Anthony Pease

Acute necrotizing enterocolitis of preterm piglets is characterized by dysbiosis of ileal mucosa-associated bacteria

Investigation of bacteria involved in pathogenesis of necrotizing enterocolitis (NEC) is limited by infant fragility, analysis restricted to feces, use of culture-based methods, and lack of clinically-relevant animal models. This study used a unique preterm piglet model to characterize spontaneous differences in microbiome composition of NEC-predisposed regions of gut. Preterm piglets (n=23) were cesarean-delivered and nurtured for 30 hours over which time 52% developed NEC. Bacterial DNA from ileal content, ileal mucosa, and colonic mucosa were PCR amplified, subjected to terminal restriction fragment length polymorphism (TRFLP) analysis and targeted 16S rDNA qPCR. Preterm ileal mucosa was specifically bereft in diversity of bacteria compared to ileal content and colonic mucosa. Preterm ileum was restricted to representation by only Proteobacteria, Firmicutes, Cyanobacteria and Chloroflexi. In piglets with NEC, ileal mucosa was uniquely characterized by increases in number of Firmicutes and diversity of phyla to include Actinobacteria and uncultured bacteria. Five specific TRFLP profiles, corresponding in closest identity to Clostridium butyricum, C. neonatale, C. proteolyticum, Streptomyces spp., and Leptolyngbya spp., were significantly more prevalent or observed only among samples from piglets with NEC. Total numbers of Clostridium spp. and C. butyricum were significantly greater in samples of NEC ileal mucosa but not ileal content or colonic mucosa. These results provide strong support for ileal mucosa as a focus for investigation of specific dysbiosis associated with NEC and suggest a significant role for Clostridium spp., and members of the Actinobacteria and Cyanobacteria in the pathogenesis of NEC in preterm piglets.

Developmental Programming: Impact of Prenatal Testosterone Excess on Insulin Sensitivity, Adiposity, and Free Fatty Acid Profile in Postpubertal Female Sheep

Prenatal T excess causes reproductive and metabolic disruptions including insulin resistance, attributes of women with polycystic ovary syndrome. This study tested whether increases in visceral adiposity, adipocyte size, and total free fatty acids underlie the insulin resistance seen in prenatal T-treated female sheep. At approximately 16 months of age, insulin resistance and adipose tissue partitioning were determined via hyperinsulinemic euglycemic clamp and computed tomography, respectively, in control and prenatal T-treated females. Three months later, adipocyte size and free fatty acid composition were determined. Results revealed that at the postpubertal time points tested, insulin sensitivity was increased, visceral adiposity and adipocyte size in both the sc and the visceral compartments were reduced, and circulating palmitic acid was increased in prenatal T-treated females relative to controls. In parallel studies, 20-month-old prenatal T-treated females tended to have increased basal insulin to glucose ratio. Relative to earlier findings of reduced insulin sensitivity of prenatal T-treated females during early life and adulthood, these findings of increased insulin sensitivity and reduced adiposity postpubertally are suggestive of a period of developmental adaptation. The disruption observed in free fatty acid metabolism a few months later correspond to a time point when the insulin sensitivity indices of prenatal T-treated animals appear to shift toward insulin resistance. In summary, current findings of improved insulin sensitivity and reduced visceral adiposity in postpubertal prenatal T-treated sheep relative to our earlier findings of reduced insulin sensitivity during early postnatal life and adulthood are indicative of a period of developmental adaptation.

Ultrasound-guided cervical centesis to obtain cerebrospinal fluid in the standing horse.

Horses with intracranial lesions and severe ataxia are not good anesthesia candidates; however, only one method to obtain cerebrospinal fluid (CSF) from the cervical region in a standing horse has been reported. This method is not performed routinely due to the difficulty for sample acquisition. Our hypothesis is that standing cervical centesis can be performed in horses without complication. Ultrasound-guided centesis of the CSF between C1 and C2 in 11 clinically normal horses and two horses with neurologic signs were performed. Horses were sedated and ultrasound was used to identify the subarachnoid space and spinal cord between C1 and C2. With ultrasound guidance, a needle was introduced into the dorsal aspect of the subarachnoid space using a lateral approach. Ten milliliters of CSF was obtained and analyzed. Two normal horses in this study had moderate red blood cell contamination in the CSF (940 and 612 RBC/microl). One horse had 11 RBC/microl and the remaining horses had < 4 RBC/microl. The total procedure time was approximately 2 min. No reaction was observed and no complications were detected up to 48 h after the procedure. Ultrasound-guided centesis between C1 and C2 is a rapid procedure that causes minimal to no reaction in standing, sedated horses used in this study. The use of ultrasound to guide a standing C1-2 centesis of the subarachnoid space provides an additional route to obtain CSF for analysis in the equine patient.

THE EFFECT OF SEQUENCE SELECTION AND FIELD STRENGTH ON DETECTION OF OSTEOCHONDRAL DEFECTS IN THE METACARPOPHALANGEAL JOINT

Six cadaver forelimbs were imaged in two high-field magnetic resonance (MR) systems and one low-field MR system following the creation of osteochondral defects on the palmar distal aspect of the third metacarpal bone. The following sequences were performed using all three systems: proton density (PD) turbo spin echo, T2* gradient echo (GRE), T2-weighted fast spin echo, and short tau inversion recovery. In addition, 3D T1 GRE sagittal standard and motion insensitive sequences were obtained using the low-field system. PD fat saturated and 3D T1-weighted spoiled GRE images with and without fat suppression were acquired with the high-field systems. Lesions were measured and assigned a confidence score. The images obtained using high-field systems (1.0 and 1.5 T) more accurately represented the osteochondral defects when compared with low-field system (0.27 T) images. The largest difference was observed when evaluating articular cartilage defects, which were not identified on the low-field images. Sequence selection affected the appearance of the lesions. On all systems the turbo and fast spin echo sequences more accurately represented the lesion size and shape when compared with the GRE sequences. The T1 GRE sequence is the only sequence that appears to allow visualization of the articular cartilage on the low-field images, but is limited in providing adequate cartilage visualization. Confidence scores were greater on the high-field systems when compared with the low-field system.

Developmental Programming: Impact of Gestational Steroid and Metabolic Milieus on Adiposity and Insulin Sensitivity in Prenatal Testosterone-Treated Female Sheep

Prenatally testosterone (T)-treated sheep present metabolic disruptions similar to those seen in women with polycystic ovary syndrome. These females exhibit an increased ratio of small to large adipocytes, which may be the earliest event in the development of adult insulin resistance. Additionally, our longitudinal studies suggest the existence of a period of compensatory adaptation during development. This study tested whether 1) in utero cotreatment of prenatally T-treated sheep with androgen antagonist (flutamide) or insulin sensitizer (rosiglitazone) prevents juvenile insulin resistance and adult changes in adipocyte size; and 2) visceral adiposity and insulin sensitivity are both unaltered during early adulthood, confirming the predicted developmental trajectory in this animal model. Insulin sensitivity was tested during juvenile development and adipose tissue distribution, adipocyte size, and concentrations of adipokines were determined during early adulthood. Prenatal T-treated females manifested juvenile insulin resistance, which was prevented by prenatal rosiglitazone cotreatment. Neither visceral adiposity nor insulin sensitivity differed between groups during early adulthood. Prenatal T-treated sheep presented an increase in the relative proportion of small adipocytes, which was not substantially prevented by either prenatal intervention. A large effect size was observed for increased leptin concentrations in prenatal T-treated sheep compared with controls, which was prevented by prenatal rosiglitazone. In conclusion, gestational alterations in insulin-glucose homeostasis likely play a role in programming insulin resistance, but not adipocyte size distribution, in prenatal T-treated sheep. Furthermore, these results support the notion that a period of compensatory adaptation of the metabolic system to prenatal T exposure occurs between puberty and adulthood.

The use of diffusion tensor imaging to evaluate the spinal cord in normal and abnormal dogs.

Diffusion tensor imaging (DTI) is a specialized magnetic resonance sequence to determine the direction of water molecule motion. Our hypothesis was that information derived from DTI will be significantly different in dogs with a spinal cord lesion compared with a normal dog. Eleven normal dogs and six dogs with a spinal cord lesions were imaged. DTI was performed along with standard T1- and T2-weighted sequences in transverse and sagittal planes. Fractional anisotrophy and apparent diffusion coefficient (ADC) were obtained using regions of interests centered on the cranial aspect, middle cranial, middle caudal, and caudal aspects of the spinal cord. In normal dogs, the DTI sequence was characterized by normal fiber tracking with no statistical difference between the four sections of spinal cord (P>0.05). In the dogs with a spinal cord lesion, there was a significant difference in fractional anisotropy between the two groups (P=0.0003) and the ADC analysis statistical significance (P=0.048) at the caudal most site. Based on these findings, DTI is a potentially useful method to evaluate the spinal cord in dogs.

Developmental programming: interaction between prenatal BPA exposure and postnatal adiposity on metabolic variables in female sheep

Among potential contributors for the increased incidence of metabolic diseases is the developmental exposure to endocrine-disrupting chemicals such as bisphenol A (BPA). BPA is an estrogenic chemical used in a variety of consumer products. Evidence points to interactions of BPA with the prevailing environment. The aim of this study was to assess the effects of prenatal exposure to BPA on postnatal metabolic outcomes, including insulin resistance, adipose tissue distribution, adipocyte morphometry, and expression of inflammatory markers in adipose tissue as well as to assess whether postnatal overfeeding would exacerbate these effects. Findings indicate that prenatal BPA exposure leads to insulin resistance in adulthood in the first breeder cohort (study 1), but not in the second cohort (study 2), which is suggestive of potential differences in genetic susceptibility. BPA exposure induced adipocyte hypertrophy in the visceral fat depot without an accompanying increase in visceral fat mass or increased CD68, a marker of macrophage infiltration, in the subcutaneous fat depot. Cohens effect size analysis found the ratio of visceral to subcutaneous fat depot in the prenatal BPA-treated overfed group to be higher compared with the control-overfed group. Altogether, these results suggest that exposure to BPA during fetal life at levels found in humans can program metabolic outcomes that lead to insulin resistance, a forerunner of type 2 diabetes, with postnatal obesity failing to manifest any interaction with prenatal BPA relative to insulin resistance and adipocyte hypertrophy.

Viral genes and cellular markers associated with neurological complications during herpesvirus infections

Despite the importance of neurological disorders associated with herpesviruses, the mechanism by which these viruses influence the central nervous system (CNS) has not been definitively established. Owing to the limitations of studying neuropathogenicity of human herpesviruses in their natural host, many aspects of their pathogenicity and immune response are studied in animal models. Here, we present an important model system that enables studying neuropathogenicity of herpesviruses in the natural host. Equine herpesvirus type 1 (EHV-1) is an alphaherpesvirus that causes a devastating neurological disease (EHV-1 myeloencephalopathy; EHM) in horses. Like other alphaherpesviruses, our understanding of virus neuropathogenicity in the natural host beyond the essential role of viraemia is limited. In particular, information on the role of different viral proteins for virus transfer to the spinal cord endothelium in vivo is lacking. In this study, the contribution of two viral proteins, DNA polymerase (ORF30) and glycoprotein D (gD), to the pathogenicity of EHM was addressed. Furthermore, different cellular immune markers, including alpha-interferon (IFN-α), gamma-interferon (IFN-γ), interleukin-10 (IL-10) and interleukin-1 beta (IL-1β), were identified to play a role during the course of the disease.

Dynamic susceptibility contrast magnetic resonance imaging protocol of the normal canine brain

Perfusion magnetic resonance imaging (MRI), specifically dynamic susceptibility MRI (DSC-MRI) is routinely performed as a supplement to conventional MRI in human medicine for patients with intracranial neoplasia and cerebrovascular events. There is minimal data on the use of DSC-MRI in veterinary patients and a DSC-MRI protocol in the veterinary patient has not been described. Sixteen normal dogs, 6 years or older were recruited for this study. The sample population included 11 large dogs (>11 kg) and 5 small dogs (<11 kg). DSC-MRI was performed on a 1.5-T MRI using an adjusted protocol inherent to the MRI. Contrast media was injected using an automatic power injector. Injections were made after five MR measurements were obtained. Following image acquisition, an arterial input function (AIF) graph mapping the transit time of contrast within the cerebral arteries was generated. The manually selected time points along this graph were used to compute perfusion maps. A dose and rate of 0.1 mmol/kg gadolinium-based contrast media at 3 ml/s followed by 10 ml saline flush at 3 ml/s was used in all dogs greater than 11 kg. In all dogs >11 kg, a useable AIF and perfusion map was generated. One dog less than 11 kg received the same contrast dose and rate. In this patient, the protocol did not generate a useable AIF. The remainder of the dogs less than 11 kg followed a protocol of 0.2 mmol/kg gadolinium-based contrast media at 1.5 ml/s with a 10 ml saline flush at 1.5 ml/s. A useable AIF and perfusion map was generated in the remaining dogs <11 kg using the higher contrast dose and slower rate protocol. This study establishes a contrast dose and administration rate for canine DSC-MRI imaging that is different in dogs greater than 11 kg compared to dogs less than 11 kg. These protocols may be used for future applications to evaluate hemodynamic disturbances in canine intracranial pathology.

Biochemical evaluation of equine articular cartilage through imaging.

The use of molecular imaging of cartilage is the next vital step in understanding, treating, and training the equine athlete. Because of the logistics of precontrast and postcontrast medium imaging, the clinical usefulness of the examination has come into question. With the large number of horses undergoing high-field magnetic resonance imaging, the use of contrast medium administration and T1 mapping or T2 imaging precontrast and postcontrast medium administration may add a limited amount of time to the scan and has the potential to provide more detailed information about the chemical composition of the articular cartilage that is not seen with routine imaging.