Yucatan Miniature Swine Research Articles

The Yucatan miniature swine as a model for post-inflammatory hyperpigmentation.

Post-inflammatory hyperpigmentation (PIH) is a hypermelanosis that often occurs secondary to skin irritation or injury, especially in darker skin tones, for which there is currently a lack of effective treatment options. Few preclinical models are available to study PIH. Here, we show that the Yucatan miniature pig consistently develops PIH after skin injuries. Skin wounds were produced on Yucatan pigs by needle punches, full-thickness excisions, or burns. Wound sites were monitored and photographed regularly. Tissue samples were collected after 24 weeks and processed for histology/immunohistochemistry. Skin pigmentation and histologic changes were quantified by computer-assisted image analyses. All injury methods resulted in hyperpigmentation. Melanin content at the histologic level was quantified in the larger (burn and excision) wounds, showing a significant increase compared to uninjured skin. Increased melanin was found for both epidermal and dermal regions. Dermal melanin deposits were primarily clustered around the papillary vasculature, and were associated not with melanocytes but with leukocytes. The Yucatan miniature pig model recapitulates key clinical and histologic features of PIH in humans, including skin hyperpigmentation at both gross and histologic levels, and persistence of dermal melanin subsequent to injury. This model could be used to further our understanding of the etiology of PIH, and for new therapy development.

Cardiovascular magnetic resonance characterization of myocardial tissue injury in a miniature swine model of cancer therapy-related cardiovascular toxicity

BackgroundLeft ventricular ejection fraction (LVEF) is the most commonly clinically used imaging parameter for assessing cancer therapy-related cardiac dysfunction (CTRCD). However, LVEF declines may occur late, after substantial injury. This study sought to investigate cardiovascular magnetic resonance (CMR) imaging markers of subclinical cardiac injury in a miniature swine model. MethodsFemale Yucatan miniature swine (n=14) received doxorubicin (2mg/kg) every 3 weeks for 4 cycles. CMR, including cine, tissue characterization via T1 and T2 mapping, and late gadolinium enhancement (LGE) was performed on the same day as doxorubicin administration and three weeks after the final chemotherapy cycle. In addition, MR spectroscopy (MRS) was performed during the 3 weeks after the final chemotherapy in 7 pigs. A single CMR and MRS exam was also performed in three Yucatan miniature swine that were age- and weight-matched to the final imaging exam of the doxorubicin-treated swine to serve as controls. CTRCD was defined as histological early morphologic changes, including cytoplasmic vacuolization and myofibrillar loss of myocytes, based on post-mortem analysis of humanely euthanized pigs after the final CMR exam. ResultsOf 13 swine completing five serial CMR scans, 10 (77%) had histological evidence of CTRCD. Three animals had neither histological evidence nor changes in LVEF from baseline. No absolute LVEF <40% or LGE were observed. Native T1, extracellular volume (ECV), and T2 at 12 weeks were significantly higher in swine with CTRCD than those without CTRCD (1178 ms vs. 1134 ms, p=0.002, 27.4% vs. 24.5%, p=0.03, and 38.1 ms vs. 36.4 ms, p=0.02, respectively). There were no significant changes in strain parameters. The temporal trajectories in native T1, ECV, and T2 in swine with CTRCD showed similar and statistically significant increases. At the same time, there were no differences in their temporal changes between those with and without CTRCD. MRS myocardial triglyceride content substantially differed among controls, swine with and without CTRCD (0.89%, 0.30%, 0.54%, respectively, ANOVA, p=0.01), and associated with the severity of histological findings and incidence of vacuolated cardiomyocytes. ConclusionsSerial CMR imaging alone has a limited ability to detect histologic CTRCD beyond LVEF. Integrating MRS myocardial triglyceride content may be useful for detection of early potential CTRCD.

Nociceptive and Transcriptomic Responses in a Swine Diabetic Wound Model Treated With a Topical Angiotensin 1 Receptor Antagonist.

Painful, treatment-resistant wounds are prevalent among diabetic patients and significantly affect health-related quality of life (HRQOL). Topical treatments may help alleviate pain without risk of dependence or side effects. However, there is a lack of topical wound compounds targeting pain-specific receptors. One possible target is proinflammatory angiotensin 1 receptor (AT1R), which is upregulated in diabetic skin and has been implicated in nociception. We investigated the effects of topical valsartan, an AT1R antagonist, on pain (nociceptive thresholds) and gene expression changes (transcriptomics) in a swine model of diabetic wounds. Eight wounds were surgically induced in diabetic, hyperglycemic Yucatan miniature swine ( n = 4). Topical AT1R antagonist was applied to wounds on one side and vehicle on the other side. Nocifensive testing was conducted at baseline and then weekly, beginning 7 days after wound induction. Mechanical and thermal stimuli were applied to the wound margins until a nocifensive reaction was elicited or a predetermined cutoff was reached. After 7 weeks of testing, tissue from the dorsal horn, dorsal root ganglion, and wounds were sequenced and analyzed with DESeq2. Unbiased pathway analyses using Metascape were conducted on differentially expressed genes. There was no significant difference in mechanical tolerance threshold between AT1R antagonist-treated and vehicle-treated wounds ( p = .106). Thermal tolerance was significantly higher in AT1R antagonist-treated wounds compared to vehicle-treated ( p = .015). Analysis of differentially expressed genes revealed enriched pathways of interest: interleukin-18 signaling in dorsal horn laminae IV-V and sensory perception of mechanical stimulus in wound tissue. In this study, wounds modeling diabetic ulcers were created in hyperglycemic swine and treated with a topical AT1R antagonist. AT1R-antagonist-treated wounds had a higher tolerance threshold than vehicle-treated wounds for thermal hyperalgesia, but not mechanical allodynia. Pathway analyses of differentially expressed genes revealed several pathways of interest for future pain research. Although further studies are needed to confirm the findings, this study can improve nursing care by providing information about a potential future treatment that may be used to decrease pain and improve HRQOL in patients with diabetic wounds.

Preclinical In-Vivo Safety and Toxicokinetics of D-PLEX100 in an Abdominal Surgery Incision Model in Juvenile Miniature Swine.

D-PLEX100 (D-PLEX) is a novel product candidate made of a polymer-lipid-based matrix (PLEX platform) which contains doxycycline that is being released at a constant rate for 30days. D-PLEX was developed to prevent surgical site infections, which are a major global health challenge. Previous studies have shown its safety in adult humans, adult swine, and adult rabbits. The aim of this study was to assess the toxicity and safety of D-PLEX also in juvenile animals to support future clinical trials in pediatric patients. Yucatan miniature swine were selected as a model, primarily due to their relatively larger mass. D-PLEX or placebo (formulation without doxycycline) was administered locally to abdominal incisions, and the animal's safety parameters were followed for 9months and compared to sham-control swine. There was no evidence of any systemic safety concern or local toxicity at the incision site in D-PLEX-treated animals. D-PLEX was detected after 1month and was fully resorbed at the 3-month time point. The surgical incision sites were fully healed at the 6-month time point in all D-PLEX-treated animals. Toxicokinetic (TK) assessments revealed that doxycycline exhibited low Cmax and therefore minimal systemic exposure following a single dose of local administration. This study provides evidence for the safety of D-PLEX and PLEX-based formulation in juvenile miniature swine and supports its further testing in clinical pediatric population. In addition, it can be used as a reference for future preclinical studies aiming to evaluate the safety of other PLEX-based product candidates for the pediatric population.

Exercise induces superoxide and NOX4 contribution in endothelium-dependent dilation in coronary arterioles from a swine model of chronic myocardial ischemia

Exercise training is an effective, nonpharmacologic therapy and preventative measure for ischemic heart disease. While recent studies have examined reactive oxygen species (ROS) as mediators of exercise training-enhanced coronary blood flow, specific oxidants and their sources have yet to be fully elucidated. We investigated the hypothesis that NADPH oxidase (NOX)-derived superoxide anion would contribute to vasodilation effects in the coronary microcirculation of swine and that these effects would be impaired by chronic ischemia and rescued with exercise training. Adult Yucatan miniature swine were instrumented with an ameroid occluder around the proximal left circumflex coronary artery, resulting in a collateral-dependent myocardial region. Eight weeks post-operatively, swine were randomly assigned to either a sedentary or exercise training (treadmill run; 5 days/week for 14 weeks) protocol. Coronary arterioles were isolated from nonoccluded and collateral-dependent myocardial regions and pressure myography was performed. Exercise training resulted in enhanced endothelium-dependent dilation after occlusion. Scavenging of superoxide via the superoxide dismutase (SOD)-mimetic, tempol, attenuated dilation in both nonoccluded and collateral-dependent arterioles of exercise-trained, but not sedentary swine. NOX1/4 inhibition with GKT136901 attenuated dilation after exercise training but only in collateral-dependent arterioles. High performance liquid chromatography revealed that neither ischemia nor exercise training significantly altered basal or bradykinin-stimulated superoxide levels. Furthermore, superoxide production was not attributable to NOX isoforms nor mitochondria. Immunoblot analyses revealed significantly decreased NOX2 protein after exercise with no differences in NOX1, NOX4, p22phox, SOD proteins. Taken together, these data provide evidence that superoxide and NOX4 independently contribute to enhanced endothelium-dependent dilation following exercise training.

Exercise training rescues impaired H2O2-mediated vasodilation in porcine collateral-dependent coronary arterioles through enhanced K+ channel activation.

We previously reported that exercise training drives enhanced agonist-stimulated hydrogen peroxide (H2O2) levels and restores endothelium-dependent dilation via an increased reliance on H2O2 in arterioles isolated from ischemic porcine hearts. In this study, we tested the hypothesis that exercise training would correct impaired H2O2-mediated dilation in coronary arterioles isolated from ischemic myocardium through increases in protein kinase G (PKG) and protein kinase A (PKA) activation and subsequent colocalization with sarcolemmal K+ channels. Female adult Yucatan miniature swine were surgically instrumented with an ameroid constrictor around the proximal left circumflex coronary artery, gradually inducing a collateral-dependent vascular bed. Arterioles (∼125 µm) supplied by the left anterior descending artery served as nonoccluded control vessels. Pigs were separated into exercise (treadmill; 5 days/wk for 14 wk) and sedentary groups. Collateral-dependent arterioles isolated from sedentary pigs were significantly less sensitive to H2O2-induced dilation compared with nonoccluded arterioles, whereas exercise training reversed the impaired sensitivity. Large conductance calcium-activated potassium (BKCa) channels and 4AP-sensitive voltage-gated (Kv) channels contributed significantly to dilation in nonoccluded and collateral-dependent arterioles of exercise-trained but not sedentary pigs. Exercise training significantly increased H2O2-stimulated colocalization of BKCa channels and PKA, but not PKG, in smooth muscle cells of collateral-dependent arterioles compared with other treatment groups. Taken together, our studies suggest that with exercise training, nonoccluded and collateral-dependent coronary arterioles better use H2O2 as a vasodilator through increased coupling with BKCa and 4AP-sensitive Kv channels; changes that are mediated in part by enhanced colocalization of PKA with BKCa channels.NEW & NOTEWORTHY The current study reveals that coronary arterioles distal to stenosis display attenuated dilation responses to H2O2 that are restored with endurance exercise training. Enhanced H2O2 dilation after exercise is dependent on Kv and BKCa channels and at least in part on in colocalization of BKCa channel and PKA and independent of PKA dimerization. These findings expand our earlier studies which demonstrated that exercise training drives beneficial adaptive responses of reactive oxygen species in the microvasculature of the ischemic heart.

Dietary Fat Saturation and Endurance Exercise Alter Lipolytic Sensitivity of Adipocytes Isolated from Yucatan Miniature Swine , ,

This study examined the effects of dietary fat saturation and endurance exercise on lipolytic sensitivity of adipocytes isolated from Yucatan miniature swine. Twenty-four female swine had free access to a high fat diet with a polyunsaturated to saturated fat ratio (P:S) of 0.3 or 1.0, and were treadmill-exercised or remained sedentary. After 3 months, biopsies were taken, adipocytes were isolated and lipolytic activity was determined. Adipocytes were incubated with adenosine deaminase followed by epinephrine, isoproterenol, or epinephrine plus phenylisopropyladenosine, and glycerol release was measured. Backfat thickness was measured by ultrasonography. Our findings revealed that 1) adipocytes from 1.0 P:S diet-fed swine released 30% more glycerol than adipocytes from 0.3 P:S diet-fed swine when stimulated by 1 micromol/L isoproterenol; 2) adipocytes from exercised swine released 45% more glycerol than adipocytes from sedentary swine when stimulated by 1 micromol/L epinephrine; 3) body weight of exercised swine was significantly lower than sedentary swine; and 4) backfat thickness was less in exercised swine than in sedentary swine (2.39 vs. 2.95 cm, P = 0.002). We conclude that ad libitum consumption of diet with a P:S of 1.0, combined with endurance exercise, increases lipolytic sensitivity, lowers body weight gain, and reduces fat accumulation in female Yucatan miniature swine.

MINIMAL IMPACT OF FOOD, DRINK, OR TEMPERATURE ON THE PHARMACOKINETICS OF A SUBLINGUAL FILM USING AA NOVEL PRODRUG OF EPINEPHRINE (DESF)

<h3>Introduction</h3> DESF is a novel alternative dosage form to Epinephrine injection. A pre-clinical study was conducted to test the effect of sublingual temperature (SL) and if exposure of the sublingual mucosa to food or drink prior to administration of DESF has an impact on the PK profile of epinephrine <i>in vivo</i>. <h3>Methods</h3> A total of 18 male Yucatan miniature swine, separated into three groups and dosed with DESF 12 mg, were used for this study. Group 1 did not receive a pre-treatment. Groups 2 and 3 were pretreated with either full-fat whole milk or extra virgin olive oil or with either warm or cool compresses at the dose site. Following dosage, the animals had Pharmacokinetics (PK) blood samples obtained at multiple timepoints, with Oral Mucositis Assessment Scale scores performed at pre-dose, 4-, and 24-hours post-dose. <h3>Results</h3> Similar PK profiles were observed for epinephrine regardless of pretreatment of food and drink. Groups pretreated with milk or oil displayed a mean Cmax and AUC similar to or higher than the control group. Tmax was similar for all treatment groups, between 25 to 30 minutes. As for the impact of temperature, pre-treatment with a warm or cool compress did not result in significantly different SL temperatures. Similar PK profiles were also observed for epinephrine regardless of pre-treatment. Simple linear regression analysis suggests that there was not a strong relationship between SL temperature and Cmax. <h3>Conclusion</h3> These results suggest that food, drink, and temperature each have minimal impact on the PK profiles of DESF.

RESULTS OF A REPEAT DOSE STUDY ON THE PHARMACOKINETICS OF A SUBLINGUAL FILM USING A NOVEL PRODRUG OF EPINEPHRINE (DESF)

<h3>Introduction</h3> DESF is the first and only orally delivered epinephrine product candidate in clinical development and has the same target indication as that for Epinephrine injection in the emergency treatment of Type 1 allergic reactions. A pre-clinical study was conducted to compare the pharmacokinetic profiles of epinephrine following administration of DESF via one or two dissolvable films in miniature swine. <h3>Methods</h3> A total of 20 male Yucatan miniature swine were used for this study. Animals were separated into two groups and received either one or two doses of DESF 12 mg 10 minutes apart. Following dose administration, the animals had pharmacokinetic (PK) blood samples obtained at multiple time-points, with Oral Mucositis Assessment Scale (OMAS) scores performed at pre-dose, 4 hours, and 24 hours post-dose. <h3>Results</h3> No mortality/moribundity occurred during the study. The group receiving a single dose had an expected lower mean Cmax (11.1 vs 24.7 ng/ml) and AUC (738.5 vs 1314 ng/ml*min) than the group receiving repeated doses. The Cmax ratio and AUC ratio were similar at 2.23 and 1.78, respectively. Tmax was established at around 30 min. for the single dose group and at around 40 min. for those receiving a repeat dose. The overall PK profiles showed similar patterns between the two groups. <h3>Conclusion</h3> DESF demonstrates dose proportional PK when a second dose is administered 10 minutes after the first done. These results suggest that a different treatment delivery modality for epinephrine is possible and could address significant unmet need in patients.

Controlling axonal regeneration with acellular nerve allograft limits neuroma formation in peripheral nerve transection: An experimental study in a swine model.

Symptomatic neuromata are a common indication for revision surgery following amputation. Previously described treatments, including traction neurectomy, nerve transposition, targeted muscle re-innervation, and nerve capping, have provided inconsistent results or are technically challenging. Prior research using acellular nerve allografts (ANA) has shown controlled termination of axonal regrowth in long grafts. The purpose of this study was to determine the ability of a long ANA to prevent neuroma formation following transection of a peripheral nerve in a swine model. Twenty-two adult female Yucatan miniature swine (Sus scrofa; 4-6months, 15-25 kg) were assigned to control (ulnar nerve transection only, n= 10), treatment (ulnar transection and coaptation of 50 mm ANA, n= 10), or donor (n= 2) groups. Nerves harvested from donor group animals were treated to create the ANA. After 20 weeks, the transected nerves including any neuroma or graft were harvested. Both qualitative (nerve architecture, axonal sprouting) and quantitative histologic analyses (myelinated axon number, cross sectional area of nerve tissue) were performed. Qualitative histologic analysis of control specimens revealed robust axon growth into dense scar tissue. In contrast, the treatment group revealed dwindling axons in the terminal tissue, consistent with attenuated neuroma formation. Quantitative analysis revealed a significantly decreased number of myelinated axons in the treatment group (1232 ± 540) compared to the control group (44,380 ± 7204) (p< .0001). Cross sectional area of nerve tissue was significantly smaller in treatment group (2.83 ± 1.53 mm2 ) compared to the control group (9.14 ± 1.19 mm2 ) (p= .0012). Aberrant axonal growth is controlled to termination with coaptation of a 50 mm ANA in a swine model of nerve injury. These early results suggest further investigation of this technique to prevent and/or treat neuroma formation.

Epigenetic regulation of BAF60A determines efficiency of miniature swine iPSC generation

Miniature pigs are an ideal animal model for translational research to evaluate stem cell therapies and regenerative applications. While the derivation of induced pluripotent stem cells (iPSCs) from miniature pigs has been demonstrated, there is still a lack of a reliable method to generate and maintain miniature pig iPSCs. In this study, we derived iPSCs from fibroblasts of Wisconsin miniature swine (WMS), Yucatan miniature swine (YMS), and Göttingen minipigs (GM) using our culture medium. By comparing cells of the different pig breeds, we found that YMS fibroblasts were more efficiently reprogrammed into iPSCs, forming colonies with well-defined borders, than WMS and GM fibroblasts. We also demonstrated that YMS iPSC lines with a normal pig karyotype gave rise to cells of the three germ layers in vitro and in vivo. Mesenchymal stromal cells expressing phenotypic characteristics were derived from established iPSC lines as an example of potential applications. In addition, we found that the expression level of the switch/sucrose nonfermentable component BAF60A regulated by STAT3 signaling determined the efficiency of pig iPSC generation. The findings of this study provide insight into the underlying mechanism controlling the reprogramming efficiency of miniature pig cells to develop a viable strategy to enhance the generation of iPSCs for biomedical research.

Exercise Training Shifts Dependency from NADPH Oxidase 2 to NADPH Oxidase 4 in Endothelium‐Dependent Dilation in Coronary Arterioles Distal to Chronic Occlusion

Previously, we have reported that exercise training enhances endothelium‐dependent dilation in coronary arterioles distal to chronic occlusion, through increased contribution of the redox signaling molecule, hydrogen peroxide (H2O2). In the present study, we tested the hypothesis that enhanced endothelium‐dependent dilation in collateral‐dependent arterioles after exercise training would be attributable to an increased contribution of NADPH oxidase (NOX) 4‐derived reactive oxygen species. Yucatan miniature swine were surgically instrumented with an ameroid constrictor around the proximal left circumflex artery, which gradually induced occlusion of the artery and created a collateral‐dependent vascular bed. Eight weeks postoperatively, swine were assigned to either a sedentary protocol consisting of normal pen activity or a progressive exercise‐training regimen comprised of treadmill running 5 days a week, for 14 weeks. Coronary arterioles were isolated from the nonoccluded and collateral‐dependent myocardial regions and bradykinin‐mediated dilation via pressure myography was performed in the presence and absence of ML‐171, gp91ds‐tat and GKT136901, inhibiting NOX1, NOX2, and NOX1/4 isoforms, respectively. Additionally, using the superoxide probe, dihydroethidium, HPLC analysis was performed on isolated microvascular endothelial cells, pretreated with NOX inhibitors, to determine candidate sources of superoxide. Inhibition of NOX2 produced a significant rightward shift in EC50 values of arterioles from sedentary animals, independent of occlusion, as well as the nonoccluded arterioles of exercise‐trained pigs. EC50 values of collateral‐dependent arterioles from exercise‐trained swine were not significantly altered by NOX2 inhibition. Contrastingly, collateral‐dependent arterioles from exercise‐trained swine exhibited a significant shift in EC50 values in the presence NOX1/4 inhibition, which was absent in other arteriole treatment groups. Importantly, NOX1 inhibition did not significantly shift EC50 values in any treatment group, suggesting responses to the NOX1/4 inhibitor, GKT136901, reflect the contribution of NOX4. Interestingly, microvascular endothelial cell superoxide levels were not different across arteriole treatment groups. Our present findings suggest that exercise training produces adaptations in collateral‐dependent arterioles that appear to shift dependence from NOX2‐ to NOX4‐derived reactive oxygen species for endothelium‐dependent dilation. Taken together with our previous findings, our data support the assertion that enhanced dilation of collateral‐dependent arterioles after exercise training is driven by enhanced NOX4‐derived H2O2 levels.

Sex Differences in Hydrogen Peroxide‐Induced K+ Channel Activation in Arterioles of Ischemic Myocardium after Exercise Training

The objective of this study was to determine sex differences in potassium (K+) channel contribution to H2O2‐mediated dilation after exercise training in arterioles distal to chronic coronary artery occlusion. We tested the hypothesis that females would have enhanced H2O2‐mediated vasodilation compared to males through enhanced coupling of H2O2 with large‐conductance, calcium‐dependent K+ (BKCa; iberiotoxin 100 nM) channels and 4‐aminopyridine (4‐AP; 1 mM)‐sensitive voltage‐gated K+ (Kv). Adult Yucatan miniature swine were surgically instrumented with an Ameroid constrictor around the proximal left circumflex coronary artery, which gradually induced occlusion and created a collateral‐dependent vascular bed. Arterioles from the left anterior descending artery myocardial region served as nonoccluded control vessels. Pigs of each sex were separated into two groups: one that underwent a progressive exercise training program (treadmill run; 5 days/week for 14 weeks) and another that remained sedentary and was limited to normal pen activity. Collateral‐dependent arterioles from female, sedentary pigs were significantly less sensitive to H2O2‐induced dilation compared with nonoccluded arterioles (EC50: ‐4.55 ± 0.15 vs. ‐4.91 ± 0.15 log M, respectively; P&lt;0.05), whereas exercise training reversed the impaired dilation so that collateral‐dependent and nonoccluded arterioles responded similarly to H2O2 (EC50: ‐4.86 ± 0.16 vs. ‐4.95 ± 0.18 log M, respectively). Conversely, male pigs demonstrated enhanced vasodilation to H2O2 in collateral‐dependent compared with nonoccluded arterioles (‐4.92 ± 0.16 vs. –4.35 ± 0.16 log M, respectively) of sedentary pigs, which persisted after exercise training (‐4.94 ± 0.11 vs. ‐4.50 ± 0.21 log M, respectively). In female sedentary pigs, H2O2‐mediated dilation in nonoccluded or collateral‐dependent arterioles was not altered by inhibitors of BKCa or Kv channels. In contrast, after exercise training, Kv channels contributed significantly to dilation in nonoccluded arterioles and both BKCa and Kv channels played a significant role in dilation of collateral‐dependent arterioles in female pigs. Blockade of BKCa or Kv channels in nonoccluded or collateral‐dependent arterioles from male pigs had no effect on H2O2‐mediated vasodilation, independent of exercise‐training. Taken together, our studies suggest that coronary arterioles in females compensate for impaired H2O2‐mediated vasodilation by modulating BKCa and 4‐AP‐sensitive Kv channels after exercise‐training. Males do not appear to utilize the particular pathway involving H2O2 action on BKCa or 4‐AP‐sensitive Kv channels.

Computed tomography myelography technique and spinal morphometry in healthy Yucatan pigs.

Porcine models of spinal cord injury (SCI) have an irreplaceable role in the development of experimental therapies. There is little literature regarding CT myelogram (CTM) techniques in swine and morphometry in miniature swine has not been established. A CT-guided method for performing myelography as well as reference values for spinal morphometry in healthy Yucatan miniature swine is lacking. The goal of this study is to describe a CT-guided method of performing CTM in a porcine model of SCI and to establish spinal morphometric reference values in mature Yucatan pigs. Six healthy, Yucatan sows, 9 months of age, weighing between 39–57.7kg, with no history of spinal disease, spinal injury, or neurologic deficits on physical exam were used in this study. CT myelography was performed in each sow under general anesthesia. CT scout images were used to guide needle placement at the L3-L4 intervertebral site. Once correct needle placement was confirmed using a 1ml test injection, a full dose of iodinated contrast (0.3ml/kg) was injected slowly over a 2-minute time period. Morphometry was performed using area measurements of the spinal cord (SC), vertebral body (VB), dural sac (DS), and vertebral canal (VC) at the mid-body and the intervertebral disc space of each spinal segment. Of the quantitative measurements, the spinal cord surface area had the widest range of values and the greatest coefficient of variance (CV) while those parameters for the vertebral canal had a low CV. Of the morphometric ratios, the DS:VC, had the lowest CV while the spinal cord ratios to DS and VC had the highest (>30). The vertebral canal surface area and the dural space: vertebral canal ratio may serve as reference values in future studies using this animal model.

Exercise Training Rescues Impaired H 2 O 2 ‐Mediated Vasodilation in Collateral‐Dependent Coronary Arterioles Through Enhanced K + Channel Activation

The objective of this study was to determine downstream cellular signaling pathways by which exercise training promotes H2O2-mediated dilation in arterioles distal to chronic coronary artery occlusion. We tested the hypothesis that exercise training would correct impaired H2O2-mediated dilation in collateral-dependent arterioles through enhanced coupling of H2O2 with voltage-gated K+ (Kv) and large-conductance, calcium-dependent K+ (BKCa) channels. Yucatan miniature swine were surgically instrumented with an Ameroid constrictor around the proximal left circumflex coronary artery, which gradually induced occlusion and created a collateral-dependent vascular bed. Arterioles from the left anterior descending artery region served as nonoccluded control vessels. Pigs were separated into two groups: one that underwent a progressive exercise training program (treadmill run; 5 days/week for 14 weeks) and another that remained sedentary and was limited to normal pen activity. Collateral-dependent arterioles from sedentary pigs were significantly less sensitive to H2O2-induced dilation compared with nonoccluded arterioles (EC50: -4.55 ± 0.15 vs. -4.91 ± 0.15 log M, respectively; P<0.05), whereas this difference was eliminated after completion of the exercise training regimen when collateral-dependent and nonoccluded arterioles responded similarly to H2O2 (EC50: -4.86 ± 0.16 vs. -4.95 ± 0.18 log M, respectively). H2O2-mediated dilation in nonoccluded or collateral-dependent arterioles from sedentary animals was not altered by inhibitors of Kv (4-aminopyridine; 1 mM) or BKCa (iberiotoxin; 100 nM) channels. In contrast, after exercise training, Kv channels contributed significantly to dilation in nonoccluded arterioles and both Kv and BKCa channels played a significant role in dilation of collateral-dependent arterioles. Interestingly, the contribution of Kv and BKCa channels to basal tone was not altered by occlusion or exercise training, suggesting similar availability of these channels in nonoccluded and collateral-dependent arterioles of sedentary and exercise-trained pigs. Immunoblot analysis was performed to assess levels of PKG dimerization, a potential mediator between H2O2 and K+ channels. These studies revealed that H2O2 treatment significantly increased PKG dimerization in all artery treatment groups, and this effect was significantly greater in nonoccluded compared with collateral-dependent arteries from sedentary pigs, but not different after exercise training. Taken together, our studies suggest that impaired H2O2-mediated vasodilation may be in part attributable to attenuated PKG dimerization in collateral-dependent arterioles of sedentary pigs. However, while exercise training appeared to correct impaired H2O2-mediated dilation via enhanced coupling to K+ channel activation, exercise-induced changes in PKG dimerization did not appear to play a role.

Exercise Training Increases the Contribution of NADPH Oxidase 4 to Endothelium‐Dependent Dilation in Collateral‐Dependent Coronary Arterioles

We have previously reported that exercise training enhances endothelium-dependent dilation in arterioles distal to chronic coronary artery occlusion through an increased contribution of the reactive oxygen species, hydrogen peroxide (H2O2). In the current study, we began to test the hypothesis that exercise training increases the role of NADPH oxidase (NOX) 4 in bradykinin-mediated, endothelium-dependent dilation, which mediates the increase in H2O2, independent of changes in superoxide. Yucatan miniature swine were instrumented with an ameroid constrictor around the proximal left circumflex coronary artery to induce gradual occlusion and create a collateral-dependent vascular bed. Eight weeks postoperatively, swine were assigned to a progressive exercise-training regimen (treadmill run; 5 days/week for 14 weeks) or remained sedentary and limited to normal pen activity. Coronary arterioles were isolated from both nonoccluded and collateral-dependent myocardial regions and cannulated and pressurized to measure vascular reactivity. Bradykinin-mediated dilation was significantly attenuated in the presence of the NOX 1/4 inhibitor, GKT136901 (1 μM), in collateral-dependent arterioles from exercise-trained pigs, which had no significant influence in other arteriole treatment groups. However, immunoblot analysis revealed no effects of occlusion or exercise training on NOX4 protein levels. In addition, superoxide levels in isolated microvascular endothelial cells, measured by HPLC and dihydroethidium (DHE; 50 μM), revealed that bradykinin-stimulated superoxide levels were not altered by occlusion or exercise training. Taken together, our data suggest that exercise training increases the contribution of H2O2 to endothelium-dependent dilation in collateral-dependent arterioles independent of changes in superoxide levels. Additional evidence suggests that increased H2O2 levels may be produced by NOX4, despite no change in NOX4 protein levels.

Use of computed tomography to assess subcutaneous drug dispersion with recombinant human hyaluronidase PH20 in a swine model

IntroductionSubcutaneous (SC) formulations of therapeutics with recombinant human hyaluronidase PH20 (rHuPH20) are currently approved across various disease indications. The rHuPH20-mediated enzymatic degradation of SC hyaluronan (HA) facilitates bulk fluid flow and dispersion of co-administered therapeutics. However, current methods of quantifying dispersion in the SC space are limited. Here, a novel method is outlined to quantify and follow rapid SC volumetric dispersion of a representative therapeutic fluid in the presence of rHuPH20 using computed tomography (CT). MethodsTen Yucatan miniature swine were randomized to three groups. Animals received simultaneous infusions of contrast agent (CA) alone (left side of the animal) or in combination with rHuPH20 (right side) at infusion rates of 2.5, 5, or 10 mL/min. Spiral CT scans (1.5 mm thickness) were conducted before and after the infusion and at regular time intervals throughout. Scans were used to create three-dimensional (3D) reconstructions of the fluid pockets and analyze surface area, volume, and sphericity. Results3D reconstruction showed increased dispersion of CA with rHuPH20 compared with CA alone, with fenestration and increased dispersion in the craniocaudal and lateromedial directions. The CA with rHuPH20 fluid pockets showed an average increase of 46% in surface area (p = 0.001), a 35% increase in volume (p = 0.001) and a 17% decrease in sphericity post-infusion compared with CA alone at 30 min post-infusion. DiscussionThis exploratory study confirms the value of CT imaging as a non-invasive method of assessing real-time spatial and temporal behavior of SC-administered fluids. This technique could help to assess the dispersion pattern of novel rHuPH20 SC co-formulations.

D-PLEX100 in an Abdominal Surgery Incision Model in Miniature Swine: Safety Study.

Surgical site infections (SSIs) are a common surgical-related complication. To avoid these complications, a new biodegradable polymer-lipid encapsulation matrix that provides controlled release of doxycycline (doxycycline/polymer-lipid encapsulation matrix [D-PLEX]) has been developed. The aim of this comprehensive study was to evaluate the potential safety of D-PLEX100 in abdominal surgical site. D-PLEX100 was administered into incisions of abdominal surgical site in Yucatan miniature swine, which were followed for up to 6 months and compared to sham-control swine. The D-PLEX100 mass did not migrate from the incisional site, and there was no evidence for systemic toxicity or other safety concerns. Surgical incision sites, including the peritoneal surface, were fully healed at 6 months in all animals. Most of the D-PLEX100 mass was absorbed during the first 3 months, and by 6 months, D-PLEX100 was fully absorbed. Toxicokinetic evaluation revealed that doxycycline concentrations were evident at 30 minutes and persisted to 8 days (71 mg/kg) or at least 15 days (284 mg/kg) and were no longer present in plasma by day 29. This study supports the safety of D-PLEX100 and its favorable degradability profile. A clinical study is being performed to assess the safety and the efficacy of D-PLEX100 to prevent human abdominal SSIs.

Exercise Training Restores K+ Channel Contributions to Hydrogen Peroxide‐Mediated Dilation Independent of PKG Dimerization in Collateral‐Dependent Coronary Arterioles

In coronary artery disease (CAD), the ability of the coronary circulation to deliver blood to meet oxygen demands of the heart is often impaired. Exercise training has been established to mitigate the negative effects of CAD by preserving reactivity of the coronary microcirculation. We have previously reported that exercise increases the contribution of hydrogen peroxide (H2O2) to enhanced endothelium‐dependent vasodilation in collateral‐dependent coronary arterioles. Others have shown that cGMP‐dependent protein kinase (PKG) can be activated via oxidation by H2O2, inducing dimer formation. The PKG dimer has been shown to act on substrates, such as K+ channels, to contribute to vascular smooth muscle relaxation and vasodilation. In the current study, we tested the hypothesis that exercise training‐enhanced H2O2‐induced vasodilation is mediated through enhanced PKG dimerization and subsequent activation of Kv and BKca channels in coronary arterioles isolated from ischemic myocardium. An ameroid occluder was surgically placed on the proximal left circumflex coronary artery of female Yucatan miniature swine to induce gradual occlusion while the left anterior descending artery was left unoccluded to serve as a control. Eight weeks postoperatively, pigs were assigned to sedentary (n=14) or exercise training (n=13; progressive treadmill training for 13 weeks) regimens. Coronary arterioles (75–150 μm) and small arteries (250–400 um) were isolated from myocardium of the left circumflex and left anterior descending regions. Immunoblot analysis indicated that H2O2 induced PKG dimer formation in both porcine coronary arterioles and small arteries. Chronic coronary artery occlusion appeared to diminish H2O2‐mediated PKG dimer formation in collateral‐dependent arterioles and the reduced dimerization persisted with exercise training. H2O2‐mediated vasodilation tended to be impaired in arterioles isolated from the collateral‐dependent regions of sedentary pigs and was partially restored with exercise training. It also appears that both Kv and BKCa channels contributed to H2O2‐mediated dilation after exercise training. Our data demonstrate that H2O2 stimulates PKG dimer formation in both porcine coronary arterioles and small arteries. Contrary to our hypothesis, H2O2‐induced PKG dimerization is not enhanced by exercise training in either nonoccluded or collateral‐dependent arterioles. However, both Kv and BKCa channels appear to contribute to exercise training‐enhanced H2O2‐mediated dilation in collateral‐dependent coronary arterioles.Support or Funding InformationNational Institutes of Health R01‐HL139903

Neo-Angiogenesis, Transplant Viability, and Molecular Analyses of Vascularized Bone Allotransplantation Surgery in a Large Animal Model.

Vascularized composite allotransplantation of bone is a possible alternative treatment for large osseous defects but requires life-long immunosuppression. Surgical induction of autogenous neo-angiogenic circulation maintains transplant viability without this requirement, providing encouraging results in small animal models [1-3]. A preliminary feasibility study in a swine tibia model demonstrated similar findings [4, 5]. This study in swine tibial allotransplantation tests its applicability in a pre-clinical large animal model. Previously, we have demonstrated bone vascularized composite allotransplantation (VCA) survival was not the result of induction of tolerance nor an incompetent immune system [1]. Fourteen tibia vascularized bone allotransplants were microsurgically transplanted orthotopically to reconstruct size-matched tibial defects in Yucatan miniature swine. Two weeks of immunosuppression was used to maintain allotransplant pedicle patency during angiogenesis from a simultaneously implanted autogenous arteriovenous bundle. The implanted arteriovenous bundle was patent in group 1 and ligated in group 2 (a neo-angiogenesis control). At twenty weeks, we quantified the neo-angiogenesis and correlated it with transplant viability, bone remodeling, and gene expression. All patent arteriovenous bundles maintained patency throughout the survival period. Micro-angiographic, osteocyte cell count and bone remodeling parameters were significantly higher than controls due to the formation of a neo-angiogenic autogenous circulation. Analysis of gene expression found maintained osteoblastic and osteoclastic activity as well as a significant increase in expression of endothelial growth factor-like 6 (EGFL-6) in the patent arteriovenous bundle group. Vascularized composite allotransplants of swine tibia maintained viability and actively remodeled over 20 weeks when short-term immunosuppression is combined with simultaneous autogenous neo-angiogenesis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:288-296, 2020.