Hyperbaric Oxygen Enhances Collagen III Formation in Wound of ZDF Rat

Diabetic foot ulcer (DFU) is a serious complication of diabetes and hyperbaric oxygen therapy (HBOT) is also considered in comprehensive treatment. The evidence supporting the use of HBOT in DFU treatment is controversial. The aim of this work was to introduce a DFU model in ZDF rat by creating a wound on the back of an animal and to investigate the effect of HBOT on the defect by macroscopic evaluation, quantitative histological evaluation of collagen (types I and III), evaluation of angiogenesis and determination of interleukin 6 (IL6) levels in the plasma. The study included 10 rats in the control group (CONT) and 10 in the HBOT group, who underwent HBOT in standard clinical regimen. Histological evaluation was performed on the 18th day after induction of defect. The results show that HBOT did not affect the macroscopic size of the defect nor IL6 plasma levels. A volume fraction of type I collagen was slightly increased by HBOT without reaching statistical significance (1.35±0.49 and 1.94±0.67 %, CONT and HBOT, respectively). In contrast, the collagen type III volume fraction was ~120 % higher in HBOT wounds (1.41±0.81 %) than in CONT ones (0.63±0.37 %; p=0.046). In addition, the ratio of the volume fraction of both collagens in the wound ((I+III)w) to the volume fraction of both collagens in the adjacent healthy skin ((I+III)h) was ~65 % higher in rats subjected to HBOT (8.9±3.07 vs. 5.38±1.86 %, HBOT and CONT, respectively; p=0.028). Vessels density (number per 1 mm2) was found to be higher in CONT vs. HBOT (206.5±41.8 and 124±28.2, respectively, p<0.001). Our study suggests that HBOT promotes collagen III formation and decreases the number of newly formed vessels at the early phases of healing.

GLP-1 alleviates NLRP3 inflammasome-dependent inflammation in perivascular adipose tissue by inhibiting the NF-κB signalling pathway

Objectives To study the effect of glucagon-like peptide 1 (GLP-1) on NLR family pyrin domain containing 3 (NLRP3) inflammasome-induced inflammation in perivascular adipose tissue (PVAT) of Zucker diabetic fatty (ZDF) rats and the underlying role of nuclear factor (NF)-κB signalling. Methods Thirty ZDF rats were randomly divided into three study groups: DM (0.9% saline, subcutaneously); DM+GLP-1 (liraglutide, s.c.); and NF-κB+GLP-1 (betulinic acid then liraglutide, s.c.). Ten Zucker lean rats were examined as normal controls. PVAT from ZDF (DM) rats was examined for inflammasome mRNA. Protein levels of NLRP3, cleaved caspase-1, caspase-1, gasdermin D (GSDMD), interleukin (IL)-1β and IL-18 in PVAT were compared between control, DM and DM+GLP-1 groups. Protein levels of NLRP3, IL-1β, IL-18 and NF-κB in PVAT were compared between control, DM, DM+GLP-1 and NF-κB+GLP-1 groups. Results The inflammasome most abundantly expressed in ZDF rat PVAT was NLRP3. NLRP3, cleaved caspase-1, IL-1β, IL-18, and GSDMD were markedly upregulated in DM versus control tissue, and GLP-1 reversed this effect. Inhibition of NLRP3 inflammasome-associated inflammation by GLP-1 was lost by activation of NF-κB with betulinic acid. Conclusion GLP-1 may alleviate NLRP3 inflammasome-dependent inflammation in PVAT by inhibiting NF-κB signalling.

Increased myofilament calcium sensitivity is associated with decreased cardiac troponin I phosphorylation in the diabetic rat heart

Background The diabetic heart has impaired systolic and diastolic function independent of other comorbidities. The availability of calcium is altered, but does not fully explain the cardiac dysfunction seen in the diabetic heart. Thus, we explored if myofilament protein regulation of contraction is altered. Methods Calcium sensitivity (pCa50) was measured in Zucker Diabetic Fatty (ZDF) rat hearts at the initial stage of diabetes (12-week-old) and after 8 weeks of uncontrolled hyperglycaemia (20-week-old) and in non-diabetic (nDM) littermates. Skinned cardiomyocytes were connected to a capacitance-gauge transducer and a torque motor to measure force as a function of pCa (-log[Ca2+]). Fluorescent gel stain (ProQ Diamond) was used to measure total protein phosphorylation. Specific phospho-sites on cardiac troponin I (cTnI) and total cTnI O-GlcNAcylation were quantified using immunoblot. Results pCa50 was greater in both 12- and 20-week-old diabetic (DM) rats compared to nDM littermates (p = 0.0005). Total cTnI and cTnI serine 23/24 phosphorylation were lower in DM rats (p = 0.003 & p = 0.01, respectively), but cTnI O-GlcNAc protein expression was not different. pCa50 is greater in DM rats and corresponds with an overall reduction in cTnI phosphorylation. Conclusions These findings indicate that myofilament calcium sensitivity is increased and cTnI phosphorylation is reduced in ZDF DM rats, which suggests an important role for cTnI phosphorylation in the DM heart.

Future Perspective of Diabetic Animal Models

Objective: The need of today’s research is to develop successful and reliable diabetic animal models for understanding the disease susceptibility and pathogenesis. Enormous success of animal models had already been acclaimed for identifying key genetic and environmental factors like Idd loci and effects of microorganisms including the gut microbiota. Furthermore, animal models had also helped in identifying many therapeutic targets and strategies for immune-intervention. In spite of a quite success, we have acknowledged that many of the discovered immunotherapies are working on animals and did not have a significant impact on human. Number of animal models were developed in the past to accelerate drug discovery pipeline. However, due to poor initial screening and assessment on inequivalent animal models, the percentage of drug candidates who succeeded during clinical trials was very low. Therefore, it is essential to bridge this gap between pre-clinical research and clinical trial by validating the existing animal models for consistency. Results and Conclusion: In this review, we have discussed and evaluated the significance of animal models on behalf of published data on PUBMED. Amongst the most popular diabetic animal models, we have selected six animal models (e.g. BioBreeding rat, “LEW IDDM rat”, “Nonobese Diabetic (NOD) mouse”, “STZ RAT”, “LEPR Mouse” and “Zucker Diabetic Fatty (ZDF) rat” and ranked them as per their published literature on PUBMED. Moreover, the vision and brief imagination for developing an advanced and robust diabetic model of 21st century was discussed with the theme of one miceone human concept including organs-on-chips.

Abstract 251: Comparison of Ischemia-Reperfusion Injury in Langendorff-Prepared Hearts From Zucker Diabetic Fatty and Lean Rats

Introduction: The Zucker Diabetic Fatty (ZDF) rat is a relevant model for assessing ischemia-reperfusion (IR) injury, due to it having comorbidities associated with a higher risk for cardiac arrest and poorer outcomes following cardiopulmonary resuscitation in humans. Hypothesis: Based on previous studies in the literature, we hypothesize that ZDF rats would have similar outcomes to lean rats subjected to IR when accounting for perfusion buffer osmolarity. Methods: ZDF (n=4) and lean control (n=6) rats were anesthetized with pentobarbital, intubated, and mechanically ventilated and monitored for 3-hours, with blood glucose (BG) readings taken before and after ventilation. They were then decapitated and a thoracotomy performed to isolate the Langendorff-prepared hearts. Krebs buffer with the osmolarity adjusted to each rat’s BG level using glucose was used to perfuse the hearts. Following a 20-minute stabilization period, the hearts were subjected to a 30-minute period of global no-flow ischemia followed by 120-minutes of reperfusion. Spontaneous heart rate, isovolumetric left ventricular pressure (LVP), and mitochondrial redox state were measured continuously. Data are mean ± SD. Statistics: two-tailed t-test, p<0.05 Results: There was no significant difference in average body weight between the two groups; however, ZDF rats showed significantly higher BG levels (504±52 vs 174±14 mg/dl) than their lean littermates. ZDF rats had a greater percent change from baseline systolic LVP upon reperfusion than lean rats (133±33.3 vs 84.9±16.5 %), with the lean rats never returning to baseline. Diastolic contracture occurred more in lean rats during ischemia, but trended towards a lower contracture during reperfusion. No significant difference was seen in developed LVP, rate pressure products, or mitochondrial redox states, although lean rats trended towards better diastolic contractility and relaxation. Conclusion: Lean rats showed marginally better outcomes in heart function than their ZDF littermates. Future studies will focus on potential mechanisms of Type 2 diabetes that may exacerbate IR injuries.

Rodent models of leptin receptor deficiency are less sensitive to amylin

The pancreatic hormone amylin is released from beta cells following nutrient ingestion and contributes to the control of body weight and glucose homeostasis. Amylin reduces food intake by activating neurons in the area postrema (AP). Amylin was also shown to synergize with the adipokine leptin, with combination therapy producing greater weight loss and food intake reduction than either hormone alone. Although amylin and leptin were initially thought to interact downstream of the AP in the hypothalamus, recent findings show that the two hormones can act on the same AP neurons, suggesting a more direct relationship. The objective of this study was to determine whether amylin action depends on functional leptin signaling. We tested the ability of amylin to induce satiation and to activate its primary target neurons in the AP in two rodent models of LepR deficiency, the db/db mouse and the Zucker diabetic fatty (ZDF) rat. When compared with wild-type (WT) mice, db/db mice exhibited reduced amylin-induced satiation, reduced amylin-induced Fos in the AP, and a lower expression of calcitonin receptor (CTR) protein, the core component of all amylin receptors. ZDF rats also showed no reduction in food intake following amylin treatment; however, unlike the db/db mice, levels of amylin-induced Fos and CTR in the AP were no different than WT rats. Our results suggest that LepR expression is required for the full anorexic effect of amylin; however, the neuronal activation in the AP seems to depend on the type of LepR mutation.

A standardised framework to identify optimal animal models for efficacy assessment in drug development

IntroductionPoor translation of efficacy data derived from animal models is a potential contributor to costly and unnecessary attrition in clinical trials.ObjectivesTo develop a tool to assess, validate and compare the clinical translatability of animal models used for the preliminary assessment of efficacy.Design and ResultsWe conducted an exploratory literature search to identify the key aspects to validate animal models. Eight aspects (Epidemiology, Pathophysiology, Genetic, Biochemistry, Aetiology, Histology, Pharmacology and Endpoints) were identified for which questions were drafted to evaluate the different faces of the human disease simulation. Features of the framework include standardised instructions, a weighting and scoring system to compare models as well as contextualising factors regarding model similarity and evidence uncertainty. We included a quality assessment of the internal validity of drug intervention studies included in the Pharmacological validation section for both effective and ineffective drugs in humans. A web-based survey was conducted with experts from different stakeholders to gather input on the framework. Finally, we present a case study of a preliminary validation and comparison of two animal models for Duchenne Muscular Dystrophy (mdx mouse and GRMD dog) and Diabetes Type 2 (ZDF rat and db/db mouse). We show that there are significant differences between the mdx mouse and the GRMD dog, the latter mimicking the human condition to a greater extent than the mouse despite the considerable lack of published data. In DT2, both the ZDF rat and the db/db mouse are comparable with minor differences in pathophysiology.ConclusionsFIMD facilitates drug development by serving as the basis to select the most relevant model that can provide meaningful and translatable results to progress drug candidates to the clinic.

Effect of type II diabetes on male rat bladder contractility

Type II diabetes is the most prevalent form of diabetes. One of the primary complications of diabetes that significantly affects quality of life is bladder dysfunction. Many studies on diabetic bladder dysfunction have been performed in models of type I diabetes; however, few have been performed in animal models of type II diabetes. Using the Zucker Diabetic Fatty (ZDF) rat model of type II diabetes, we examined the contractility and sensitivity of bladder smooth muscle in response to mediators of depolarization-induced contraction, muscarinic receptor-mediated contraction, ATP-induced contraction, and neurogenic contraction. Studies were performed at 16 and 27 wk of age to monitor the progression of diabetic bladder dysfunction. Voiding behavior was also quantified. The entire bladder walls of diabetic rats were hypertrophied compared with that of control rats. Contractility and sensitivity to carbachol and ATP were increased at 27 wk in bladder smooth muscle strips from diabetic rats, suggesting a compensated state of diabetic bladder dysfunction. Purinergic signaling was increased in response to exogenous ATP in bladders from diabetic animals; however, the purinergic component of neurogenic contractions was decreased. The purinergic component of neurogenic contraction was reduced by P2X receptor desensitization, but was unchanged by P2X receptor inhibition in diabetic rats. Residual and tetrodotoxin-resistant components of neurogenic contraction were increased in bladder strips from diabetic animals. Overall, our results suggest that in the male ZDF rat model, the bladder reaches the compensated stage of function by 27 wk and has increased responsiveness to ATP.