Porcine Explants Research Articles

Magnetically Guided Adeno-Associated Virus Delivery for the Spatially Targeted Transduction of Retina in Eyes.

Adeno-associated viruses (AAVs) are intensively explored for gene therapies in general and have found promising applications for treating retina diseases. However, controlling the specificity (tropism) and delivery of AAVs to selected layers, cell types, and areas of the retina is a major challenge to further develop retinal gene therapies. Magnetic nanoparticles (MNPs) provide effective delivery platforms to magnetically guide therapeutics to target cells. Yet, how MNPs can deliver AAVs to transfect particular retina layers and cells remains elusive. Here, it is demonstrated that MNPs can be used to transport different AAVs through the retina and to modulate the selective transduction of specific retinal layers or photoreceptor cells in ex vivo porcine explants and whole eyes. Thereby, transduction is triggered by bringing the viruses in close proximity to the target cell layer and by controlling their interaction time. It is shown that this magnetically guided approach to transport AAVs to selected areas and layers of the retina does not require the cell-specific optimization of the AAV tropism. It is anticipated that the new approach to control the delivery of AAVs and to selectively transduce cellular systems can be applied to many other tissues or organs to selectively deliver genes of interest.

Nanosecond multipulse retinal damage thresholds of elongated irradiance profiles in explant measurements and simulations.

The database for multipulse retinal damage thresholds for the laser safety standard (IEC 60825-1:2014) is confined, especially for elongated irradiation profiles. To ensure eye safety, retinal damage thresholds ( values) need to be determined. This study aims to examine nanosecond multipulse scenarios. To determine values in ex vivo measurements, an optical laser setup is presented. Porcine explant tissue is irradiated with rectangular top-hat profiles. Thermal simulations are carried out on a validated computer model and retinal injury thresholds are obtained. The measurements resulted in values from 8.46 to with a slope from 1.15 to 1.4. A thermal damage in the measurements can be excluded due to the level value in combination with a different type of declining behavior for increasing pulses compared to the simulations. A dependence with increasing elongation or area of the retinal image emerges in the simulations but could not be confirmed in the measurements due to the influencing factors (biological variability, focusing, and measuring procedure). Using slit apertures for beam shaping, variable rectangular spot geometries are realized without changing elements in the setup. For further evaluation of the behavior of elongated irradiation profiles, additional measurements to improve the measurement accuracy are necessary.

Efficacy and toxicity of hydrogen peroxide producing electrochemical bandages in a porcine explant biofilm model.

Effects of H2 O2 producing electrochemical-bandages (e-bandages) on methicillin-resistant Staphylococcus aureus colonization and biofilm removal were assessed using a porcine explant biofilm model. Transport of H2 O2 produced from the e-bandage into explant tissue and associated potential toxicity were evaluated. Viable prokaryotic cells from infected explants were quantified after 48 h treatment with e-bandages in three ex vivo S. aureus infection models: (1) reducing colonization, (2) removing young biofilms and (3) removing mature biofilms. H2 O2 concentration-depth profiles in explants/biofilms were measured using microelectrodes. Reductions in eukaryotic cell viability of polarized and nonpolarized noninfected explants were compared. e-Bandages effectively reduced S. aureus colonization (p =0.029) and reduced the viable prokaryotic cell concentrations of young biofilms (p =0.029) with limited effects on mature biofilms (p >0.1). H2 O2 penetrated biofilms and explants and reduced eukaryotic cell viability by 32-44% compared to nonpolarized explants. H2 O2 producing e-bandages were most active when used to reduce colonization and remove young biofilms rather than to remove mature biofilms. The described e-bandages reduced S. aureus colonization and young S. aureus biofilms in a porcine explant wound model, supporting their further development as an antibiotic-free alternative for managing biofilm infections.

Activity of a hypochlorous acid-producing electrochemical bandage as assessed with a porcine explant biofilm model.

The activity of a hypochlorous acid-producing electrochemical bandage (e-bandage) in preventing methicillin-resistant Staphylococcus aureus infection (MRSA) infection and removing biofilms formed by MRSA was assessed using a porcine explant biofilm model. e-Bandages inhibited S. aureus infection (p = 0.029) after 12 h (h) of exposure and reduced 3-day biofilm viable cell counts after 6, 12, and 24 h exposures (p = 0.029). Needle-type microelectrodes were used to assess HOCl concentrations in explant tissue as a result of e-bandage treatment; toxicity associated with e-bandage treatment was evaluated. HOCl concentrations in infected and uninfected explant tissue varied between 30 and 80 µM, decreasing with increasing distance from the e-bandage. Eukaryotic cell viability was reduced by an average of 71% and 65% in fresh and day 3-old explants, respectively, when compared to explants exposed to nonpolarized e-bandages. HOCl e-bandages are a promising technology that can be further developed as an antibiotic-free treatment for wound biofilm infections.

3D-Printed Replica and Porcine Explants for Pre-Clinical Optimization of Endoscopic Tumor Treatment by Magnetic Targeting.

Simple SummaryAnimal models are often needed in cancer research but some research questions may be answered with other models, e.g., 3D replicas of patient-specific data, as these mirror the anatomy in more detail. We, therefore, developed a simple eight-step process to fabricate a 3D replica from computer tomography (CT) data using solely open access software and described the method in detail. For evaluation, we performed experiments regarding endoscopic tumor treatment with magnetic nanoparticles by magnetic hyperthermia and local drug release. For this, the magnetic nanoparticles need to be accumulated at the tumor site via a magnetic field trap. Using the developed eight-step process, we printed a replica of a locally advanced pancreatic cancer and used it to find the best position for the magnetic field trap. In addition, we described a method to hold these magnetic field traps stably in place. The results are highly important for the development of endoscopic tumor treatment with magnetic nanoparticles as the handling and the stable positioning of the magnetic field trap at the stomach wall in close proximity to the pancreatic tumor could be defined and practiced. Finally, the detailed description of the workflow and use of open access software allows for a wide range of possible uses.Background: Animal models have limitations in cancer research, especially regarding anatomy-specific questions. An example is the exact endoscopic placement of magnetic field traps for the targeting of therapeutic nanoparticles. Three-dimensional-printed human replicas may be used to overcome these pitfalls. Methods: We developed a transparent method to fabricate a patient-specific replica, allowing for a broad scope of application. As an example, we then additively manufactured the relevant organs of a patient with locally advanced pancreatic ductal adenocarcinoma. We performed experimental design investigations for a magnetic field trap and explored the best fixation methods on an explanted porcine stomach wall. Results: We describe in detail the eight-step development of a 3D replica from CT data. To guide further users in their decisions, a morphologic box was created. Endoscopies were performed on the replica and the resulting magnetic field was investigated. The best fixation method to hold the magnetic field traps stably in place was the fixation of loops at the stomach wall with endoscopic single-use clips. Conclusions: Using only open access software, the developed method may be used for a variety of cancer-related research questions. A detailed description of the workflow allows one to produce a 3D replica for research or training purposes at low costs.

Asurvey of physician training and credentialing in endoscopic submucosal dissection in the United States.

Endoscopic submucosal dissection (ESD) is technically challenging, has a longer learning curve and a greater complication rate than most new endoscopic procedures. Formal training and credentialing guidelines for ESD are currently lacking in the United States (US). To survey ESD experts across the US to determine their learning process and obtain their opinion on how training and credentialing for ESD should develop. Anonymous electronic survey. ASGE members who are practicing US endoscopists. The survey was developed by iterative revisions of questions administered to three investigators who have been performing ESD for > 5years. The final survey was distributed electronically to US endoscopists who had previously attended ESD expert conferences. Thirty-five (58.3%) of 60 practicing ESD experts responded to the survey. A majority (91%) were in university-based, community, or tertiary care hospitals. All practitioners practiced on porcine explants and observed live ESD procedures as part of the training. Out of the participants, 75.8% received formal supervised hands-on training on porcine explants and/or humans before performing ESD independently. Fifty percent indicated that their facility had written guidelines specifically for ESD credentialing. Four out of 5 felt that credentialing requirements should include attending weekend ESD courses, observing live procedures, practicing on explants, and advanced endoscopic training in interventional endoscopy such as an additional year of fellowship. Survey completion rate of 58.3%. ESD training should include practicing on explants, observation of live procedures, training in interventional endoscopy, and attending educational courses. Credentialing guidelines for ESD based on expert opinion need to be developed in the US.

Evaluation of a Novel Three-Dimensional Emergency Cricothyrotomy Task Trainer.

BackgroundEmergency cricothyrotomy is a critical, yet infrequently performed and time-sensitive procedure that requires practice in order to be reliably completed in emergent airway situations. Many physicians never have the opportunity to practice this rarely performed but highly impactful procedure during their training due to a lack of an affordable, high-fidelity training model. In this study, the educational impact and realism of a new synthetic cricothyrotomy training model (high-fidelity emergency cricothyrotomy, HiFEC) were compared with those of a standard porcine explant model.MethodologyA total of thirty-one attending physicians from four medical specialties were recruited on a volunteer basis to participate in a cricothyrotomy simulation workshop. Participants were randomly assigned to complete the initial workshop using one of the two models but had the opportunity to practice on both trainers. Pre- and post-workshop comfort level as well as the realism of the models were surveyed using questionnaires and evaluated using a five-point Likert scale.ResultsImprovements in self-reported comfort levels were seen in both the porcine group (p = 0.0014) and HiFEC group (p = 0.0036) as well as overall (p < 0.001). The realism rating of both training models was similar with a median score of 4 on a five-point Likert scale. When comparing the cost of conducting our workshop using these models, the synthetic model saved over $650.ConclusionsGiven the similar realism of the models and the improvement in participant comfort level, the synthetic HiFEC trainer is an effective and more affordable alternative training model for emergency cricothyrotomies.

Ovarian toxicity by fusariotoxins in pigs: Does it imply in oxidative stress?

Mycotoxins are natural contaminants of food and feed occurring worldwide. Deoxynivalenol (DON) and fumonisin B1 (FB1) are the most frequent fusariotoxins and induce immune and intestinal toxicity in humans and animals. Recently, an association between mycotoxins exposure and impaired fertility has been suggested. However, the effects of these mycotoxins on the reproductive system are not well established. This study aimed to evaluate the effects of FB1 and DON, in combination or alone, on the ovarian morphology and oxidative responses using porcine explants. Seventy-two explants were obtained from six pigs and submitted to the following treatments: control (MEM medium), DON (10 μM), FB1 (100 μM FB1), and DON + FB1 (10 μM + 100 μM). Histological and immunohistochemical assays were performed to evaluate ovarian changes, cell proliferation, and apoptosis. Oxidative stress response was evaluated through lipid peroxidation and antioxidant capacity response assays. The exposure to mycotoxins induced significant histological changes in the ovaries, which were characterized by a decrease in viable follicles and increase in degenerated follicles. A significant decrease in granulosa cell proliferation was observed in explants exposed to all mycotoxins. In addition the multi-contaminated treatment was responsible for an increase in the cell apoptosis index of growing follicles. On the other hand, the FB1 and multi-contaminated treatments induced a significant decrease in lipid peroxidation accompanied by an increase in antioxidant responses. Altogether, our results indicate a reproductive toxicity induced by fusariotoxins. Moreover, mycotoxins, alone or in combination, modulate oxidative stress response, interfering with the production of free radicals and affecting the reproductive capacity of pigs.

Kappa opioids modulate hedgehog signaling to attenuate osteoarthritis

Purpose: Osteoarthritis (OA) affects 50 million Americans, at an annual cost of $100 billion. Pain management is currently a hallmark of OA treatment. One of the most frequently employed treatment strategies involves opioid analgesics. Opioids are highly addictive and have led to an opioid epidemic. One strategy to reduce opioid addiction is to implement kappa-opioid receptor (KOR) agonist medications which have been documented to provide pain-relief without the addictive potential of commonly used opioid medications (which target the mu-opioid receptor). Our previous work showed that KOR is expressed in chondrocytes and synovial cells. Previous studies have also shown that inhibition of the Hedgehog (Hh) signaling pathway prevents cartilage degeneration and promotes repair in animal models of OA. Current study was designed to explore if modulation of kappa opioid receptor (KOR) signaling may have a therapeutic potential for achieving disease-modifying activity in OA models through Hh modulation. Methods: Normal human primary tissues were obtained from the National Disease Research Interchange (NDRI). Human OA samples were collected from patients with severe osteoarthritis undergoing total joint replacements. Normal porcine articular cartilage tissue was used for explant studies. For in vivo studies a partial medial meniscectomy (PMM) rat model was used. A novel automated 3D indentation mapping mechanical tester (Mach-1) was used to quantify articular cartilage thickness and stiffness properties. Results: We have demonstrated that activation of KOR by a selective peptide agonist JT09 inhibits Hh signaling in normal and osteoarthritic human chondrocytes via cAMP/CREB pathway. JT09 has also markedly decreased matrix degeneration in porcine explants induced by a Hh agonist SAG. Moreover, in vivo application of JT09 via intra-articular injection significantly attenuated rat articular cartilage degeneration after PMM. In JT09 treated rats, cartilage content and structure were largely maintained, and osteophyte formation was greatly reduced. In addition, treatment with JT09 prevented functional compressive tissue degeneration following PMM. Conclusions: Current study defines a novel molecular mechanism for the role of the KOR in articular cartilage homeostasis and disease, providing a potential unifying mechanistic basis for the overlap in processes and pathologies shown to involve opioid and Hh signaling. Moreover, this study identifies a potential novel therapeutic strategy in which KOR modulation in cartilage and/or synovial tissue can improve outcomes in patients with arthritis.

Facile and efficient encapsulation of antimicrobial peptides via crosslinked DNA nanostructures and their application in wound therapy

There is growing interest in the development of nucleic acid nanostructures as smart functional materials for applications in drug delivery. Inspired by the diverse physical interactions that exist in nature, crosslinked DNA nanostructures can serve as attractive affinity binding networks that interact with therapeutic cargos or living cells. Herein we report a strategy that addresses the challenges of topical oligopeptide therapy by exploiting high binding affinity between polyanionic DNA nanostructures and cationic antimicrobial peptides (AMPs) to fabricate hydrogels that release a model antimicrobial L12 peptide in response to pathogenic S. aureus infections. We further demonstrated controlled peptide release profiles via the DNA hydrogels that were biocompatible and delivered superior antimicrobial activity against nuclease-releasing susceptible and methicillin-resistant S. aureus infections. Single application of the L12-loaded DNA hydrogels on porcine explant S. aureus infections revealed potent efficacy after 24h. As a result of the capacity of the crosslinked DNA nanostructures to elicit a strong anti-inflammatory response, in vivo treatment of mice excision wounds translated into faster healing rates. Overall, the crosslinked DNA nanostructures reported in this study offer significant advantage as functional wound dressings and their future adaptation holds equally great promise for the delivery of cationic antimicrobials.

Technological features of advanced skin protectants and an examination of the evidence base.

Products that provide a protective skin barrier play a vital role in defending the skin against the corrosive effect of bodily fluids, including wound exudate, urine, liquid faeces, stoma output and sweat. There are many products to choose from, which can be broadly categorised by ingredients. This article describes the differences in mechanisms of action between barrier products comprising petrolatum and/or zinc oxide, silicone film-forming polymers and cyanoacrylates, and compares the evidence on them. The literature indicates that all types of barrier product are clinically effective, with little comparative evidence indicating that any one ingredient is more efficacious than another, although film-forming polymers and cyanoacrylates have been found to be easier to apply and more cost-effective. However, laboratory evidence, albeit limited, indicates that a concentrated cyanoacrylate produced a more substantial and adherent layer on a porcine explant when compared with a diluted cyanoacrylate and was more effective at protecting skin from abrasion and repeated exposure to moisture than a film-forming polymer. Finally, a silicone-based cream containing micronutrients was found to significantly reduce the incidence of pressure ulceration when used as part of a comprehensive prevention strategy.

Acute tissue reactions, inner segment pathology, and effects of the antioxidant α1-microglobulin in an in vitro model of retinal detachment

The purpose of this study was to explore acute tissue reactions, ultrastructural photoreceptor morphology with emphasis on inner segments, and the effect of antioxidant treatment in an in vitro model of rhegmatogenous retinal detachment (RRD). A previously described method of RRD simulation was used with adult retinal porcine explants kept free-floating in culture medium with or without treatment with the radical scavenger α1-microglobulin (A1M). Explants were examined at 5 time points from 1 to 24 h using transmission electron microscopy as well as quantitative real-time PCR (RT-PCR) to quantify gene expression of the cell stress marker heat shock protein 70 (Hsp70) and oxidative stress marker heme oxygenase (HO-1). The culture medium level of the cell damage marker lactate dehydrogenase (LDH) and oxidative stress DNA damage marker 8-Oxo-2′-deoxyguanosine (8-OHdG) was also assessed at each time point. We found that the levels of Hsp70 and LDH rapidly increased in both groups, and at 3 and 6 h, Hsp70 was significantly higher in A1M treated retinas. At 24 h, Hsp70 and LDH, as well as 8-OHdG were significantly lower compared with controls, whereas the tissue level of HO-1 was significantly higher. Progressive ultrastructural photoreceptor changes were seen in untreated control explants from 1 h and onwards including outer segment shortening and loss, disruption of organelles within the inner segments and loss of perikarya in the outer nuclear layer. Inner segment pathology was more rapid and extensive in rods compared with in cones. In A1M treated counterparts, damage to rod inner segment mitochondria was significantly higher after 1 h of culture, but after this time, no statistical difference was found. At 24 h, cone inner segment mitochondrial disruption was significantly higher in control retinas and the number of surviving perikarya lower. From our results, we conclude that retinal explants elicit acute cell stress reactions when placed in culture without physical support simulating a detached retina floating in the vitreous space. Photoreceptors rapidly display degenerative changes including extensive damage to inner segment mitochondria indicating loss of energy transduction as an early key event. A1M increases initial mitochondrial stress in the rods, however, subsequent pathology is attenuated by the treatment, highlighting the dynamics of protective as well as disruptive oxidative stress reactions in the detached retina.

Bone marrow mechanotransduction in porcine explants alters kinase activation and enhances trabecular bone formation in the absence of osteocyte signaling

Bone is a dynamic tissue that can adapt its architecture in response to mechanical signals under the control of osteocytes, which sense mechanical deformation of the mineralized bone. However, cells in the marrow are also mechanosensitive and may contribute to load-induced bone adaptation, as marrow is subjected to mechanical stress during bone deformation. We investigated the contribution of mechanotransduction in marrow cells to trabecular bone formation by applying low magnitude mechanical stimulation (LMMS) to porcine vertebral trabecular bone explants in an in situ bioreactor. The bone formation rate was higher in stimulated explants compared to unloaded controls which represent a disuse condition (CNT). However, sclerostin protein expression in osteocytes was not different between groups, nor was expression of osteocytic mechanoregulatory genes SOST, IGF-1, CTGF, and Cyr61, suggesting the mechanoregulatory program of osteocytes was unaffected by the loading regime. In contrast, c-Fos, a gene indicative of mechanical stimulation, was upregulated in the marrow cells of mechanically stimulated explants, while the level of activated c-Jun decreased by 25%. The activator protein 1 (AP-1) transcription factor is a heterodimer of c-Fos and c-Jun, which led us to investigate the expression of the downstream target gene cyclin-D1, a gene associated with cell cycle progression and osteogenesis. Cyclin-D1 gene expression in the stimulated marrow was approximately double that of the controls. The level of phosphorylated PYK2, a purported inhibitor of osteoblast differentiation, also decreased in marrow cells from stimulated explants. Taken together, mechanotransduction in marrow cells induced trabecular bone formation independent of osteocyte signaling. Identifying the specific cells and signaling pathways involved, and verifying them with inhibition of specific signaling molecules, could lead to potential therapeutic targets for diseases characterized by bone loss.

Urocortin protects chondrocytes from acute trauma in articular cartilage

Purpose: Single injurious mechanical impact to joints, as seen in sports injury, is known to be associated with a high risk of later development of osteoarthritis (OA). Irreversible chondrocyte death is initiated following impact and results in progressive breakdown of articular cartilage. Promotion of chondrocyte survival is therefore a necessary target in the prevention of development of further OA. Urocortin1 (Ucn) has recently been shown to have a pro-survival effect on chondrocytes in both the presence and absence of pro-apoptotic stimuli. The aim of this study was to investigate the role of Ucn in response to mechanical impact. Methods: Full thickness porcine cartilage explants from lateral and medial femoral condyles were impacted with a 500g weight using a custom-made drop tower device. Impacted explants were treated with human Ucn (10-7M, 30 minutes prior to or following impact), non-selective cation channel blocker Gadolinium (500μM, 30 minutes following impact), or mechanosensitive ion channel blocker GsMTx4 (40μM, 2 hours prior to impact) and cultured for 72hrs. Cell viability was assessed with a CMFDA:PI live dead assay, using both macro confocal microscopy, as a means of avoiding bias, and high magnification confocal microscopy techniques. Lactate dehydrogenase (LDH) assays were conducted at 72hrs to provide a quantitative read out of cell death for each explant. Calcium influx was assessed 1hr post impact by Fluo-4 AM staining and visualised by multiphoton microscopy. Results: Porcine explants were impacted from a range of heights (10-70mm) in order to establish the correct energy of impact for inducing cell death without excess damage and fracture of the cartilage explant. Cell death increased in a dose dependent manner with height. 50mm was chosen as optimal height for all further experiments. Pre-treatment of explants with Ucn had a significant increase on cell viability when compared to impacted alone, as seen by quantification of the CMFDA:PI ratio on macro confocal (p=0.0042) and by high magnification confocal microscopy (p=0.0015). This increase in cell viability was confirmed by LDH assay (p=0.0063). Post-impact treatment of the explants with Ucn also resulted in a significant increase in cell survival, seen in both CMFDA:PI ratio (p=0.0009) and LDH assay (p=0.0053). We have previously found in primary human chondrocytes that Ucn exerts its protective effect on chondrocytes by the inhibition of excessive calcium influx. Calcium influx was therefore visualised in impacted explants treated pre- and post-impact with Ucn. A significant decrease in the levels of calcium staining was seen in both of these conditions when compared to explants impacted in the absence of Ucn (p=0.0073, p=0.05). In vitro work also highlighted a role for the mechanosensitive ion channel Piezo1 in cell death associated with the absence of Ucn. Explants were therefore treated with both Gadolinium and GsMTx4 post-impact and cell viability assessed by multiphoton microscopy. An increase in cell viability was observed in both conditions, comparable to those seen with addition of Ucn. Conclusions: A novel role for Ucn in the protection of articular chondrocytes maintained in cartilage explants has been shown and validated by two microscopy techniques and LDH assay. Ucn has also been shown to reduce calcium influx in impacted explants at 60 minutes post impact, and increased cell viability was observed by blocking mechanosensitive ion channels. These data suggest the importance of regulation of calcium influx in Ucn-mediated cell protection. The pro-survival effect of Ucn was seen both pre- and post-impact, highlighting the potential prophylactic and therapeutic use of Ucn for treating sudden injury to cartilage, and subsequent progression to post-traumatic OA.

607. Organotypic Culture of Adult Porcine Retina as an In Vitro Screening Model for AAV-Mediated Gene Therapy in Ophthalmology

Introduction: Gene therapy studies in vivo using animal models provide critical information regarding vector specificity, therapeutic efficacy, and safety. However, long durations to observe transgene expression, high degree of variability between animals, and reduced control of experimental conditions often leads to large sample sizes and expense. Organotypic cultures of the retina can provide an efficient in vitro model with a high degree of throughput and control that can be used for pharmacological screening. These explants preserve complex intercellular processes and communications among the neural retinal layers with minimal manipulation of tissue architecture and molecular pathways, making it a useful model in target-tissue validation of adeno-associated virus (AAV) vector variants. Porcine retina has similar anatomical and physiologic features to the human retina and is therefore a suitable surrogate for preclinical testing in ophthalmology. Methods: We have developed a porcine explant culture system from retinas isolated from adult pigs as a model to screen novel rAAV vectors for transduction efficiency and cellular tropism. Pig eyes were enucleated and the neural retina dissected away from the pigment epithelium within 1-2 hours postmortem. Tissue was cut into 4 mm pieces and placed photoreceptor layer down on polycarbonate transwells, and cultured in Neurobasal-A medium supplemented with B-27. Viability of the explant cultures was examined over time by gross morphology, quantification of glucose-6-phosphate dehydrogenase release from dying cells, and TUNEL assay. Transductions with AAV vectors were performed on retinal explants on the day the cultures were started, and transgene expression was analyzed by live cell imaging and immunofluorescence (IF) staining, or ELISA from tissue supernatants. Results: Retinal explants were viable for up to 4 weeks and IF staining of retinal protein markers including β-III-tubulin, Chx10, glutamine synthetase, rhodopsin and L/M opsin suggested that most retinal cell types were preserved in vitro, with tissue architecture closely resembling that of intact in vivo retina. Transduction with different rAAV serotypes encoding GFP displayed protein expression at 3 days, with gradually rising levels during the first 2 weeks following transduction. Furthermore, IF staining using retinal cell markers revealed differential cellular transgene expression from the AAV vectors encoding various cell-specific or ubiquitous promoters under investigation. Finally, high levels of secreted soluble VEGFR-1 protein expressed from rAAV were detected from explant culture media samples with expression increasing over a period of 3 weeks. Conclusions: Our results demonstrate viability of retinal explants for up to 4 weeks and efficient rAAV transduction with various serotypes. Our organotypic porcine retinal explant model facilitates the evaluation of efficacy, cellular tropism, and promoter selectivity of rAAV vectors in a fast, reproducible, and economical manner, contributing to a reduction in the use of animals for the development of therapies in ophthalmology.

In vitro biomechanical modulation--retinal detachment in a box.

To illustrate the importance of biomechanical impact on tissue health within the central nervous system (CNS), we herein describe an in vitro model of rhegmatogenous retinal detachment (RRD) in which disruption and restoration of physical tissue support can be studied in isolation. Adult retinal porcine explants were kept in culture for 3 or 12 hours without any tissue support, simulating clinical RRD, after which they were either maintained in this state or reattached to the culture membrane for an additional 48 hours. In vitro detachment resulted in gliosis and severe progressive loss of retinal neurons. In contrast, if the explant was reattached, gliosis and overall cell death was attenuated, ganglion cell death was arrested, and the number of transducin-expressing cone photoreceptors increased. These results support the hypothesis that removal of the elastic retina from its normal physical environment results in degenerative damage, and, if restored, rescues retinal neurons. Our study reinforces the notion of a strong relationship between the biomechanical environment and homeostasis within the retina, which has significant bearing on pathologic events related to RRD, and may also have impact on other regions within the CNS under biomechanical influence.

Electrochemical scaffold generates localized, low concentration of hydrogen peroxide that inhibits bacterial pathogens and biofilms.

We hypothesized that low concentrations of H2O2 could be generated through the electrochemical conversion of oxygen by applying an electric potential to a conductive scaffold and produce a low, but constant, concentration of H2O2 that would be sufficient to destroy biofilms. To test our hypothesis we used a multidrug-resistant Acinetobacter baumannii strain, because this species is often implicated in difficult-to-treat biofilm infections. We used conductive carbon fabric as the scaffold material (“e-scaffold”). In vitro experiments demonstrated the production of a maximum constant concentration of ~25 μM H2O2 near the e-scaffold surface. An e-scaffold was overlaid onto an existing A. baumannii biofilm, and within 24 h there was a ~4-log reduction in viable bacteria with an ~80% decrease in biofilm surface coverage. A similar procedure was used to overlay an e-scaffold onto an existing A. baumannii biofilm that was grown on a porcine explant. After 24 h, there was a ~3-log reduction in viable bacteria from the infected porcine explants with no observable damage to the underlying mammalian tissue based on a viability assay and histology. This research establishes a novel foundation for an alternative antibiotic-free wound dressing to eliminate biofilms.

Low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments affect degeneration of cultured articular cartilage explants

Articular cartilage has some capacity for self-repair. Clinically used low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments were compared in their potency to prevent degeneration using an explant model of porcine cartilage. Explants of porcine cartilage and human osteoarthritic cartilage were cultured for four weeks and subjected to daily LIPUS or PEMF treatments. At one, two, three and four weeks follow-up explants were prepared for histological assessment or gene expression (porcine only). Non-treated porcine explants showed signs of atrophy of the superficial zone starting at one week. Treated explants did not. In LIPUS-treated explants cell clusters were observed. In PEMF-treated explants more hypertrophic-like changes were observed at later follow up. Newly synthesized tissue was present in treated explants. Gene expression profiles did indicate differences between the two methods. Both methods reduced expression of the aggrecan and collagen type II gene compared to the control. LIPUS treatment of human cartilage samples resulted in a reduction of degeneration according to Mankin scoring. PEMF treatment did not. LIPUS or PEMF prevented degenerative changes in pig knee cartilage explants. LIPUS reduced degeneration in human cartilage samples. LIPUS treatment seems to have more potency in the treatment of osteoarthritis than PEMF treatment.

Endoscopic suture fixation of self-expanding metallic stents with and without submucosal injection.

Self-expanding metallic stents (SEMS) are useful for treating leaks after bariatric procedures but stent migration (12 to 40 %) remains a problem. Suture fixation has been used to minimize migration but has attendant risks of transmural penetration. We hypothesized that submucosal injection would decrease the risk of full thickness suture penetration while still providing greater pullout forces than endoscopic clipping. Porcine explant models and 155 mm SEMS were used with a force meter to measure the pullout forces required to dislodge the stent. Stents were first deployed without fixation and this pullout force acted as a control. The explants were then randomized to receive fixation with clips, endoscopic suturing with submucosal elevation, or endoscopic suturing without submucosal elevation. The pullout force was again measured and the ratio of the experimental to control pullout forces was used as a measure of the efficacy of the fixation. Endoscopic suture fixation after submucosal injection resulted in statistically significant increased pullout force compared to clip fixation (n = 5; mean force ratio 462 %; 95 % confidence interval [CI] 281-643 %; p < 0.01). Endoscopic suturing fixation without submucosal injection also resulted in statistically significant increased pullout force (n = 5; mean force ratio 765 %; 95 % CI 258-632 %; p < 0.01). Fixation with clips did not result in significantly increased pullout force compared to no fixation (n = 5; mean force ratio 108 %; 95 % CI 56-159 %; p < 0.01). Submucosal injection also eliminated full thickness suture penetration (0/10 submucosal injection; 7/10 no injection). Endoscopic suture fixation of SEMS resulted in a statistically significant increase in pullout force necessary to displace the stents. Submucosal injection prior to suture fixation preserves the increased pullout force while minimizing the risk of transmural penetration with the potential for less risk of injury to mediastinal structures.

Abstract 221: PDGF-Mediated Activation of CREB in Vascular Smooth Muscle Cells Alters Cell Cycling via Rb, p27kip1 and Fox01

Introduction: Vascular injury initiates signals which lead to the secretion of humoral factors and ultimately to vessel repair. PDGF is one such factor that promotes conversion of smooth muscle cells (SMC) from their contractile state to the synthetic phenotype, which is characterized by the ability to migrate and proliferate. If not properly controlled, such changes can lead to neointimal hyperplasia or restenosis. As CREB has been shown to participate in vascular remodeling, it is plausible that it is an important player in PDGF-mediated phenotypic switch. Hypothesis and aim: We assessed the hypothesis that a relationship exists between CREB and PDGF-dependent proliferation of coronary artery SMCs. We also aimed to identify the pathways both upstream and downstream of CREB activation. Methods: Vascular SMCs from porcine explants and Western blotting were used to monitor protein levels and changes in phosphorylation resulting from various treatments. Multiple cell growth methods were applied to study cell proliferation. A dominant-negative CREB mutant and constitutive expression of p27kip1 were used to assess the involvement of these proteins in mediating the actions of PDGF. Results: Phosphorylation of CREB by PDGF was dependent on both Src and PI3 kinase, and partially dependent on MAPK. Dominant-negative CREB decreased PDGF-dependent cell proliferation by approximately 75%, and both PCNA expression and Ser-780 phosphorylation of Rb were inhibited. While no change in either cyclin D or cdk4 levels was observed, PCNA and Rb mediate the signals transduced through CREB. These cell cycle proteins likely controlled via p27kip1 expression in response to CREB-dependent Fox01 acetylation. Conclusion: CREB phosphorylation is required for SMC proliferation in response to PDGF. Additionally, p27kip1 mediates the actions of CREB in SMCs, likely as a result of changes in Fox01 activity.