Time Of Euthanasia Research Articles

Intrathecal mepivacaine after general anesthesia is an effective method of equine euthanasia when compared to intravenous pentobarbital.

This study aims to assess intrathecal mepivacaine for euthanasia in anesthetized horses and compare it to a traditional euthanasia method using a single intravenous injection of pentobarbital in sedated horses. Client-owned horses and horses requiring euthanasia due to involvement in concurrent research projects were used. Horses were randomly assigned to 1 of 2 groups: intrathecal mepivacaine after anesthesia or intravenous pentobarbital after sedation. All horses had normal vital parameters and no signs of infectious disease at the time of euthanasia. The intrathecal mepivacaine group was anesthetized before the intrathecal injection of mepivacaine. The pentobarbital group was sedated, concurrently anesthetized and euthanized using intravenous pentobarbital, then received an intrathecal saline (0.9% NaCl) solution injection to a blind observer. Both groups were sedated with detomidine and the time from sedation to the cessation of vital parameters (respirations, pulse, corneal reflex, and ECG) was recorded. Euthanasias were recorded for review by a blinded anesthesiologist, using an independent scale to assess the quality of sedation, anesthesia induction, and lateral recumbency. Time from detomidine administration to cessation of each vital parameter was significantly longer in the intrathecal mepivacaine group. There was no statistically significant difference in qualitative scores between groups for sedation or induction, but lateral recumbency was subjectively superior in the anesthetized intrathecal mepivacaine group. Intrathecal mepivacaine provided a safe, effective, alternative method of euthanasia to intravenous pentobarbital and addresses concerns about barbiturate availability. This study also informs practitioners of what to expect (ie, longer cessation of vital parameters) when using the intrathecal mepivacaine method.

Experimental infection of sheep at mid-pregnancy with archetypal type II and type III Toxoplasma gondii isolates exhibited different phenotypic traits

Toxoplasma gondii is a major cause of reproductive failure in small ruminants. Genotypic diversity of T. gondii strains has been associated with variations in phenotypic traits in in vitro and murine models. However, whether such diversity could influence the outcome of infection in small ruminants remains mostly unexplored. Here, we investigate the outcome of oral challenge in sheep at mid-pregnancy with 10 sporulated oocysts from three different T. gondii isolates belonging to archetypal II and III and selected according to their genetic and phenotypic variations shown in previous studies. Seventy-three pregnant sheep were divided in four groups: G1 infected with TgShSp1 isolate (type II, ToxoDB#3), G2 with TgShSp16 isolate (type II, ToxoDB#3), G3 with TgShSp24 isolate (type III, ToxoDB#2) and G4 of uninfected control sheep. Two different approaches were carried out within this study: (i) the outcome for the pregnancy after infection (n = 33) and (ii) the lesions and parasite tropism and burden at 14 and 28 days post infection (dpi) (n = 40). The onset of hyperthermia and seroconversion occurred one and two days later, respectively in G1 when compared to G2 and G3. However, sheep that suffered from reproductive failure, either by abortion, foetal dead at the time of euthanasia or stillbirth were similar among infected groups (50%, 40% and 47%, respectively). Histological lesions in placentomes and foetal tissues from euthanized animals from the second approach were only detected at 28 dpi and mainly in G1. At 14 dpi, T. gondii-DNA was only detected in G1 in the 11% of the placentomes. However, at 28 dpi the frequency of detection in placentomes was higher in G1 (96%) than in G2 and G3 (7% and 47%, respectively) besides in foetuses was lower in G2 (20%) than in G1 and G3 (100% and 87%, respectively). Regarding late abortions, stillbirths, and lambs of G1, G2 and G3, the frequency of microscopic lesions was similar between groups (79%, 78% and 67%, respectively) whereas T. gondii-DNA was evidenced in 100%, 55% and 100%, respectively. These recently obtained T. gondii isolates led to similar reproductive losses but intra- and inter-genotype variations in the rise of hyperthermia, dynamics of antibodies, frequency of lesions and parasite detection and distribution. Thus, the different phenotypic traits of the isolates could influence the outcome of the infection and mechanisms responsible for it, and further investigations are warranted.

Abstract WMP114: Changes In Gut Microbiome Precede Cognitive Impairment In A Mouse Model Of Vascular Cognitive Impairment And Dementia

Introduction: Vascular cognitive impairment (VCI) is the second most common cause of clinical dementia after Alzheimer’s disease. VCI results from injury to the cerebral blood vessels. Cerebral perfusion is diminished in elderly individuals and additional reduction of cerebral blood flow increases the risk of developing VCI. These findings have been successfully modeled in mice with bilateral common carotid artery stenosis (BCAS). Age is associated with gut dysbiosis and transplantation of aged microbiome leads to cognitive decline in young animals. However, there are large gaps in our understanding of the molecular mechanisms induced by chronic hypoperfusion contributes to impaired cognitive function. Methods: C57Bl6 aged (~18m) male mice were subjected to a sham or a BCAS surgery using 0.18mm titanium coils placed on both common carotid arteries. Mice were followed for 90d after surgery to assess both gut microbial content and behavioral changes. 16S ribosomal RNA (rRNA) sequencing analysis was performed on fecal samples collected from aged baseline, sham and BCAS animals at 7 and 28 days. Cognition was assessed using Y-maze. Tissues were collected at the time of euthanasia for metabolomics and histological analysis. Results: BCAS resulted in significant reduction of cerebral blood flow, measured using laser speckle (p<0.05; n=4/grp; t test). 16s rRNA analysis revealed BCAS led to a remarkable shift in bacterial diversity as early as day 7 in aged male mice, measured by using unweighted UniFrac analysis. These differences remained significant (p<0.05; n=4/grp) in BCAS compared to sham mice on day 28. In depth analysis revealed significant changes at the genus level between both groups. Interestingly, BCAS mice did not show significant difference in Y-maze at day 14, but significant cognitive impairment was found at day 90 compared to sham mice (n=8-9/grp). Conclusions: We found that chronic cerebral hypoperfusion is associated with significant changes in gut microbiome and cognitive impairment in aged mice. Importantly, shifts in the microbiome preceded cognitive decline. These findings suggest that targeting these detrimental changes in the gut microbiome might be a novel therapeutic strategy to delay or prevent progression of VCI.

Electrolyte imbalances and dehydration play a key role in Batrachochytrium salamandrivorans chytridiomycosis.

One of the most important emerging infectious diseases of amphibians is caused by the fungal pathogen Batrachochytrium salamandrivorans (Bsal). Bsal was recently discovered and is of global concern due to its potential to cause high mortality in amphibians, especially salamander species. To date, little has been reported on the pathophysiological effects of Bsal; however, studies of a similar fungus, B. dendrobatidis (Bd), have shown that electrolyte losses and immunosuppression likely play a key role in morbidity and mortality associated with this disease. The goal of this study was to investigate pathophysiological effects and immune responses associated with Bsal chytridiomycosis using 49 rough-skinned newts (Taricha granulosa) as the model species. Taricha granulosa were exposed to a 1 × 107 per 10 mL dose of Bsal zoospores and allowed to reach various stages of disease progression before being humanely euthanized. At the time of euthanasia, blood was collected for biochemical and hematological analyses as well as protein electrophoresis. Ten standardized body sections were histologically examined, and Bsal-induced skin lesions were counted and graded on a scale of 1-5 based on severity. Results indicated that electrolyte imbalances and dehydration induced by damage to the epidermis likely play a major role in the pathogenesis of Bsal chytridiomycosis in this species. Additionally, Bsal-infected, clinically diseased T. granulosa exhibited a systemic inflammatory response identified through alterations in complete blood counts and protein electrophoretograms. Overall, these results provide foundational information on the pathogenesis of this disease and highlight the differences and similarities between Bsal and Bd chytridiomycosis.

Rosuvastatin Attenuates Progression of Atherosclerosis and Reduces Serum IL6 and CCL2 Levels in Apolipoprotein-E-deficient Mice.

Apolipoprotein E-deficient (Apoe-/-) mice develop atherosclerotic lesions that closely resemble metabolic syndrome in humans. We sought to investigate how rosuvastatin mitigates the atherosclerotic profile of Apoe-/- mice over time and its effects on certain inflammatory chemokines. Eighteen Apoe-/- mice were allocated into three groups of six mice each receiving: standard chow diet (SCD; control group); high-fat diet (HFD); and HFD and rosuvastatin at 5 mg/kg/d orally via gavage for 20 weeks. Analysis of aortic plaques and lipid deposition was conducted by means of en face Sudan IV staining and Oil Red O staining. Serum cholesterol, low-density lipoprotein, high-density lipoprotein, plasma glucose and triglyceride levels were determined at baseline and after 20 weeks of treatment. Serum interleukin 6 (IL6), C-C motif chemokine ligand 2 (CCL2) and tumor necrosis factor-α (TNFα) levels were measured by enzyme-linked immunosorbent assay at the time of euthanasia. The lipidemic profile of Apoe-/- mice on HFD deteriorated over time. Apoe-/- mice on HFD developed atherosclerotic lesions over time. Sudan IV and Oil Red O-stained sections of the aorta revealed increased plaque formation and plaque lipid deposition in HFD-fed mice compared with SCD-fed mice and reduced plaque development in HFD-fed mice treated with rosuvastatin compared with mice not receiving statin treatment. Serum analysis revealed reduced metabolic parameters in HFD-fed mice on rosuvastatin compared with non-statin, HFD-fed mice. At the time of euthanasia, HFD-fed mice treated with rosuvastatin had significantly lower IL6 as well as CCL2 levels when compared with HFD-fed mice not receiving rosuvastatin. TNFα levels were comparable among all groups of mice, irrespective of treatment. IL6 and CCL2 positively correlated with the extent of atherosclerotic lesions and lipid deposition in atherosclerotic plaques. Serum IL6 and CCL2 levels might potentially be used as clinical markers of progression of atherosclerosis during statin treatment for hypercholesterolemia.

Comparison of Corticosterone Concentrations in Dermal Secretions and Urine in Free-Ranging Marine Toads (Rhinella marina) in Human Care.

Corticosterone concentrations have been measured in amphibians by collecting blood or urine samples. However, blood sampling is invasive, and urine can be difficult to collect. A novel method of swabbing the skin of an amphibian has been utilized in numerous species but has not been verified in marine toads (Rhinella marina). This pilot study tested dermal swabs as a noninvasive method for collecting and measuring dermal corticosterone secretions. Swabs were used to collect dermal secretion samples from sixty-six free-ranging marine toads collected on Zoo Miami grounds. The subsequent day the toads were shipped to the North Carolina Zoo where dermal samples were collected again. Additional dermal and urine samples were collected on days 9, 15, 32, and 62 under human care to measure corticosterone concentrations. There was no significant correlation (P ≥ 0.05) noted between corticosterone concentrations reported in dermal swabs and those in urine samples at all four of the euthanasia time points or between the corticosterone concentrations reported in either urine or dermal swabs and the weight of the toads. Dermal swab concentrations (ng/mL) were significantly higher (P ≤ 0.05) on the day of capture (0.64 ± 0.03) and the day of arrival (0.67 ± 0.03) than on day 15 (0.47 ± 0.03). The urine corticosterone concentrations decreased while the toads were in human care with a significant decrease (P ≤ 0.05) between days 9 (0.45 ± 0.07) and 32 (0.21 ± 0.06). This study demonstrated that dermal swabs can be used to collect marine toad corticosterone concentration samples.

RBIO-04. STEREOTACTIC RADIOSURGERY REPROGRAMS MACROPHAGE, MICROGLIA, AND CD8+ T CELL POPULATIONS IN THE GLIOBLASTOMA IMMUNE MICROENVIRONMENT

Abstract INTRODUCTION Fractionated radiotherapy is a first-line treatment for glioblastoma, but daily ionizing radiation prevents durable immune infiltration of the tumor microenvironment. Hypofractionated radiotherapy is used to treat glioblastomas at recurrence, but the impact of hypofractionated radiotherapy on the glioblastoma immune microenvironment is incompletely understood. Here we define immune microenvironment changes across multiple immunocompetent intracranial mouse models of glioblastoma after treatment with ionizing radiation mimicking stereotactic radiosurgery (SRS) in humans. METHODS Syngeneic GL261 (3x105 cells/mouse) or SB28 glioblastoma cells (3x104 cells/mouse) were implanted into the frontal lobe of immunocompetent C57BL/6J mice (18 mice/arm x 4 arms). Intracranial bioluminescence was used to assess glioblastoma growth. After tumor engraftment, glioblastomas were treated with conformal SRS (18Gy/1Fx) or sham. Glioblastomas were collected for histologic, single-cell, or molecular analyses 5 days after treatment (6 mice/arm) or at the time of euthanasia after monitoring for survival (12 mice/arm). Glioblastoma immune microenvironment responses were assessed using (1) H&E, (2) single cell mass cytometry (CyTOF) or IHC to define or validate immune cell changes, respectively, or (3) multiplexed cytokine assays to elucidate molecular mechanisms reprograming the glioblastoma immune microenvironment in response to SRS. RESULTS SRS attenuated glioblastoma growth and prolonged survival compared to sham treatment in both immunocompetent intracranial mouse models (GL261: 14 days versus 27 days, p< 0.001, SB28: 19 days versus 22 days, p=0.001). CyTOF showed SRS decreased immunosuppressive macrophage infiltration and increased microglia or CD8+ T cell infiltration of the glioblastoma immune microenvironment. Histologic analyses validated T cell and microglia infiltration after SRS. Glioblastoma cytokine analysis revealed inhibition of pro-tumor/anti-inflammatory cytokines (IL6, LIF) after SRS. CONCLUSIONS Single-fraction SRS durably reprograms glioblastoma macrophage, microglia, and CD8+ T cell populations in preclinical models, suggesting SRS or inhibition of pro-tumor/anti-inflammatory mechanisms underlying the immunosuppressive glioblastoma microenvironment represent immunogenic therapies that may offer a benefit to patients with glioblastoma.

The Novel Use of a Nanofiber Hydrogel Composite for Perineural Adhesion Prevention in a Rodent Model

INTRODUCTION: Perineural adhesions can form after any surgical intervention involving peripheral nerves. Adhesion formation may then lead to nerve entrapment and compressive neuropathy, which can result in a wide variety of symptoms ranging from sensory deficits to motor weakness. Previously, we created a novel poly(ε -caprolactone) (PCL) nanofiber/ hyaluronic acid hydrogel composite that was shown to mimic the microarchitecture and mechanical properties of soft tissue extracellular matrix.1 We hypothesize that the use of this novel nanofiber hydrogel composite (NHC) will reduce perineural adhesion formation in a rodent hindlimb model. METHODS: This study was performed with Institutional Animal Care and Use Committee approval. Male Lewis rats underwent bilateral circumferential mechanical irritation of the sciatic nerve to induce adhesion formation with subsequent primary closure. Animals then underwent a secondary neurolysis 8 weeks post-operatively. At the time of neurolysis, the experimental group (n=6) were treated with circumferentially application of NHC around the sciatic nerve before closure and the control group were closed without treatment (n=6). Both groups were sacrificed 8 weeks after their secondary surgery. At the time of euthanasia, all rodents underwent unilateral biomechanical force testing to assess the breaking point of the perineural adhesions surrounding the sciatic nerve (measured in Newtons). In the contralateral limb, the sciatic nerve, surrounding muscle, and NHC in experimental animals was harvested to assess perineural collagen deposition using hematoxylin and eosin (H&E) and Masson’s Trichrome staining. RESULTS: Significant perineural adhesions were visually apparent after sciatic nerve irritation in the control group. In the experimental group, the sciatic nerve was grossly encapsulated by the NHC which closely resembled subcutaneous fat with visible neovascularization. Biomechanical testing demonstrated the average force required to remove the nerve from the wound bed in the experimental group was 2.02±0.43 N. In the control group, the sciatic nerve could not be removed from the wound bed and the average force prior to failure was 2.77±0.18 N. Collagen deposition, a measure of scar formation around the sciatic nerve, was assessed via H&E and MT staining. Minimal collagen deposition was seen in the experimental group compared to control, indicating a decrease in scar formation in the animals treated with perineural application of the NHC. CONCLUSION: We found that the use of a novel PCL nanofiber/hyaluronic acid hydrogel composite resulted in a decrease in perineural adhesion and scar formation in a rat model. 1. Li X, Cho B, Martin R, Seu M, Zhang C, Zhou Z, Choi JS, Jiang X, Chen L, Walia G, Yan J, Callanan M, Liu H, Colbert K, Morrissette-McAlmon J, Grayson W, Reddy S, Sacks JM, Mao HQ. Nanofiber-hydrogel composite-mediated angiogenesis for soft tissue reconstruction. Sci Transl Med. 2019 May 1;11(490):eaau6210. doi: 10.1126/scitranslmed.aau6210. PMID: 31043572.

Genotoxic and cytotoxic potential of food azodyes: preclinical safety assessment using the in vivo micronucleus assay

This study evaluated the genotoxic effects of azodyes (ponceau 4R, red 40, sunset yellow and tartrazine) by the use of in vivo micronucleus assays. Swiss albinus young adult mice of both sexes, healthy and heterogeneous, were used in the micronucleus assay. Groups of animals were treated using a single dosing regimen and euthanized at 24 and 48 hours. The study design included treatment groups (0.5, 1.0 and 2 g/kg of azodyes) and negative (150 mM NaCl) and positive (50 mg/kg of NEU) control groups. Bone marrow polychromatic (PCE) and normochromatic (NCE) erythrocytes and micronucleated PCE (MNPCE) were statistically analyzed: frequency and PCE:NCE ratio. For animal groups treated with all azodyes, analyses of the frequency of MNPCEs and PCE/NCE ratio showed significant differences between the treatment groups (0.5-2 g/kg) and the control groups (NaCl and NEU). Each azodye exhibited genotoxic and systemic toxic effects correlated to treatment dose, time of euthanasia, and sex of the animal. The data suggest potential clastogenic and/or aneugenic effects that can potentiate systemic toxic risks associated with the azodyes whether the genotoxicity be dependent on dose (ponceau 4R, sunset yellow, and tartrazine), time (ponceau 4R, red 40, and sunset yellow), or sex (red 40 and tartrazine).

Intranasal instillation of Pasteurella multocida lipopolysaccharide in rabbits causes interstitial lung damage

In order to characterize the in vivo lesions in the nasal cavities and lungs, twenty-eight rabbits were intranasally instilled with lipopolysaccharide (LPS) from P. multocida and then divided into seven groups according to euthanasia time. The nasal cavities and the lungs were processed for light microscopy, lectin histochemistry and transmission electron microscopy. Increased goblet cell activation and neutrophil infiltration were relevant changes in the nasal cavity. A predominantly interstitial pattern of diffuse alveolar damage and bronchopneumonic foci were the main lesions found in the lungs. LPS was found in the cytoplasm of ciliated cells, goblet cells, glandular cells, venular endothelial cells and neutrophils in the nasal cavity and in club cells, capillary endothelial cells and neutrophil in the lung. This study demonstrates that the LPS is able to cause lesions in the upper and lower respiratory tract, it binds to and is internalized by respiratory epithelial cells. Furthermore, it also traverses the intercellular spaces to reach the blood vessels, where it binds to and is internalized by neutrophil and red blood cells. These cells may then travel to the lungs where the LPS induces typical diffuse alveolar damage. This route of lung interstitial damage, to our knowledge, has not been described for this molecule or any known pathogen.

EGFL6 is an important growth factor involved in endometrial cancer tumorigenesis (073)

EGFL6 is an important growth factor involved in endometrial cancer tumorigenesis (073)

SARS-CoV-2 Doggybone DNA Vaccine Produces Cross-Variant Neutralizing Antibodies and Is Protective in a COVID-19 Animal Model.

To combat the COVID-19 pandemic, an assortment of vaccines has been developed. Nucleic acid vaccines have the advantage of rapid production, as they only require a viral antigen sequence and can readily be modified to detected viral mutations. Doggybone™ DNA vaccines targeting the spike protein of SARS-CoV-2 have been generated and compared with a traditionally manufactured, bacterially derived plasmid DNA vaccine that utilizes the same spike sequence. Administered to Syrian hamsters by jet injection at two dose levels, the immunogenicity of both DNA vaccines was compared following two vaccinations. Immunized hamsters were then immunosuppressed and exposed to SARS-CoV-2. Significant differences in body weight were observed during acute infection, and lungs collected at the time of euthanasia had significantly reduced viral RNA, infectious virus, and pathology compared with irrelevant DNA-vaccinated controls. Moreover, immune serum from vaccinated animals was capable of neutralizing SARS-CoV-2 variants of interest and importance in vitro. These data demonstrate the efficacy of a synthetic DNA vaccine approach to protect hamsters from SARS-CoV-2.

35. Human Adipose-derived Stem Cells Decrease Skin Damage in Mice After Scalp Irradiation: A Pilot Experiment

Purpose: The treatment for radiation dermatitis ulcers is surgical reconstruction. We aimed to characterize the effects of human adipose-derived stem cells (hAdMSCs) to prevent complications of radiation dermatitis of the scalp in an immunocompetent mouse model. Methods: Two 4-week-old female C57BL/6 mice were delivered a dose of 60 Gy to a skin area between the coronal and lambdoid sutures using a 5 mm circular collimator. On day 12 after irradiation, 600,000 luciferase- and green fluorescent protein-labeled hAdMSCs in 300 µL of PBS were injected subcutaneously into the scalp of one mouse. Mice were photographed under standardized conditions and weighted daily. Bioluminescence imaging (BLI) was performed daily from the day after injection until there was no detectable signal. Both were euthanized on day 27 after irradiation, and the scalp tissue was collected. Staining with hematoxylin and eosin, Masson’s trichrome, and immunohistochemistry for CD31 were performed. The histological slides were digitalized and analyzed using ImageJ (NIH). Statistical analyses were performed using Excel 2010 (Microsoft Corp., United States). Results: Bioluminescence images showed that hAdMSCs survived in vivo for five days. While desquamation and ulceration appeared on day 14 and lasted for 12 days in the treated mouse, the control mouse did not show signs of improvement by the time of euthanasia. The treated mouse had a greater mean erythematous area than the control mouse (0.38 cm2 vs. 0.23 cm2, p<0.05). The mean ulceration areas did not differ between the treated and control mouse (0.12 cm2 vs. 0.14 cm2, p>0.05). The epidermis, total skin thickness, and hypodermis of the irradiated regions were significantly thicker than the non-irradiated regions in both the treatment and control mice. The vessel size (treated = 67.25 µm2, control = 72.27 µm2, p>0.05) and the difference in tissue area occupied by vessels (treated = 2.58%, control = 2.42%, p>0.05) of the radiated regions did not differ between mice. Collagen deposition in the irradiated region was decreased in the treated mouse compared to the control mouse (11% vs. 42%). Conclusion: Human adipose-derived stem cells decrease the duration of ulceration and improve the macroscopic quality of the skin, survive for only 5 days after transplantation, decrease collagen deposition in the irradiated area, increase micro vessel area and size, and decrease the thickness of the irradiated area of the skin compared to controls two weeks after transplantation.

Biodistribution and toxicity of antimicrobial ionic silver (Ag+) and silver nanoparticle (AgNP+) species after oral exposure, in Sprague-Dawley rats

Although antimicrobial nanosilver finds numerous applications in the health and food industries, the in vivo toxicity of positively charged silver nanoparticles (AgNPs+) and relevant controls are largely unexplored. This study investigates the relationship between the biodistribution and toxicity of the well-known cetyltrimethylammonium bromide (CTAB)-capped AgNPs+ in 6-weeks old female Sprague-Dawley rats, at sublethal doses. Amounts comparative to those leaked from food products or considered for animal feed were administered through daily water intake, for an 18-day period: AgNPs+ (40 μg mL−1), Ag+ (40 μg mL−1), antimicrobial CTAB+ (24 μg mL−1) and tap water. All exposures except for the water control had adverse effects on the health and systemic functions of rats (e.g., lethargy, hepatomegaly, splenomegaly, impediment of bone development, and/or heightened immune response). Although the total Ag accumulation in tissues (1.4–1.6 μg of Ag/g of liver, spleen, jejunum, and brain) was comparable for the two Ag species, AgNPs+ were generally more toxic than Ag+, particularly in spleen (0.8 μg Ag/g). Significantly reduced euthanasia time, alopecia, inflammatory responses in spleen, fragile veins, and enhanced lymphocytosis were observed only for AgNPs+. Overall, this study raises health concerns about the ingestion of capped-AgNPs+ or Ag+ by first-hand consumers and industry workers.

MO284: Dipeptidyl-Peptidase-4 Inhibitor, Teneligliptin, Ameliorates Folic Acid-Induced Acute Kidney Injury in Mice

Abstract BACKGROUND AND AIMS Acute kidney injury (AKI) is histologically characterized by tubular cell death and inflammation. It has demonstrated that cell death in renal tubule cells on AKI could be introduced by regulated necrosis or apoptosis, but the molecular mechanisms are reported to be different between AKI models [1]. Recently, Dipeptidyl -peptidase-4(DPP4) inhibitors have been reported to ameliorate cisplatin-induced AKI and ischemia-reperfusion-induced AKI [2, 3]. In this study, we examined the effect of teneligliptin, a DPP4 inhibitor, on folic acid (FA)-induced AKI(FA-AKI) model in mice, which is reported to be mainly induced by ferroptosis, one of the regulated necrotic pathways [4]. METHOD C57BL/6J mice (12–14 weeks old) received a single intraperitoneal injection of FA of 250 mg/kg in 0.3 mol/L sodium bicarbonate, and were euthanized 48 h later. Teneligliptin or vehicle were orally administered just before and 24 h after FA infusion, respectively. Blood samples and renal tissue samples were collected at the time of euthanasia for evaluation. RESULTS The increase of serum creatinine (mean 0.297 mg/dL in teneligliptin-treated mice versus 0.833 mg/dL in vehicle-treated mice; P &amp;lt; 0.01) and blood urea nitrogen (mean 72.22mg/dL in teneligliptin-treated mice versus 177.7mg/dL in teneligliptin-treated mice; P = 0.016) were suppressed in teneligliptin-treated mice, compared to vehicle-treated mice (Fig. 1A and B). Histologically, teneligliptin treatment reduced tubular injury score, tubular cell death evaluated with the terminal uridine nick-end labeling (TUNEL) and kidney injury molecule-1 (kim-1) expression in the kidney (Fig. 2A-C). In addition, number of infiltrating Ly6G + neutrophil and number of infiltrating F4/80 + macrophages were decreased in the kidney of teneligliptin-treated mice, compared with that of vehicle-treated mice (Fig. 2D and E). CONCLUSION We found that teneligliptin ameliorated FA-AKI in mice by suppressing necroinflammation in the kidney. Further studies are needed to clarify the underlying mechanisms of the renoprotection.

Neuregulin‐1β promotes myoblast differentiation and is anti‐cachexic in respiratory muscles of post‐infarcted swine

Neuregulin‐1β (NRG‐1β) is a growth and differentiation factor with pleiotropic systemic effects. Because NRG‐1β has therapeutic potential for heart failure and known growth effects in skeletal muscle, we hypothesized that it might affect heart failure‐associated cachexia, a severe co‐morbidity characterized by a loss of muscle mass. We therefore assessed NRG‐1β’s effect on skeletal muscle gene expression in a swine model of heart failure using recombinant Glial Growth Factor 2 (USAN ‐ cimaglermin alfa), a version of NRG‐1β currently being tested in humans with systolic heart failure. Animals received one of two intravenous doses (0.67 or 2 mg/kg) of NRG‐1β bi‐weekly for 4 weeks, beginning one week after infarct. Skeletal muscle was collected at the time of euthanasia from the intercostal space and paired‐end RNA sequencing performed. NRG‐1β treatment altered expression of 583 transcripts including 242 transcripts altered at both doses. These included genes required for myofiber growth, maintenance and survival such as MYH3, MYHC, MYL6B, KY and HES1. Importantly, NRG‐1β altered the directionality of at least 85 genes associated with cachexia, including myostatin, which negatively regulates myoblast differentiation by down‐regulating MyoD expression. Consistent with this, MyoD was increased in NRG‐1β treated animals. In vitro experiments with myoblast cell lines confirmed that NRG‐1β induces skeletal muscle differentiation with an absolute requirement for ERBB signaling on myoblast differentiation. These findings suggest a NRG‐1β‐mediated anti‐atrophic, anti‐cachexia effect that may provide additional benefits to this potential therapy in heart failure.

Prior Angiotensin Converting Enzyme Inhibition Differentially Alters Angiotensin II‐Induced Oxidative Stress in Male and Female Hypertensive Rats

Hypertension promotes fibrotic cardiac remodeling that involves oxidative stress and inflammation that contribute to heart failure. Transient angiotensin converting enzyme inhibitor (ACEi) treatment in male hypertensive rats (SHR) produces persistent changes in the left ventricle (LV) that render it resistant to future fibrosis and inflammation. Oxidative stress produced by NADPH oxidase (NOX) enzymes is linked to angiotensin II (Ang II)‐mediated fibrotic signaling. Ang II also promotes secretion of macrophage‐recruiting cytokines including monocyte chemoattractant protein 1 (MCP1) which can perpetuate oxidative stress. Thus the present study investigated the impact of transient ACEi‐induced cardio‐protection on subsequent Ang II‐induced inflammatory and oxidative stress responses. After a 2‐week ACEi treatment (enalapril, 30mg/kg/day) followed by a 2‐week washout period male and female SHRs (11 wk old) were infused with Ang II (400ng/kg/min, s.c.) or vehicle (saline, n=5‐11/group/sex) for 2 weeks. At time of euthanasia, a mid‐myocardial section was fixed and paraffin embedded and remaining LV tissue was collected for protein, RNA and DNA extraction. LV macrophage infiltration was determined histologically by staining for ED‐1. MCP1 gene expression was evaluated by RTqPCR, and pro‐oxidants (NOX2 and NOX4) and antioxidants (catalase and superoxide dismutase 2; SOD2) were measured by immunoblotting. Oxidative DNA damage was quantified using an 8‐hydroxy‐2’‐deoxyguanosine (8‐OHdG) ELISA. Cardiac MCP1 gene expression was increased by Ang II in both sexes with no impact of transient ACEi. However, Ang II induced LV macrophage infiltration (ED‐1) in males only which was attenuated by prior transient ACEi. LV NOX2 was increased by Ang II and attenuated by prior transient ACEi in both sexes. NOX4 was reduced in females previously treated with an ACEi, but not males. The antioxidant, catalase, was increased by Ang II in both sexes, but attenuated by transient ACEi in females only. Further, both catalase and SOD2 were positively correlated to NOX2 in females, but not males. This suggests that Ang II‐induced pro‐oxidant responses may be more tightly coupled with antioxidant responses in females. DNA damage as measured by 8‐OHdG was not significantly impacted by Ang II in males or females. However 8‐OHdG was reduced in males previously treated with an ACEi suggesting that an alternative antioxidant mechanism may be active in males previously treated with an ACEi. At this dose and duration of Ang II there is not significant oxidative DNA damage, but MCP1 and pro‐oxidants are increased in both sexes. Males exhibit greater macrophage infiltration, while females exhibit greater antioxidant responses; both of which are prevented by prior transient ACEi. In conclusion, there are sex‐specific inflammatory and oxidative stress responses to Ang II and transient ACEi that may impact future cardiac remodeling. Current and future studies will aim to identify mechanisms involved in transient ACEi‐mediated cardio‐protection and how these effects differ between males and females.

Influence of the use of autogenous bone particles to close the access window after maxillary sinus floor augmentation: a micro‐computed tomography and positron emission tomography study in rabbits

AimThe purpose of this study was to evaluate using microCT and positron emission tomography (PET) analysis, the influence on bone healing of the placement of particulate autogenous bone in the antrostomy, and in the subjacent region after maxillary sinus elevation with xenograft.Material and methodsThe sinus mucosa was elevated in sixteen male New Zealand rabbits and they were both grafted with a collagenated cortico-cancellous porcine bone. The antrostomy and the near subjacent region were filled with either the same xenograft (control site) or with particulate autogenous bone (test site) harvested from the tibia. The antrostomies were covered with collagen membranes. MicroCT (measured in Hounsfield Units) and microPET (kBq/cm3) using sodium fluoride infiltration (18F-NaF) were performed at the time of euthanasia that was performed after 1 and 8 weeks of healing, using 8 animals in each group. The Wilcoxon test was used for analysis.ResultsAt the microCT analysis, after 1 and 8 weeks of healing, no statistically significant differences were found between groups. Bone increased and xenograft decreased significantly between the two periods of healing. At the microPET analysis, the percentage of bone increased significantly over time in both test and control groups and no significant differences were found between groups.ConclusionThe placement of autogenous bone in the antrostomy and the subjacent region after maxillary sinus elevation did not enhance bone formation compared with sites where only xenograft was used. Both microCT and microPET showed increase bone formation over time.

An Antibiotic-Loaded Hydrogel Demonstrates Efficacy as Prophylaxis and Treatment in a Large Animal Model of Orthopaedic Device-Related Infection.

Local antibiotic therapy is increasingly being recognised for its role in preventing and treating orthopaedic device-related infection (ODRI). A bioresorbable, injectable gentamicin-loaded hydrogel has been developed to deliver local antibiotics at the time of surgery with potential for both prevention and treatment of ODRI. In a prophylaxis model, the antibiotic hydrogel was compared with systemic perioperative antibiotic prophylaxis alone in twelve sheep (six per group) at the time of intramedullary (IM) nail insertion to the tibia, which was inoculated with methicillin-sensitive Staphylococcus aureus (MSSA). In a treatment model of single-stage revision surgery, adjunctive antibiotic-loaded hydrogel was compared with systemic antibiotics alone in a single stage revision of MSSA infection associated with a tibia intramedullary nail in eleven sheep (five/six per group). The primary endpoint was quantitative microbiological results of soft tissue, bone and sonicate fluid from explanted hardware at the time of euthanasia. At euthanasia, the control sheep that received no local antibiotics in the prophylaxis model were all culture-positive (median 1x108, range 7x106-3x108 colony forming units, CFU) while only two of six sheep receiving local gentamicin had any culture positive biopsies (median 1x101, range 0 - 1x105 CFU). For the treatment model, sheep receiving only systemic antibiotics were all culture-positive (median 8x105, range 2x103- 9x106 CFU) while only two of six sheep treated with gentamicin-loaded hydrogel had any culture positive biopsies (median 3x102, range 0 - 7x104 CFU). Local gentamicin concentrations measured in extracellular fluid in the tibial canal show a burst release of gentamicin from the hydrogel. Serum gentamicin concentrations peaked in both models at one day post application and were below detection limit thereafter. This study has demonstrated the effective use of a locally delivered antibiotic hydrogel for both the prevention and treatment of ODRI that is superior to that of systemic antibiotics alone. Future studies will endeavour to translate from preclinical to clinical research trials.

A collagen membrane for periosteal expansion osteogenesis using a timed-release system in rabbit calvaria

BackgroundThe purpose of this study was to evaluate the effects of resorbable membranes, combined with a shape memory alloy (SMA) mesh device, on bone formation using a timed-release system for periosteal expansion osteogenesis (TIME-PEO).Materials and methodsTwelve Japanese white rabbits were used in this study. An SMA device was inserted under the forehead periosteum, pushed and bent for attachment to the bone surface, and then fixed using resorbable thread. The rabbits were divided into four groups: C1 (5 weeks postoperatively without membrane), C2 (8 weeks postoperatively without membrane), E1 (5 weeks postoperatively with membrane), and E2 (8 weeks postoperatively with membrane). The rabbits were killed 5 or 8 weeks after the operation and the newly formed bone was assessed histologically and radiographically.ResultsSMA devices, concealed under soft tissue until the time of euthanasia, did not cause active inflammation. The mean activation height, from the original bone surface to the midpoint of the mesh, was 3.1 ± 0.6 mm. Newly formed bone was observed, and most of the subperiosteal space underneath the device was occupied by fibrous tissue. Immature bone was present at the outer surface of the original skull bone in all groups. On histomorphometric analysis, there was no significant difference in the volume of the new bone between C1 and E1 (p = 0.885), and C2 and E2 (p = 0.545).ConclusionsPEO using an SMA mesh device, which is based on guided bone regeneration (in atrophic alveolar bone), shows promise as an alternative for bone augmentation, irrespective of whether a resorbable membrane is used.