Sacrifice Time Points Research Articles

Differential Immunogenicity and Lung Disease-Inducing Potential of Mycobacterium immunogenum Genotypes and Impact of Co-Exposure with Pseudomonas: Optimizing a Mouse Model of Chronic Hypersensitivity Pneumonitis.

Mycobacterium immunogenum (MI) colonizing metalworking fluids (MWFs) has been associated with chronic hypersensitivity pneumonitis (HP) in machinists. However, it is etiologically unclear why only certain mycobacteria-contaminated fluids induce this interstitial lung disease. We hypothesized that this may be due to differential immunogenicity and the HP-inducing potential of MI strains/genotypes as well as the confounding effect of co-inhaled endotoxin-producers. To test this hypothesis, we optimized a chronic HP mouse model in terms of MI antigen dose, timepoint of sacrifice, and form of antigen (cell lysates vs. live cells) and compared six different field-isolated MI strains. Overall, MJY10 was identified as the most immunogenic and MJY4 (or MJY13) as the least immunogenic genotype based on lung pathoimmunological changes as well as Th1 cellular response (IFN-γ release). Infection with MI live cells induced a more severe phenotype than MI cell lysate. Co-exposure with Pseudomonas fluorescens caused a greater degree of lung innate immune response and granuloma formation but a diminished adaptive (Th1) immune response (IFN-γ) in the lung and spleen. In summary, this study led to the first demonstration of differential immunogenicity and the disease-inducing potential of field strains of MI and an interfering effect of the co-contaminating Pseudomonas. The improved chronic MI-HP mouse model and the identified polar pair of MI strains will facilitate future diagnostic and therapeutic research on this poorly understood environmental lung disease.

Toll-Like Receptor 4 Induction During Renal Ischemia Correlates with Serum Creatinine in a Pig Model.

Objectives: To determine whether toll-like receptor 4 (TLR4), a mediator of organ ischemia-reperfusion injury, is overexpressed during warm ischemia in a porcine solitary kidney model, and whether its expression correlates with creatinine, a surrogate for kidney function. Materials and Methods: Eight adult Yorkshire pigs underwent initial laparoscopic nephrectomy. After 1 week, animals were randomized into two groups: group 1 underwent laparoscopic renal hilar dissection, renal ischemia by cross-clamping, and reperfusion (ischemia group); group 2 underwent laparoscopic renal hilar dissection alone (sham group). Animals were survived to day 7 postrandomization. Peripheral blood was sampled for serum creatinine (sCr) and TLR4 expression at the following time points or corresponding intervals: prenephrectomy, 1-week postnephrectomy (preischemia), after 90 minutes of ischemia, 30 minutes postreperfusion, and at sacrifice. Intragroup TLR4 expression changes were analyzed using repeated measures ANOVA. Intergroup TLR4 expression was compared using Mann-Whitney's test. Correlation between sCr and TLR4 was assessed using Spearman's test. Results: Seven animals completed the experiment (four ischemia and three sham). Relative TLR4 expression significantly increased from baseline levels during ischemia, reperfusion, and sacrifice time points only in the ischemia group, and was significantly higher for the ischemia group after 90 minutes of ischemia (p = 0.034). sCr was significantly higher for the ischemia group during the reperfusion phase (p = 0.048). Relative TLR4 expression level significantly correlated with sCr in the overall cohort (Spearman's rho = 0.69) and in the ischemia group (Spearman's rho = 0.82; p < 0.0001 for each). Conclusions: Warm ischemia in a porcine solitary kidney induces acute overexpression of TLR4 in peripheral blood leukocytes, which is detectable. Relative TLR4 expression level strongly correlated with sCr but had an observable change sooner than change in sCr. Pending further investigation, TLR4 overexpression during renal ischemia may represent a sensitive quantitative marker of unilateral renal injury sustained during nephron-sparing surgery.

Intramuscular mitochondrial and lipid metabolic changes of rats after regular high-intensity interval training (HIIT) of different training periods.

High-intensity Interval Training (HIIT) is a time-efficient form of exercise and has gained popularity in recent years. However, at molecular level, the understanding about the effects of HIIT is not comprehensive, and even less is elucidated about HIIT of different training duration cycles, although different durations always lead to different post-training consequences. In this study, by training SD rats using HIIT protocols lasting for different training duration cycles, we investigated the adaptive response of intramuscular triglyceride abundance as well as mitochondrial and lipid metabolic changes after HIIT training (2, 4, 6, 8, and 10weeks). We selected 72h after the last session of training as the time point of sacrifice. The suppressed activation of the cAMP-PKA pathway indicates that skeletal muscle was in the recovery phase at this time point. Intramuscular triglyceride abundance was significantly elevated after 2, 4, and 10weeks of HIIT. However, the lipid metabolism-related proteins inconsistently changed in a chaotic trend (see Table 1). The expression levels of PGC1-α and COX IV decreased after 2 and 4weeks of training and raised after 6 and 8weeks of training. The expression level of citrate synthase (CS) decreased after 2, 4, 8, and 10weeks of training, and showed an upward trend after 6weeks of training. While the activity of CS decreased after 2 and 8weeks of training and showed an upward trend after 6weeks of HIIT. Given the abovementioned changing trends, we propose two speculations: (A) the damaged mitochondria oxidation capacity might be one of the causes of IMTG accumulation observed after 2 and 4weeks of HIIT. This phase might be similar to the condition of type 2 diabetes. (B) after 6-week HIIT, mitochondria function and biogenesis might be improved and the IMTG contents declined to baseline. This might be explained as: mitochondrial enhancement increased the capacity of lipid oxidation and then offset the increase in IMTG achieved during the first 4weeks. For HIIT Rat Modelling, if the aim is to observe HIIT-induced positive effects, caution should be exercised when considering 2 and 4weeks of training under our HIIT frame. Also, implementing six-week training is at least effective for mitochondrial enhancement when using similar HIIT frame of this study.

Does Sex Matter in Liver Surgery? Comparison of Severity Assessments between Female and Male Rats after Partial Hepatectomy: A Pilot Study

Introduction: Current animal-based biomedical research, including studies on liver function and disease, is conducted almost exclusively on male animals to mitigate confounding effects of the estrous cycle. However, liver diseases afflict both men and women, so translational research findings should also be applicable to female patients. This pilot study investigated sex differences in objective and subjective severity assessment parameters in rats following 50% partial hepatectomy. Materials and Methods: This study was performed using Wistar Han rats, in which measurements of body weight, spontaneous motor activity in the open field (OF) (movement distance, movement velocity, rearing frequency), and fecal corticosterone metabolites were conducted at baseline and at multiple times after partial hepatectomy. Subjective postsurgical severity assessments were conducted using modified score sheets. Blood parameters such as leukocyte count and serum aspartate aminotransferase, as well as estrogens and testosterone were measured from samples obtained during partial hepatectomy and at sacrifice. In addition, the amount of resected liver tissue was measured at partial hepatectomy, and the proliferated liver was weighed at sacrifice. Results: Fecal corticosterone metabolite concentrations differed significantly between males and females at baseline and following hepatectomy. Also, leukocyte counts and estrogen concentrations were significantly different between sexes before partial hepatectomy. Alternatively, there were no sex differences in severity assessments, body weight changes, and behavior in the OF at any measurement time point. Liver weight was significantly different in males and females at the time point of partial hepatectomy and sacrifice. Conclusion: The results of this pilot study suggest that males and females respond similarly following partial hepatectomy. Examination of both sexes is very important for translation to humans, where both men and women suffer from liver disease. Furthermore, the use of both sexes in animal-based research would improve the utilization of the animal breeding in terms of the 3 Rs. However, due to some limitations, larger scale investigations including a broader spectrum of pathophysiolological, behavioral, and pharmacokinetic measures are planned.

Biocompatibility and feasibility of VisiPlate, a novel ultrathin, multichannel glaucoma drainage device

BackgroundGlaucoma is the leading cause of blindness worldwide. Glaucoma drainage devices and minimally invasive glaucoma surgeries (MIGS) often present with tradeoffs in safety and durability of efficacy. Using a rabbit model, we examined the biocompatibility and feasibility of VisiPlate, a novel, ultrathin, tubeless subconjunctival shunt comprised of a network of microchannels.MethodsSix naive female New Zealand White rabbits received implants (three only in the right eye with contralateral eye untreated and three in both eyes) composed of a 400-nm-thick aluminum oxide core coated with 2 µm of parylene-C, manufactured with microelectromechanical systems (MEMS) techniques. Tonometry, slit lamp exam, clinical exam, fluorescein patency testing, and histopathology were performed.ResultsVisiPlate demonstrated IOP-lowering of 20–40% compared to baseline at each time point over the course of 3 months in the nine implanted eyes. All eyes developed blebs over the implant, and fluorescein testing demonstrated fluid patency at 22 days post-implantation. Slit lamp and clinical observations showed that VisiPlate was well tolerated, with low levels of conjunctival congestion, conjunctival swelling, aqueous flare, hyphema, and iris involvement from surgery that resolved over time. At sacrifice time points of 93 days and 180 days, the only notable observations were mild levels of conjunctival congestion in implanted eyes. Histopathology showed minimal tissue response and no obvious inflammation, fibrosis, or necrosis around the implant.ConclusionsThe results of this in vivo study demonstrate the biocompatibility and IOP-lowering effect of a multichannel, ultrathin subconjunctival shunt in a rabbit model. The data suggest that VisiPlate may safely enhance aqueous outflow and significantly reduce intraocular pressure.

Mechanical Overload Followed by Consecutive Collagenase Injections: Developing a Multifactorial and Long-Lasting Animal Model of Induced Achilles Tendinopathy

Category:Basic Sciences/Biologics; Hindfoot; SportsIntroduction/Purpose:Different animal models of Achilles tendinopathy have been proposed in the literature. They usually involve the induction of tendinopathic findings by either chemical stress (most commonly with one or more injections of collagenase, mimicking intrinsic factors) or mechanical stress (by repetitive exercise-induced stress with treadmill running exercises, simulating extrinsic risk factors). To date, no study has evaluated the combination of a mechanical trigger followed by collagenase injections, replicating the logical and sequential steps involved in the development the human pathology. Our goal was to develop this novel animal model of Achilles tendinopathy and to compare histological and functional findings with animals subjected to isolated mechanical or chemical stress, as well as to controls.Methods:Sixty-four Sprague-Dawley rats were divided into four groups (n=16): isolated treadmill running protocol (15o uphill running, 20meters/minute, 1hour/day, 3 weeks duration, weeks 2-4); isolated injections of collagenase (0.1mg each, 3 injections total, weeks 5-7); treadmill protocol (weeks 2-4) followed by three consecutive collagenase injections (weeks 5-7); and controls, no running and three injections of normal saline (weeks 5-7). Five animals from each group were sacrificed at weeks 8 and 10. Six animals by group were sacrificed at week 12. Gait analysis was performed at weeks one (after acclimation), five (following running protocol), eight (following injection protocol) and twelve (just before latest sacrifice time-point). Histological findings were assessed by the Movin Tendinopathy Score (eight parameters, scored from 0-3, total score 0-24), assessing collagen arrangement, structure, and stainability, cellularity, vascularity, nuclear rounding, hyalinization and presence of glycosaminoglycans. Gait parameters included stand and swing times, stride length, duty cycle and swing length.Results:After 8 weeks, significantly increased tendinopathic scores (p<0.001) were found in animals subjected to collagenase injections (16, CI 13.1-18.9) and to running/collagenase (17.4, CI 14.4-20.3), when compared to controls (1.6, CI -1.3-4.50) and running (3, CI 0.1-5.9). Similarly, after 10 weeks significantly increased scores were found in the same groups, with slight severity regression: controls (1, CI -0.8-2.8), running (2.2, CI 0.4-4.0), collagenase (10, CI 8.2-11.8) and running/collagenase (17.6, CI 15.8- 19.4). After 12 weeks, collagenase group demonstrated reversion of the findings (3.3, CI 1.6-5.1), and wasn’t different than control (2.1, CI 0.4-3.9) and running groups (2.5, CI 0.3-4.7). However, significantly increased pathological findings were noted in the running/collagenase group (20.0, CI 18.2-21.8) consistent with chronic tendinopathic process. Gait analysis results presented in Figure1.Conclusion:When compared to other models of induced Achilles tendinopathy and to controls, the novel animal model induced by a mechanical trigger and sustained by chemical stress demonstrated progressively increased histological tendinopathic scores after 12 weeks. Findings observed after isolated mechanical or chemical stresses were temporary, not maintained at latest follow- up. Steps involved in tendinopathy development, as well as the observed histological results of the combined running/collagenase model, replicate better the findings of human chronic Achilles tendinopathy. Applications for this novel model are promising, potentially supporting a better understanding of early/late findings as well as treatment options for Achilles tendinopathy.Figure 1.

Characterizing Vocal Fold Injury Recovery in a Rabbit Model With Three-Dimensional Virtual Histology.

In animal studies of vocal fold scarring and treatment, imaging-based evaluation is most often conducted by tissue slicing and histological staining. Given variation in anatomy, injury type, severity, and sacrifice timepoints, planar histological sections provide limited spatiotemporal details of tissue repair. Three-dimensional (3D) virtual histology may provide additional contextual spatial information, enhancing objective interpretation. The study's aim was to evaluate the suitability of magnetic resonance imaging (MRI), microscale computed tomography (CT), and nonlinear laser-scanning microscopy (NM) as virtual histology approaches for rabbit studies of vocal fold scarring. A unilateral injury was created using microcup forceps in the left vocal fold of three New Zealand White rabbits. Animals were sacrificed at 3, 10, and 39 days postinjury. ex vivo imaging of excised larynges was performed with MRI, CT, and NM modalities. The MRI modality allowed visualization of injury location and morphological internal features with 100-μm spatial resolution. The CT modality provided a view of the injury defect surface with 12-μm spatial resolution. The NM modality with optical clearing resolved second-harmonic generation signal of collagen fibers and two-photon autofluorescence in vocal fold lamina propria, muscle, and surrounding cartilage structures at submicrometer spatial scales. Features of vocal fold injury and wound healing were observed with MRI, CT, and NM. The MRI and CT modalities provided contextual spatial information and dissection guidance, whereas NM resolved extracellular matrix structure. The results serve as a proof of concept to motivate incorporation of 3D virtual histology techniques in future vocal fold injury animal studies. NA Laryngoscope, 131:1578-1587, 2021.

Exploring the Potential of Mesenchymal Stem Cell-Based Therapy in Mouse Models of Vascular Cognitive Impairment.

Closely linked to Alzheimer’s disease (AD), the pathological spectrum of vascular cognitive impairment (VCI) is known to be wide and complex. Considering that multiple instead of a single targeting approach is considered a treatment option for such complicated diseases, the multifaceted aspects of mesenchymal stem cells (MSCs) make them a suitable candidate to tackle the heterogeneity of VCI. MSCs were delivered via the intracerebroventricular (ICV) route in mice that were subjected to VCI by carotid artery stenosis. VCI was induced in C57BL6/J mice wild type (C57VCI) mice by applying a combination of ameroid constrictors and microcoils, while ameroid constrictors alone were bilaterally applied to 5xFAD (transgenic AD mouse model) mice (5xVCI). Compared to the controls (minimal essential medium (MEM)-injected C57VCI mice), changes in spatial working memory were not noted in the MSC-injected C57VCI mice, and unexpectedly, the mortality rate was higher. In contrast, compared to the MEM-injected 5xVCI mice, mortality was not observed, and the spatial working memory was also improved in MSC-injected 5xVCI mice. Disease progression of the VCI-induced mice seems to be affected by the method of carotid artery stenosis and due to this heterogeneity, various factors must be considered to maximize the therapeutic benefits exerted by MSCs. Factors, such as the optimal MSC injection time point, cell concentration, sacrifice time point, and immunogenicity of the transplanted cells, must all be adequately addressed so that MSCs can be appropriately and effectively used as a treatment option for VCI.

Comparative analysis of the ovarian transcriptome reveals novel insights into fertility differences in Large White sows.

Fertility is the most important economic trait in sows, as it is critical for profitability. Considerable phenotypic variation in litter size exists in Large White sows. However, relatively little is known about the underlying molecular and genetic bases. An experiment was conducted to screen key genes that affect the fecundity of pigs during the luteal (L) and follicular phases (F) of the estrous cycle. Eight sows (n = 4 for high fertility sows and n = 4 for low fertility sows) were sacrificed on day 14 (day 1 = first day of estrus) after estrus in the L phase. Another eight sows were slaughtered on day 20 of the estrous cycle in the F phase. Sixteen ovarian tissue samples were collected at the different sacrifice time points. Total RNA extracted was used to construct the library and then sequence on an Illumina HiSeq X10 system. Differentially expressed genes (DEGs) between high and low fertility in Large White sows were identified, and their potential biological functions were analyzed using bioinformatics analysis. In total, 457 DEGs (161 up-regulated and 296 down-regulated genes) were detected in the ovarian tissues of the high and low fertility groups in the L phase of the estrous cycle. Furthermore, 475 DEGs (253 up-regulated and 222 down-regulated genes) were identified in the F phase. Twenty-nine DEGs were common to both comparisons. Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the DEGs were mainly associated with steroid biosynthesis, the Hippo signaling pathway, and lysosomes. Others, such as MSMO1, CYP27B1, and CTSB, were related to reproduction. These results will contribute to a better understanding of the individual differences in fertility at the transcriptome level, which may provide useful information to explore new ways to improve fertility in pigs.

Long-Term Evaluation of Gadolinium Retention in Rat Brain After Single Injection of a Clinically Relevant Dose of Gadolinium-Based Contrast Agents.

PurposeThe aim of this study was to investigate the presence and chemical forms of residual gadolinium (Gd) in rat brain after a single dose of Gd-based contrast agent.MethodsFour groups of healthy rats (2 sacrifice time-points, n = 10/group, 80 rats in total) were randomized to receive a single intravenous injection of 1 of the 3 Gd-based contrast agents (GBCAs) (gadoterate meglumine, gadobenate dimeglumine, or gadodiamide) or the same volume of 0.9% saline solution. The injected concentration was 0.6 mmol/kg, corresponding to a concentration of 0.1 mmol/kg in humans after body surface normalization between rats and humans (according to the US Food and Drug Administration recommendations). Animals were sacrificed at 2 washout times: 1 (M1) and 5 (M5) months after the injection. Total Gd concentrations were determined in cerebellum by inductively coupled plasma mass spectrometry. Gadolinium speciation was analyzed by size-exclusion chromatography coupled to inductively coupled plasma mass spectrometry after extraction from cerebellum.ResultsA single injection of a clinically relevant dose of GBCA resulted in the detectable presence of Gd in the cerebellum 1 and 5 months after injection. The cerebellar total Gd concentrations after administration of the least stable GBCA (gadodiamide) were significantly higher at both time-points (M1: 0.280 ± 0.060 nmol/g; M5: 0.193 ± 0.023 nmol/g) than those observed for macrocyclic gadoterate (M1: 0.019 ± 0.004 nmol/g, M5: 0.004 ± 0.002 nmol/g; P < 0.0001). Gadolinium concentrations after injection of gadobenate were significantly lower at both time-points (M1: 0.093 ± 0.020 nmol/g; M5: 0.067 ± 0.013 nmol/g; P < 0.05) than the Gd concentration measured after injection of gadodiamide. At the 5-month time-point, the Gd concentration in the gadoterate group was also significantly lower than the Gd concentration in the gadobenate group (P < 0.05). Gadolinium speciation analysis of the water-soluble fraction showed that, after injection of the macrocyclic gadoterate, Gd was still detected only in its intact, chelated form 5 months after injection. In contrast, after a single dose of linear GBCAs (gadobenate and gadodiamide), 2 different forms were detected: intact GBCA and Gd bound to soluble macromolecules (above 80 kDa). Elimination of the intact GBCA form was also observed between the first and fifth month, whereas the amount of Gd present in the macromolecular fraction remained constant 5 months after injection.ConclusionsA single injection of a clinically relevant dose of GBCA is sufficient to investigate long-term Gd retention in the cerebellar parenchyma. Administration of linear GBCAs (gadodiamide and gadobenate) resulted in higher residual Gd concentrations than administration of the macrocyclic gadoterate. Speciation analysis of the water-soluble fraction of cerebellum confirmed washout of intact GBCA over time. The quantity of Gd bound to macromolecules, observed only with linear GBCAs, remained constant 5 months after injection and is likely to represent a permanent deposition.

A novel UHPLC-HRMS-based metabolomics strategy enables the discovery of potential neuroactive metabolites in mice plasma, following i.p. administration of the main Crocus sativus L. bioactive component

Trans-crocin 4 (TC4) is an important carotenoid constituent of saffron showing potential activity against Alzheimer’s Disease (AD) due to its antioxidant and antiamyloidogenic properties. Metabolomics is an emerging scientific field that enhances biomarker discovery and reveals underlying biochemical mechanisms aiming towards the early subclinical diagnosis of diseases. To date, there are no reports on the changes induced to mice plasma metabolome after TC4 administration. We report a novel untargeted UHPLC-ESI HRMS metabolomics strategy to determine the alteration of the metabolic fingerprint following i.p. administration of TC4 in male and female mice. Blood samples from fiftysix mice treated with TC4 as well as from control animals were analyzed with UHPLC-ESI HRMS. Statistical evaluation of the results was achieved by multivariate analysis (MVA), i.e., principal component analysis (PCA), Partial Least Squares–Discriminant Analysis (PLS-DA) in order to discover the variables that contributed to the discrimination between treated and untreated groups which were identified by online database searching (e.g., Metlin, HMDB, KEGG) aided by chemometric processing, e.g., covariance searching etc. Due to the high variability imposed by various factors, e.g., sex of the animals participating in the study, administration dose and time-points of sacrifice, multilevel sparse PLS-DA analysis, e.g., splitting variation to each individual component, has been employed as a more efficient approach for such designs.This methodology allowed the identification of the time sequence of metabolome changes due to the administration of TC4, whereas a sex-related effect on the metabolome is indicated, denoting that the administration in both sexes is indispensable in order to acquire safe conclusions as reliable metabolome pictures. The results demonstrated a number of annotated metabolites playing a potential role in neuroprotection while they are closely related to AD. Moreover, five additional annotated metabolites were involved in the steroid biosynthesis pathway while two of them may be considered as putative neuroprotective agents.

Riboflavin/ultraviolet A-induced collagen cross-linking in rabbit corneal scar.

To evaluate the biomechanical stability of the corneal scar treating with riboflavin and ultraviolet A (UVA). Totally 86 New Zeal rabbits were divided into control group (group A, n=8) and trauma groups [group B (n=27), group C (n=24) and group D (n=27)]. Then groups B, C and D were divided into three sub-groups according to the time points of sacrifice, i.e. groups Ba, Ca and Da (4wk, n=8); Bb, Cb and Db (6wk, n=8); Bc (n=11), Cc (n=8) and Dc (8wk, n=11). The right corneas of these 78 rabbits in the trauma groups were penetrated. Group B were only sutured. Group C were treated with corneal cross-linking (CXL) immediately after suturing. Group D were treated with CXL seven days after suturing. The corneal scar strips of 4.0×10.0 mm2 were cut and the stress and Young's modulus at 10% strain were evaluated. Samples from the three rabbits of group Bc and three of group Dc were used to measure the expression of alpha smooth muscle action (α-SMA). The mechanical strength of the corneal scar increased with time, and was strongest at 8wk after the injury. The ultimate stress of corneal scar (group D) were 2.17±0.52 MPa, 2.92±0.63 MPa, and 4.21±0.68 Mpa at 4wk, 6wk and 8wk, respectively; Young's modulus were 10.94±1.57 MPa, 11.16±2.50 MPa, and 13.36±2.10 Mpa, which were higher than that of other groups except for normal control. The expression of α-SMA in group B and group D were 0.28±0.11 and 0.65±0.20, respectively, and the difference was statistically significant (P=0.048). CXL with riboflavin/UVA at seven days after suturing improved the biomechanical properties of corneal scars most effectively in the present study.

Wednesday, September 26, 2018 7:35 AM–9:00 AM ePosters

BACKGROUND CONTEXT Pseudarthrosis or failure-to-fuse occurs in approximately 70% of lumbar interbody fusion procedures. To improve the success of fusion, various surgical adjuncts such as bone grafts and bone morphogenic protein (BMP) are commonly used. Unfortunately, BMP has been associated with serious complications including local inflammation and ectopic bone formation in the spinal canal. To overcome the challenges of BMP, direct current electric stimulation (DCES) has been examined as a nonpharmacologic means of inducing local bone formation and spinal fusion. Direct current electric stimulation has been demonstrated to up-regulate osteoinductive factors which govern new bone formation and lead to fusion. The principal innovation behind the technology is the concept of using existing metallic spinal instrumentation (pedicle screws, rods, etc.) to focally deliver bone forming DCES directly to the fusion site. The system of instrumentation thereby functions simultaneously as both conventional mechanical fixation and internal osteogenic stimulator. PURPOSE The present study was designed to determine whether novel osteogenic spinal instrumentation is capable of improving mechanical stabilization and bony interbody fusion of the lumbar spine in a large animal model. METHODS A posterolateral interbody fusion across L4–L5 was performed on 36 skeletally mature sheep. The treated functional spinal unit (FSU) was stabilized posteriorly using four osteogenic pedicle screws and connecting bars (INDOSTM, OsteoVantage, Inc. Fayetteville, AR, USA), with a PEEK interbody device (IBD), placed via a lateral approach, across the disc space. Three treatment groups were studied: (Group 1) IBD filled with tricalcium phosphate (TCP), (Group 2) IBD filled with TCP with DCES, and (Group 3) IBD filled with BMP (InfuseTM, Medtronic Spine, Memphis, TN). Active osteogenic spinal instrumentation (ie, Group 2) consisted of standard titanium pedicle screws and rods that have been modified with proprietary anodization patterns and power supplies to conduct and deliver therapeutic DCES to the fusion site (ie, the disc space). Eighteen (n=18) animals were sacrificed at 13 and 26-weeks. Following euthanasia, kinematic, radiographic and histological analyses were performed on explanted FSUs. RESULTS No in-life complications were observed, and all osteogenic spinal instrumentation were confirmed to be in operation over the study period. For all 3 kinematic loading cases Group 2 and Group 3 demonstrated significantly lower ROM compared to Group 1 at the 13-week sacrifice time point. Reduction in ROM magnitudes was observed in all directions for Group 2 as compared to Group 1 at the 13-week time point; these reductions were significant in lateral bending and flexion–extension. For all treatment groups, the ROM results from the 26-week group were significantly less than the 13-week data. The significant differences at the 13-week time point between Group 2 and Group 1 clearly demonstrate that DCES causes increased reduction in ROM during the early healing cascade. Histomorphometric data demonstrated a significant increase in the percent (%) bone within the IBD graft window between the 13 and 26-week time points for Group 2 only (p CONCLUSIONS Previous research has demonstrated that DCES up-regulates osteoinductive factors which govern new bone formation and fusion. It has been shown to enhance normal physiologic expression of BMP-2, BMP-6 and BMP-7 in osteoblasts. In addition, DCES stimulates macrophages to release VEG-F. This study indicates that DCES can have a significant effect on local bone formation and spinal fusion as measured by ROM kinematics and bony response as measured by radiographic analysis and histopathology. In total, osteogenic spinal instrumentation delivering focused DCES at the fusion site promoted both mechanical stabilization of the spine and osteogenic stimulation of new bone formation and bony fusion.

MP23-03 IL-6/STAT3 SIGNALING IS A HOST DETERMINANT OF UPEC VIRULENCE VIA ALTERED BACTERIAL INVASION AND INTRACELLULAR BACTERIAL COMMUNITY FORMATION

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder II1 Apr 2018MP23-03 IL-6/STAT3 SIGNALING IS A HOST DETERMINANT OF UPEC VIRULENCE VIA ALTERED BACTERIAL INVASION AND INTRACELLULAR BACTERIAL COMMUNITY FORMATION Christina Ching, Sudipti Gupta, and Brian Becknell Christina ChingChristina Ching More articles by this author , Sudipti GuptaSudipti Gupta More articles by this author , and Brian BecknellBrian Becknell More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.735AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Our understanding of bacterial virulence in urinary tract infection (UTI) is primarily limited to identification of bacteria-specific determinants. Host mediators are less well known. IL-6 is a cytokine that is induced during UTI and results in subsequent Stat3 phosphorylation, but without readily apparent functional significance. We sought to evaluate the impact of IL-6/Stat3 signaling on host susceptibility to UTI via urothelial bacterial invasion and intracellular bacterial community formation. METHODS We performed in vivo and in vitro studies in order to evaluate the impact of IL-6 and Stat3 in bacterial invasion. We infected 6 week old female wild type (WT) C57BL/6J, IL-6 knock out (KO), or tamoxifen-inducible Stat3 conditional KO mice transurethrally with UPEC. We evaluated bladder and urine bacterial burden at varying time points of sacrifice by serial plating of tissue homogenates on LB agar and IBC formation via beta-galactosidase staining. We similarly evaluated the impact of recombinant IL-6 rescue in IL-6 KO mice on IBC formation and antimicrobial formation. We evaluated the impact of recombinant IL-6 incubation and Stat3 inhibition on intracellular bacterial invasion with gentamicin protection assay in serum starved 5637 urothelial cells. Results were evaluated by Mann-Whitney U test with p <0.05 being significant. RESULTS We observed an early transient (6 hours post inoculation - hpi) increase in bladder bacterial burden in our IL-6 KO mice compared to the WT controls, with a subsequent and persistent increase in urine bacterial burden starting at 24 hpi. This matched an initial significant difference in bladder IBC number in the IL-6 KO vs. WT that resolved by 24 hpi and was confirmed in our Stat3 CKO mice. Recombinant intravesical IL-6 rescued this phenotype in the IL-6 KO mouse and also significantly increased antimicrobial peptide expression. Similarly, we found that there was a significant decrease in bacterial invasion in vitro when 5637 cells were preincubated with IL-6. Stat3 inhibition resulted in a reversal of this phenotype with an increase in bacterial invasion on par with if not above unstimulated cells. CONCLUSIONS IL-6 plays an important role in host susceptibility to UTI as a mediator of bacterial invasion and IBC formation, potentially via antimicrobial peptide formation. Given IBC formation represents a significant bottleneck in UPEC survival during cystitis, these findings could suggest a role of IL-6 in UTI persistence and chronicity. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e282 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Christina Ching More articles by this author Sudipti Gupta More articles by this author Brian Becknell More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Bony ingrowth potential of 3D-printed porous titanium alloy: a direct comparison of interbody cage materials in an in vivo ovine lumbar fusion model

Background ContextThere is significant variability in the materials commonly used for interbody cages in spine surgery. It is theorized that three-dimensional (3D)-printed interbody cages using porous titanium material can provide more consistent bone ingrowth and biological fixation. PurposeThe purpose of this study was to provide an evidence-based approach to decision-making regarding interbody materials for spinal fusion. Study DesignA comparative animal study was performed. MethodsA skeletally mature ovine lumbar fusion model was used for this study. Interbody fusions were performed at L2–L3 and L4–L5 in 27 mature sheep using three different interbody cages (ie, polyetheretherketone [PEEK], plasma sprayed porous titanium-coated PEEK [PSP], and 3D-printed porous titanium alloy cage [PTA]). Non-destructive kinematic testing was performed in the three primary directions of motion. The specimens were then analyzed using micro-computed tomography (µ-CT); quantitative measures of the bony fusion were performed. Histomorphometric analyses were also performed in the sagittal plane through the interbody device. Outcome parameters were compared between cage designs and time points. ResultsFlexion-extension range of motion (ROM) was statistically reduced for the PTA group compared with the PEEK cages at 16 weeks (p-value=.02). Only the PTA cages demonstrated a statistically significant decrease in ROM and increase in stiffness across all three loading directions between the 8-week and 16-week sacrifice time points (p-value≤.01). Micro-CT data demonstrated significantly greater total bone volume within the graft window for the PTA cages at both 8 weeks and 16 weeks compared with the PEEK cages (p-value<.01). ConclusionsA direct comparison of interbody implants demonstrates significant and measurable differences in biomechanical, µ-CT, and histologic performance in an ovine model. The 3D-printed porous titanium interbody cage resulted in statistically significant reductions in ROM, increases in the bone ingrowth profile, as well as average construct stiffness compared with PEEK and PSP.

Abstract A077: Sustained immune response to anti-PD-1 therapy for oral premalignant lesions (OPLs) in a carcinogen-induced oral cancer mouse model

Abstract PD-L1 expression occurs in OPLs in humans and is associated with increased cancer risk (William et al., ASCO 2017). These data suggest a contribution of the PD-1/PD-L1 axis to immune evasion of OPLs, and warrant evaluation of anti-PD-1 immunotherapy for cancer prevention. We conducted this preclinical study in a carcinogen-induced mouse model of oral cancer to test the hypothesis that a short course of anti-PD-1 therapy, given at a time point when only OPLs are present (and before invasive cancer development), could lead to enhanced and sustained immune responses. We treated C57BL/6 mice with the carcinogen 4-nitroquinoline 1-oxide (4-NQO) for 8 weeks in drinking water. Eight weeks after 4-NQO discontinuation, a group of mice (N=6) was sacrificed for assessment of invasive and noninvasive tongue lesions. A second group of mice was treated with either anti-PD-1 antibody (N=10) or IgG (N=10) for a total of 3 doses every 3 days initiating 8 weeks after the cessation of 4-NQO. We sacrificed these animals 56 days after the last dose of treatment, harvested cervical lymph nodes, and prepared and stained cell suspensions with fluorochrome-labeled antibodies against CD45, CD3, CD8, Granzyme B (GZB), PD-1, TIM-3, Lag-3, ZombieNirt (live/dead discrimination) for flow cytometric evaluation. We also assessed serial H&amp;E-stained cross-sections of the tongues harvested at that same time point for development of OPLs and cancers. Student’s t-test and Fisher's exact test were used for statistical comparisons between groups. At 8 weeks after 4NQO cessation, 6 out of 6 mice developed tongue hyperplasia or dysplasia. No invasive lesions were identified, indicating this to be an ideal time point to study/initiate treatment strategies addressing OPLs. Anti-PD-1 therapy led to a sustained increase in the proportion of CD8+|GZB+ cytotoxic T lymphocytes (CTLs) when compared to IgG (mean 26.9% ± 4.4% versus 14.4% ± 2.3%, respectively; p= 0.025) in cervical lymph nodes harvested 56 days after treatment discontinuation. We also observed an increase in the proportion of CD8+|PD-1+ CTLs (mean 22.3% ± 2.08% versus 13.3% ± 1.42%, respectively; p= 0.0026) and a trend towards an increase in CD8+|TIM-3+|LAG-3+ CTLs (mean 5.9% ± 1.5% versus 2.5% ± 0.7%, respectively; p= 0.0733). This immune profile is indicative of activated CTLs with limited/no signs of exhaustion. At this late sacrifice time point, frankly invasive oral cancers had developed in 70% vs 40% of IgG and anti-PD1-treated mice, respectively (p=0.36). Short-term anti-PD-1 immune checkpoint inhibitor therapy in the context of OPLs led to a sustained increase in the population of granzyme B+ CTLs in cervical lymph nodes, accompanied by a trend towards a reduction in the incidence of oral cancer. These results suggest that immune checkpoint-targeted therapy can reverse immune suppression already present at the preinvasive stage, and support the ongoing Immune Prevention of Oral Cancer (IPOC) clinical trial evaluating pembrolizumab in high-risk OPLs (NCT02882282). Additional preclinical studies are under way to assess if an immune response to anti-PD1 therapy can occur at earlier time points, and lead to pronounced prevention of oral cancers at a stage when the (irreversible) malignant transformation process induced by the potent 4-NQO carcinogen is not yet far advanced. Citation Format: Jose Augusto Monteiro de Oliveira Novaes, Taghreed Hirz, Marlese A. Pisegna, Patrick Hwu, John V. Heymach, William N. William. Sustained immune response to anti-PD-1 therapy for oral premalignant lesions (OPLs) in a carcinogen-induced oral cancer mouse model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A077.

Comparison of the Local Tolerability to 5 Long-acting Drug Nanosuspensions with Different Stabilizing Excipients, Following a Single Intramuscular Administration in the Rat

To investigate the effects of common nanosuspension-stabilizing excipients on the nature and temporal evolution of histopathological changes at intramuscular (i.m.) administration sites, 5 groups of 39 male rats per group received a single injection of 1 of the 5 analogous crystalline drug nanosuspensions containing 200 mg/ml of an antiviral compound with particle sizes of ±200 nm and identical vehicle compositions, except for the type of nanosuspension stabilizer. The investigated stabilizers were poloxamer 338, poloxamer 407, d-α-tocopherol polyethylene glycol 1,000-succinate (TPGS), polysorbate 80, and polysorbate 80 combined with egg phosphatidylglycerol. Histopathology and immunohistochemistry revealed progressive inflammatory changes at the i.m. administration sites and the draining lymph nodes that differed according to the time point of sacrifice and the type of stabilizer. Although the overall time course of inflammatory changes was similar across the groups, differences in the nature, severity, and timing of the inflammatory response were observed between animals injected with poloxamer- or TPGS-containing nanosuspensions and those injected with formulations containing polysorbate 80. A more severe and prolonged active inflammatory phase, the presence of multinucleate giant cells, prolonged macrophage infiltration of the formulation depot, and more persistent histiocytic infiltrates in the lymph nodes were observed in the polysorbate 80-containing nanosuspension groups. Such vehicle-mediated effects could influence the overall tolerability profile of long-acting nanosuspensions.

Autophagy in long propriospinal neurons is activated after spinal cord injury in adult rats

Spinal cord injury (SCI) is a common disease worldwide that causes permanent neuronal dysfunction without an effective treatment. Long propriospinal neurons (LPSNs) that are spared from injury play a key role in spontaneous recovery after SCI. Traumatic injury of the central nervous system can activate autophagy, which could be a target in the development of a new therapeutic strategy to prevent neuronal loss. Our research focused on whether autophagy is involved in the loss of LPSNs after introducing spinal cord injury in adult rats. Different sacrifice time points were chosen to characterize autophagy and apoptosis. Autophagy and a blocked autophagy flux reached their peaks at 3 d after injury, while apoptosis reached its peak at 7 d after injury when the number of LPSNs significantly decreased. Both autophagy and apoptosis contributed to the loss of LPSNs, and apoptosis was the main cause of cell death. However, autophagy may prevent programmed LPSN cell death (apoptosis), which could promote cell survival.

Does Citrulline Have Protective Effects on Liver Injury in Septic Rats?

Citrulline (Cit) supplementation was proposed to serve as a therapeutic intervention to restore arginine (Arg) concentrations and improve related functions in sepsis. This study explored whether citrulline had positive effects on liver injury and cytokine release in the early stages of sepsis. The cecal ligation and puncture (CLP) model was utilized in our study. Rats were divided into four groups: normal, Cit, CLP, and CLP+Cit. The CLP group and CLP+Cit group were separated into 6-, 12-, and 24-hour groups, according to the time points of sacrifice after surgery. Intragastric administration of L-citrulline was applied to rats in Cit and CLP+Cit groups before surgery. Serum AST and ALT levels and levels of MDA, SOD, NO, and iNOS in the liver tissues were evaluated. Plasma concentrations of Cit and Arg were assessed using HPLC-MS/MS. Serum concentrations of cytokines and chemokines were calculated by Luminex. Results showed SOD activities of CLP+Cit groups were significantly higher than that of CLP groups, contrasting with the MDA and NO levels which were significantly lower in CLP+Cit groups than in CLP groups. In addition, plasma concentrations of TNF-α, IL-6, and IL-1β were significantly lower in the CLP+Cit 6-hour group than in the CLP 6-hour group.

Characterization of a nose-only inhaled phosgene acute lung injury mouse model

Context: Phosgene’s primary mode of action is as a pulmonary irritant characterized by its early latent phase where life-threatening, non-cardiogenic pulmonary edema is typically observed 6‐24 h post-exposure.Objective: To develop an inhaled phosgene acute lung injury (ALI) model in C57BL/6 mice that can be used to screen potential medical countermeasures.Methods: A Cannon style nose-only inhalation exposure tower was used to expose mice to phosgene (8 ppm) or air (sham). An inhalation lethality study was conducted to determine the 8 ppm median lethal exposure (LCt50) at 24 and 48 h post-exposure. The model was then developed at 1.2 times the 24 h LCt50. At predetermined serial sacrifice time points, survivors were euthanized, body and lung weights collected, and lung tissues processed for histopathology. Additionally, post-exposure clinical observations were used to assess quality of life.Results and discussion: The 24-hour LCt50 was 226 ppm*min (8 ppm for 28.2 min) and the 48-hour LCt50 was 215 ppm*min (8 ppm for 26.9 min). The phosgene exposed animals had a distinct progression of clinical signs, histopathological changes and increased lung/body weight ratios. Early indicators of a 1.2 times the 24-hour LCt50 phosgene exposure were significant changes in the lung-to-body weight ratios by 4 h post-exposure. The progression of clinical signs and histopathological changes were important endpoints for characterizing phosgene-induced ALI for future countermeasure studies.Conclusion: An 8 ppm phosgene exposure for 34 min (1.2 × LCt50) is the minimum challenge recommended for evaluating therapeutic interventions. The predicted higher mortality in the phosgene-only controls will help demonstrate efficacy of candidate treatments and increase the probability that a change in survival rate is statistically significant.