Preclinical research performed on reanimated/perfused swine kidneys: The Visible Kidney™ methodologies.

Preclinical research remains the essential platform in the development and optimization of medical therapies and advancements in translational medicines. However, specifically to animal research, federal laws, and institutional policies require investigators to apply the principles of the 3R's (replacement, reduction, and refinement). The concept of benchtop models utilizing isolated organs, in which multiple variables can be controlled to recreate human function, has been innovative advancements in preclinical research models that adhere to these principles. More specifically, isolated perfused kidney (IPK) models have been invaluable preclinical tools that have led to numerous advancements over the decades, including understanding renal physiology, pharmacologic therapies, and improvements in renal transplantation. However, pre-existing IPK models are not without their own limitations, leaving areas for improvement. An isolated perfused kidney apparatus was designed to best recreate human use conditions as a preclinical tool. Porcine renal blocks were chosen over the more commonly used rodent models, due to their greater similarities to human anatomies. Sixteen porcine kidney pairs obtained en bloc were extracted and placed onto an apparatus where aortic flows, pressures, and overall systemic temperatures were controlled. Organ viability was assessed in 10 renal blocks (n = 8 fresh and n = 2 previously frozen specimens) via both urinary flows and compositions at timepoints up to 180 min. Multimodality imaging, which included fluoroscopy, ultrasound, optical coherence tomography (OCT), and video scopes, was also employed to capture internal and external images to determine renal artery orientations and dimensions. Anatomical measurements and viability assessments of porcine renal blocks were successfully achieved in our perfusion model. Renal main artery diameters averaged smaller in our sample size than in human anatomy while also having more superior takeoff angles. Yet, the average lengths of each main segment were comparable to human anatomy: 32.09 ± 7.97 mm and 42.23 ± 7.33 mm in the left and right renal main artery, respectively. Urine production and urine composition of the fresh renal blocks, when compared to the frozen blocks and baseline perfusate, showed kidney viabilities of up to 3 h via excretion and retention of various metabolites. In this paper, we described a protocol for an isolated perfused kidney apparatus using large mammalian renal blocks. We believe this protocol to be an improvement from similar pre-existing models in better representing human physiologic function while allowing for multimodal imaging. The resulting Visible Kidney™ preclinical model, which has shown viability after isolation and reperfusion, can be a fast and reliable tool for the development of medical devices while also reducing the unnecessary use of animals for research.

Translational value of preclinical models for renal denervation: a histological comparison of human versus porcine renal nerve anatomy.

BACKGROUND: Preclinical models have provided key insights into the response of local tissues to radiofrequency (RF) renal denervation (RDN) that is unobtainable from human studies. However, the anatomic translatability of these models to the procedure in humans is incompletely understood.  Aims: We aimed to compare the renal arterial anatomy in normotensive pigs treated with RF-RDN to that of human cadavers to evaluate the suitability of normotensive pigs for determining the safety of RF-RDN. METHODS: Histopathologic analyses were performed on RF-treated renal arteries in a porcine model and untreated control renal arteries. Similar analyses were performed on untreated renal arteries from human cadavers.  Results: In both human and porcine renal arteries, the median number of nerves was lower in the more distal sections (the numbers in the proximal, middle, distal, 1st bifurcation, and 2nd bifurcation sections were 65, 58, 47, 22.5, and 14.7 in humans, respectively, and 39, 26, 29, 16.5, and 9.3 in the porcine models, respectively). Renal nerves were common in the regions between arteries and adjacent veins, but only 3% and 13% of the renal nerves in humans and pigs, respectively, were located behind the renal vein. The semiquantitative score of RF-induced renal arterial nerve necrosis was significantly greater at 7 days than 28 days (0.98 vs 0.75; p=0.01), and injury to surrounding organs was rarely observed. CONCLUSIONS: The distribution of nerve tissue and the relative distribution of extravascular anatomic structures along the renal artery was similar between humans and pigs, which validates the translational value of the normotensive porcine model for RDN.

Histological evidence supporting the durability of successful radiofrequency renal denervation in a normotensive porcine model.

BACKGROUND: Sustained blood pressure reductions after radiofrequency (RF) renal denervation (RDN) have been reported to 3 years in patients with uncontrolled hypertension. However, mechanistic data to support procedural durability are lacking. We aimed to quantify the long-term nerve anatomic and functional effects of RF RDN in a preclinical model. METHODS: Bilateral RF RDN was performed in 20 normotensive swine. Renal tissue samples were obtained in the RDN-treated groups at 7 ( n  = 6), 28 ( n  = 6), and 180 days ( n  = 8) postprocedure for quantification of cortical norepinephrine (NE) levels and renal cortical axon density. Tissue fibrosis, necrosis and downstream nerve fiber atrophy (axonal loss) were also scored for each sample. Three additional untreated groups ( n  = 6, n  = 6 and n  = 8, respectively) served as control. RESULTS: Pathologic nerve changes were characterized by necrosis in the ablated region at 7 days that partially resolved by 28 days and fully resolved at 180 days. Axonal loss was apparent within and downstream to the ablation regions and was evident at 7, 28 and 180 days in the main vessel and branch vessels. Consequently, renal cortical axon density and corresponding cortical NE levels were significantly reduced at 7 days in the RDN vs. control group and remained suppressed at 180 days. CONCLUSIONS: Reductions in renal NE, cortical axon density and downstream axonal loss caused by axonal destruction persisted through 180 days post-RDN in a normotensive swine model. These results suggest functional nerve regrowth after RF RDN is unlikely and support published clinical evidence that the procedure results in durable blood pressure reduction.

Scientific and Regulatory Policy Committee Points to Consider for Medical Device Implant Site Evaluation in Nonclinical Studies.

Nonclinical implantation studies are a common and often critical step for medical device safety assessment in the bench-to-market pathway. Nonclinical implanted medical devices or drug-device combination products require complex macroscopic and microscopic pathology evaluations due to the physical presence of the device itself and unique tissue responses to device materials. The Medical Device Implant Site Evaluation working group of the Society of Toxicologic Pathology's (STP) Scientific and Regulatory Policy Committee (SRPC) was tasked with reviewing scientific, technical, and regulatory considerations for these studies. Implant site evaluations require highly specialized methods and analytical schemes that should be designed on a case-by-case basis to address specific study objectives. Existing STP best practice recommendations can serve as a framework when performing nonclinical studies under Good Laboratory Practices and help mitigate limitations in standards and guidances for implant evaluations (e.g., those from the International Organization for Standardization [ISO], ASTM International). This article integrates standards referenced by sponsors and regulatory bodies with practical pathology evaluation methods for implantable medical devices and combination products. The goal is to ensure the maximum accuracy and scientific relevance of pathology data acquired during a medical device or combination drug-device implantation study.

Time, Temperature, Power, and Impedance Considerations for Radiofrequency Catheter Renal Denervation.

Radio frequency (RF) based percutaneous catheter renal denervation systems offer an additional clinical tool, along with lifestyle modification and drug therapy, to address the global epidemic of uncontrolled hypertension. The most widely applied RF system has been designed to optimize both procedural and safety and efficacy. Lesion size, shape, and depth result from a complex interaction of device design, anatomy, and tissue electrical conduction properties. Power control algorithms must be carefully designed, incorporating feedback to maximize nerve destruction while minimizing collateral damage. Physical and numerical modelling as well as analysis of sensor feedback provide insight into design performance that cannot be derived from clinical trials. This review is focused on key design and performance aspects of the most widely applied renal denervation system meant to optimize safety and efficacy of the procedure.

Downstream Paclitaxel Released Following Drug-Coated Balloon Inflation and Distal Limb Wound Healing in Swine.

The authors evaluated the presence of paclitaxel and healing of distal hind limb wounds created in 27 swine using biopsy punches followed by paclitaxel-coated balloon (PCB) use in the iliofemoral arteries of healthy swine. After 14 and 28 days, no differences were seen in time course, appearance, and histopathology of wound healing between the single or triple PCB and uncoated balloon treatment despite clinically relevant paclitaxel concentrations in the skin adjacent to the healing wounds. Presence of paclitaxel downstream from the PCB treatment site does not impair the wound healing response of preexisting distal cutaneous lesions in healthy swine.

Thromboresistance and endothelial healing in polymer-coated versus polymer-free drug-eluting stents: Implications for short-term dual anti-platelet therapy.

BACKGROUND: Short-term dual antiplatelet therapy (DAPT) is a suitable strategy after stent implantation especially in patients at high risk for bleeding. The thromboresistant characteristics and the healing profile permanent polymer stents such as the Resolute Onyx- drug-eluting stent (DES) has never been tested against the current approved stents for short-term DAPT, the polymer free (PF) biolimus-eluting stent (PF-BES) and bare metal stents (BMS) in dedicated preclinical models. METHODS: An ex-vivo porcine arteriovenous shunt and in-vivo flow loop model were used to evaluate thromboresistance. The healing profile was assessed in the rabbit model at 28 days by confocal microscopy (CM), scanning electron microscopy (SEM) and histology. Onyx-DES was separately compared with Onyx-BMS in first experiment and PF-BES in second experiment. RESULTS: In an ex-vivo shunt model, CM and SEM showed significantly less platelet adhesion for Onyx-DES relative to Onyx-BMS and PF-BES. In a flow loop model using human blood, platelet adhesion was also significantly less in Onyx-DES as compared to PF-BES and Onyx-BMS. In the healing study, Onyx-BMS showed significantly greater healing profile relative to Onyx-DES as expected, whereas Onyx-DES showed equivalent endothelial coverage by SEM and significantly less Evan's blue uptake and comparable colocalization of p120 and vascular endothelial-cadherin when compared with PF-BES. CONCLUSIONS: Onyx-DES showed qualities of thomboresistance and healing which appear to be compatible with short-term DAPT. Thromboresistance was superior to PF-BES and healing was equivalent to PF-BES in this pre-clinical study. Onyx-DES might provide advantages when considering short-term DAPT especially in patients at high risk of bleeding.

Rate of Drug Coating Dissolution Determines In-Tissue Drug Retention and Durability of Biological Efficacy.

Two different drug-coated balloons (DCBs) possessing different coating formulations were compared for rate of coating dissolution in vitro, in addition to tissue drug concentration and histological responses of treated vascular tissue in vivo, to determine if the rate of drug bioavailability to vascular tissue can impact the degree and duration of the observed pharmacological response to locally delivered drug. In vitro dissolution comparison demonstrated that a urea/paclitaxel-based coating formulation (IN.PACT™ Admiral™) released drug from solid to soluble phase at a slower and constant rate, yielding approximately 7% solubilized drug in 24 h. In contrast, a coating formulated from polysorbate/sorbitol/paclitaxel (Lutonix™) released 51% of solid phase drug to soluble phase in 1 h of dissolution with the remainder solubilizing in 24 h. In vivo evaluation of tissue drug concentration of both products showed significantly different tissue pharmacokinetic profile, with a higher concentration of paclitaxel in tissue at 90 days with a urea-based formulation excipient. Histological comparison of smooth muscle cell loss in response to drug exposure revealed contrasting trends of smooth muscle cell loss from 28 to 90 days with significantly higher response to drug observed at 90 days with the urea-based formulation. Rapid dissolution of drug from the polysorbate/sorbitol coating formulation was associated with an early increase in local cellular response to drug which diminished over 90 days with clearance of local drug from tissue. Sustained long-term drug-in-tissue concentration associated with the urea-based formulation demonstrated sustained pharmacological activity at 90 days, suggesting that slow coating dissolution provides a sustainable long-term tissue response.

Historical Incidence of Spontaneous Lesions in Kidneys from Naïve Swine Utilized In Interventional Renal Denervation Studies.

The use of preclinical animal models is integral to the safety assessment, pathogenesis research, and testing of diagnostic technologies and therapeutic interventions. With inherent similarity to human anatomy and physiology, various porcine models have been the preferred preclinical model in some research areas such as medical devices, wound healing, and skin therapies. The porcine model has been the cornerstone for interventional cardiology for the evaluation and development of this catheter-based renal denervation (RDN) therapy. The porcine model provides similar vascular access and renal neurovascular anatomy to humans. In these preclinical studies, the downstream kidneys from treated arteries are assessed for possible histopathological changes in the vessel dependent territories. In assessing renal safety following RDN, it becomes critical to distinguish treatment-related changes from pre-existing background pathologies. The incidence of background pathological changes in porcine kidneys has not been previously established in normal clinically healthy. Samples from the cranial, middle, and caudal portion of 331 naïve kidneys from 181 swine were processed histologically to slides and evaluated microscopically. The most commonly encountered spontaneous changes were chronic pyelonephritis found in nearly half of the evaluated naïve kidneys (∼40 %; score 1 = 91 %, score 2 = 8.4 %, score 3 = 0.76 %) followed by chronic interstitial inflammation in 9.7 % of the kidneys (score 1 = 90.6 %, score 2 = 9.4 %). Interestingly, there were a few rare spontaneous vascular changes that could potentially affect data interpretation in interventional and toxicology studies: arteritis and arteriolar dissection. The presence of pelvic cysts was a common occurrence (6.3 %) in the kidney. The domestic swine is a widely used preclinical species in interventional research, namely in the emerging field of transcatheter renal denervation. This retrospective study presents the historical incidence of spontaneous lesions recorded in the kidneys from naive pigs enrolled in renal denervation studies. There were commonly encountered changes of little pathological consequence such as pyelonephritis or pelvic cysts and rare vascular changes such as arteritis and arteriolar dissection that were of greater potential impact on study data interpretation. These results offer a benchmark by which to gage the potential effect of a procedure or treatment on renal histopathology in swine and assist in data interpretation.

Impact of Lesion Placement on Efficacy and Safety of Catheter-Based Radiofrequency Renal Denervation.

BACKGROUND: Insufficient procedural efficacy has been proposed to explain nonresponse to renal denervation (RDN). OBJECTIVES: The aim of this study was to examine the impact of different patterns of lesion placements on the efficacy and consistency of catheter-based radiofrequency RDN in pigs. METHODS: The impact of increasing number of lesions versus location of RDN was investigated in a porcine model (Group 1; n = 51). The effect of treating the main artery, the branches, and the 2 combined was compared in Group 2 (n = 48). The durability of response and safety of combined treatment of the main artery plus branches was examined in Group 3 (n = 16). Renal norepinephrine (NE) tissue content and renal cortical axon density were assessed. RESULTS: Increasing the number of RF lesions (4, 8, and 12) in the main renal artery was not sufficient to yield a clear dose-response relationship on NE content and axon density. In contrast, targeted treatment of the renal artery branches or distal segment of the main renal artery resulted in markedly less variability of response and significantly greater reduction of both NE and axon density than conventional treatment of only the main renal artery. Combination treatment (main artery plus branches) produced the greatest change in renal NE and axon density with the least heterogeneity. The changes were durable through 28 days post-treatment. CONCLUSIONS: These data provide the rationale for investigation of an optimized approach for RDN in future clinical studies. This may have profound implications for the clinical application of RDN, as this approach may not only achieve greater reductions in sympathetic activity but also reduce treatment effect variability.

Comparison of histopathologic analysis following renal sympathetic denervation over multiple time points.

BACKGROUND: The pathology of radiofrequency-derived sympathetic renal denervation has not been studied over time and may provide important understanding of the mechanisms resulting in sustained blood pressure reduction. The purpose of this study was to investigate chronological changes after radiofrequency-renal denervation in the swine model. METHODS AND RESULTS: A total of 49 renal arteries from 28 animals with 4 different time points (7, 30, 60, and 180 days) were examined. Semiquantitative histological assessment of arteries and associated tissue was performed to characterize the chronological progression of the radiofrequency lesions. Arterial medial circumferential injury (%) was greatest at 7 days (38±13%), followed by 30 days (31±6%) and 60 days (31±15%), and least at 180 days (21±12%) (P=0.046). Nerve injury score was significantly greater (P<0.001) at 7 days (3.9±0.4) compared with 30 days (2.5±0.5), 60 days (2.6±0.5), and 180 days (1.9±0.9). Tyrosine hydroxylase score, which assesses functional nerve damage, was significantly less after 7 (1±1) and 30 days (0.7±0.6) compared with 60 (2.7±0.6) and 180 days (2.7±0.6; P=0.01). Focal nerve regeneration at the sites of radiofrequency ablation was observed in 17% of renal arteries at 60 days and 71% of 180 days. CONCLUSIONS: Nerve injury after radiofrequency ablation was greatest at 7 days, with maximum functional nerve damage sustained ≤30 days. Focal terminal nerve regeneration was observed only at the sites of ablation as early as 60 days and continued to 180 days. Renal artery and peri-arterial soft tissue injury is greatest in the subacute phase, and least in the chronic phase, suggesting gradual recovery of the renal arterial wall and surrounding tissue.

Efficacy and safety of catheter-based radiofrequency renal denervation in stented renal arteries.

BACKGROUND: In selected patients with hypertension, renal artery (RA) stenting is used to treat significant atherosclerotic stenoses. However, blood pressure often remains uncontrolled after the procedure. Although catheter-based renal denervation (RDN) can reduce blood pressure in certain patients with resistant hypertension, there are no data on the feasibility and safety of RDN in stented RA. METHODS AND RESULTS: We report marked blood pressure reduction after RDN in a patient with resistant hypertension who underwent previous stenting. Subsequently, radiofrequency ablation was investigated within the stented segment of porcine RA, distal to the stented segment, and in nonstented RA and compared with stent only and untreated controls. There were neither observations of thrombus nor gross or histological changes in the kidneys. After radiofrequency ablation of the nonstented RA, sympathetic nerves innervating the kidney were significantly reduced, as indicated by significant decreases in sympathetic terminal axons and reduction of norepinephrine in renal tissue. Similar denervation efficacy was found when RDN was performed distal to a renal stent. In contrast, when radiofrequency ablation was performed within the stented segment of the RA, significant sympathetic nerve ablation was not seen. Histological observation showed favorable healing in all arteries. CONCLUSIONS: Radiofrequency ablation of previously stented RA demonstrated that RDN provides equally safe experimental procedural outcomes in a porcine model whether the radiofrequency treatment is delivered within, adjacent, or without the stent struts being present in the RA. However, efficacious RDN is only achieved when radiofrequency ablation is delivered to the nonstented RA segment distal to the stent.

Lymphoadenopathy during lyme borreliosis is caused by spirochete migration-induced specific B cell activation.

Lymphadenopathy is a hallmark of acute infection with Borrelia burgdorferi, a tick-borne spirochete and causative agent of Lyme borreliosis, but the underlying causes and the functional consequences of this lymph node enlargement have not been revealed. The present study demonstrates that extracellular, live spirochetes accumulate in the cortical areas of lymph nodes following infection of mice with either host-adapted, or tick-borne B. burgdorferi and that they, but not inactivated spirochetes, drive the lymphadenopathy. The ensuing lymph node response is characterized by strong, rapid extrafollicular B cell proliferation and differentiation to plasma cells, as assessed by immunohistochemistry, flow cytometry and ELISPOT analysis, while germinal center reactions were not consistently observed. The extrafollicular nature of this B cell response and its strongly IgM-skewed isotype profile bear the hallmarks of a T-independent response. The induced B cell response does appear, however, to be largely antigen-specific. Use of a cocktail of recombinant, in vivo-expressed B. burgdorferi-antigens revealed the robust induction of borrelia-specific antibody-secreting cells by ELISPOT. Furthermore, nearly a quarter of hybridomas generated from regional lymph nodes during acute infection showed reactivity against a small number of recombinant Borrelia-antigens. Finally, neither the quality nor the magnitude of the B cell responses was altered in mice lacking the Toll-like receptor adaptor molecule MyD88. Together, these findings suggest a novel evasion strategy for B. burgdorferi: subversion of the quality of a strongly induced, potentially protective borrelia-specific antibody response via B. burdorferi's accumulation in lymph nodes.

Antibody-mediated disease remission in the mouse model of lyme borreliosis.

In the mouse model of Lyme borreliosis, the host immune response during infection with Borrelia burgdorferi results in the remission of carditis and arthritis, as well as global reduction of spirochete numbers in tissues, without elimination of infection. These events were recapitulated by passive transfer of immune serum from infected immunocompetent mice or T-cell-deficient mice to severe combined immunodeficient (SCID) mice. Previous studies have shown that immune serum is reactive against arthritis-related protein (Arp) and that Arp antiserum induces arthritis remission. However, although immune serum from T-cell-deficient mice induced disease remission, it was not reactive against Arp, suggesting that antibody to another antigen may be responsible. T-cell-deficient mouse immune serum was reactive to decorin binding protein A (DbpA). Therefore, DbpA antiserum was tested to determine its ability to induce disease remission in SCID mice. Antisera to Arp or DbpA induced both carditis and arthritis remission but did not significantly reduce spirochete numbers in tissues, based upon quantitative flaB DNA analysis, nor did treatment affect RNA levels of several genes, including arp and dbpA. Immunohistochemical labeling of spirochetes in hearts and joints during disease remission induced by adoptive transfer of lymphocytes, passive transfer of immune serum, or passive transfer of DbpA antiserum revealed that such treatment resulted in elimination of spirochetes from heart base and synovium but not vascular walls, tendons, or ligaments. These results suggest that Arp and DbpA antibodies may be active as disease-resolving components in immune serum but antibody against other antigens may be involved in reductions of spirochetes in tissues.

Immunoglobulin-regulated expression of Borrelia burgdorferi outer surface protein A in vivo.

Borrelia burgdorferi, the agent of Lyme disease, down-regulates outer surface protein A (OspA), which is abundantly expressed in ticks, during infection of the mammalian host. In this study we examined the signals that may be responsible for maintaining the OspA-negative state of spirochetes during infection. Transcription of ospA mRNA was found in tissues of C3H-severe combined immunodeficient (C3H-scid) mice, but not immunocompetent C3H mice, inoculated with cultured B. burgdorferi, tick-borne spirochetes, and host-adapted spirochetes. Transcription was more frequent at 4 weeks than at 1 week. Transcription was present at the host-tick interface as early as 24 h after tick attachment but declined at 48 and 72 h. Thus, ospA mRNA transcription in distant tissues and at later times in C3H-scid mice is probably due to up-regulation during infection. Adoptive lymphocyte transfer from naive C3H mice to infected C3H-scid mice resulted in OspA seroconversion, confirming OspA expression in the host. Passive transfer of normal mouse serum, immunoglobulin M (IgM) from normal mouse serum, or IgG from normal mouse serum into infected C3H-scid mice resulted in down-regulation of ospA, but transfer of normal mouse serum depleted of immunoglobulin did not influence ospA mRNA transcription. Collectively, our results indicate that ospA mRNA transcription in the host is regulated by nonspecific immunoglobulin, which may be a natural antibody.