9-mm endotracheal tube. Tracheal cautery (40/60 watts) was performed usinga rigid bronchoscopic electrocoagulator. After intervention, the pigs were observed for 3 weeks andbronchoscopy was performed every 7 days. When the cross-sectional area decreased by > 50%, itwas confirmed that tracheal stenosis was established. The time for tracheal stenosis was 14 days inthe TC group and 7 days in the COI-TC combination group. In the COI group, no stenosis occurred.In the COI-TC group, electrocautery (40 watts) immediately after intubation for>1 h with a cufpressure of 200 mmHg or more resulted in suficient tracheal stenosis within 7 days. Moreover, thedegree of tracheal stenosis increased in proportion to the cuff pressure and tracheal intubation time.The combined use of cuf overpressure and electrocautery helped to establish tracheal stenosis inpigs rapidly.

Gut microbiota absence and transplantation affect growth and intestinal functions:An investigation in a germ-free pig model

This study was conducted to investigate host-microbiota interactions and explore the effects of maternal gut microbiota transplantation on the growth and intestinal functions of newborns in a germ-free(GF)pig model.Twelve hysterectomy-derived GF Bama piglets were reared in 6 sterile isolators.Among them,6 were considered as the GF group,and the other 6 were orally inoculated with healthy sow fecal suspension as fecal microbiota transplanted(FMT)group.Another 6 piglets from natural birth were regarded as the conventional(CV)group.The GF and FMT groups were hand-fed with Co60-y-irradiated sterile milk powder,while the CV group was reared by lactating Bama sows.All groups were fed for 21 days.Then,all piglets and then were switched to sterile feed for another 21 days.Results showed that the growth performance,nutrient digestibility,and concentrations of short-chain fatty acids in the GF group decreased(P<0.05).Meanwhile,the serum urea nitrogen concentration and digesta pH values in the GF group increased compared with those in the FMT and CV groups(P<0.05).Compared with the CV group,the GF group demonstrated upregulation in the mRNA expression levels of intestinal barrier function-related genes in the small intestine(P<0.05).In addition,the mRNA abundances of intestinal development and absorption-related genes in the small intestine and colon were higher in the GF group than in the CV and FMT groups(P<0.05).The FMT group exhibited greater growth performance,lipase activity,and nutrient digestibility(P<0.05),higher mRNA expression levels of intestinal development and barrier-related genes in the small intestine(P<0.05),and lower mRNA abundances of pro-inflammatory factor in the colon and jejunum(P<0.05)than the CV group.In conclusion,the absence of gut microbes impaired the growth and nutrient digestibility,and healthy sow gut microbiota transplantation increased the growth and nutrient digestibility and improved the intestinal development and barrier function of newborn piglets,indicating the importance of intestinal microbes for intestinal development and functions.

Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression

Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process.Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline.Previous pig models with tetracycline regulatory elements were generated through random integration.This process often resulted in uncertain expression and unpredictable phenotypes,thus hindering their applications.Here,by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus,respectively,a double knock-in reporter pig model was generated.We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo.Two att P sites were arranged to flank the td Tomato to switch reporter gene.Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos.To display the flexible application of this system,we generated a pig strain with Dox-inducing h KRASexpression through phiC31 integrase-mediated cassette exchange.After eight months of Dox administration,squamous cell carcinoma developed in the nose,mouth,and scrotum,which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis.Overall,the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.

A harlequin ichthyosis pig model with a novel ABCA12 mutation can be rescued by acitretin treatment

Harlequin ichthyosis (HI) is a severe genetic skin disorder and caused by mutation in the ATP-binding cassette A12 (ABCA12) gene. The retinoid administration has dramatically improved long-term survival of HI, but improvements are still needed. However, the ABCA12 null mice failed to respond to retinoid treatment, which impedes the development of novel cure strategies for HI. Here we generated an ethylnitrosourea mutagenic HI pig model (named Z9), which carries a novel deep intronic mutation IVS49-727 A>G in the ABCA12 gene, resulting in abnormal mRNA splicing and truncated protein production. Z9 pigs exhibit significant clinical symptom as human patients with HI. Most importantly, systemic retinoid treatment significantly prolonged the life span of the mutant pigs via improving epidermal maturation, decreasing epidermal apoptosis, and triggering the expression of ABCA6. Taken together, this pig model perfectly resembles the clinical symptom and molecular pathology of patients with HI and will be useful for understanding mechanistic insight and developing therapeutic strategies.

Viscosity and degradation controlled injectable hydrogel for esophageal endoscopic submucosal dissection

Endoscopic submucosal dissection(ESD)is a common procedure to treat early and precancerous gastrointestinal lesions.Via submucosal injection,a liquid cushion is created to lift and separate the lesion and malignant part from the muscular layer where the formed indispensable space is convenient for endoscopic incision.Saline is a most common submucosal injection liquid,but the formed liquid pad lasts only a short time,and thus repeated injections increase the potential risk of adverse events.Hydrogels with high osmotic pressure and high viscosity are used as an alternate;however,with some drawbacks such as tissue damage,excessive injection resistance,and high cost.Here,we reported a nature derived hydrogel of gelatin-oxidized alginate(G-OALG).Based on the rheological analysis and compare to commercial endoscopic mucosal resection(EMR)solution(0.25%hyaluronic acid,HA),a designed G-OALG hydrogel of desired concentration and composition showed higher performances in controllable gelation and injectability,higher viscosity and more stable structures.The G-OALG gel also showed lower propulsion resistance than 0.25%HA in the injection force assessment under standard endoscopic instruments,which eased the surgical operation.In addition,the G-OALG hydrogel showed good in vivo degradability biocompatibility.By comparing the results acquired via ESD to normal saline,the G-OALG shows great histocompatibility and excellent endoscopic injectability,and enables create a longer-lasting submucosal cushion.All the features have been confirmed in the living both pig and rat models.The G-OALG could be a promising submucosal injection agent for esophageal ESD.