Patients with acute respiratory distress syndrome (ARDS) exhibit elevated levels of interleukin-6 (IL-6), which correlate with increased morbidity and mortality. The exact role of IL-6 in ARDS has proven difficult to study because it exhibits either pro- or anti-inflammatory actions in mouse models of lung injury, depending on the model utilized. In order to improve understanding of the role of this complex cytokine in ARDS, we evaluated IL-6 using the clinically relevant combination of lipopolysaccharide (LPS) and ventilator-induced lung injury (VILI) in IL-6(-/-) mice. Bronchoalveolar lavage fluid (BAL), whole-lung tissue, and histology were evaluated for inflammatory markers of injury. Transendothelial electrical resistance was used to evaluate the action of IL-6 on endothelial cells in vitro. In wild-type mice, the combination model showed a significant increase in lung injury compared to either LPS or VILI alone. IL-6(-/-) mice exhibited a statistically significant decrease in BAL cellular inflammation as well as lower histologic scores for lung injury, changes observed only in the combination model. A paradoxical increase in BAL total protein was observed in IL-6(-/-) mice exposed to LPS, suggesting that IL-6 provides protection from vascular leakage. However, in vitro data showed that IL-6, when combined with its soluble receptor, actually caused a significant increase in endothelial cell permeability, suggesting that the protection seen in vivo was likely due to complex interactions of IL-6 and other inflammatory mediators rather than to direct effects of IL-6. These studies suggest that a dual-injury model exhibits utility in evaluating the pleiotropic effects of IL-6 in ARDS on inflammatory cells and lung endothelium.

Background: Microfracture or bone marrow stimulation (BMS) is often the first choice for clinical treatment of cartilage injuries; however, fibrocartilage, not pure hyaline cartilage, has been reported because of the development of fibrosis in the repair tissue. Transforming growth factor β1 (TGF-β1), which can promote fibrosis, can be inhibited by losartan and potentially be used to reduce fibrocartilage. Hypothesis: Blocking TGF-β1 would improve cartilage healing in a rabbit knee BMS model via decreasing the amount of fibrocartilage and increasing hyaline-like cartilage formation. Study Design: Controlled laboratory study. Methods: An osteochondral defect was made in the patellar groove of 48 New Zealand White rabbits. The rabbits were divided into 3 groups: a defect group (defect only), a BMS group (osteochondral defect + BMS), and a BMS + losartan group (osteochondral defect + BMS + losartan). For the rabbits in the BMS + losartan group, losartan was administrated orally from the day after surgery through the day of euthanasia. Rabbits were sacrificed 6 or 12 weeks postoperatively. Macroscopic appearance, microcomputed tomography, histological assessment, and TGF-β1 signaling pathway were evaluated at 6 and 12 weeks postoperatively. Results: The macroscopic assessment of the repair revealed that the BMS + losartan group was superior to the other groups tested. Microcomputed tomography showed superior healing of the bony defect in the BMS + losartan group in comparison with the other groups. Histologically, fibrosis in the repair tissue of the BMS + losartan group was significantly reduced when compared with the other groups. Results obtained with the modified O’Driscoll International Cartilage Repair Society grading system yielded significantly superior scores in the BMS + losartan group as compared with both the defect group and the BMS group ( F value: 15.8, P < .001, P = .012, respectively). TGF-β1 signaling and TGF-β-activated kinase 1 of the BMS + losartan group were significantly suppressed in the synovial tissues. Conclusion: By blocking TGF-β1 with losartan, the repair cartilage tissue after BMS was superior to the other groups and consisted primarily of hyaline cartilage. These results should be easily translated to the clinic because losartan is a Food and Drug Administration–approved drug and it can be combined with the BMS technique for optimal repair of chondral defects. Clinical Relevance: Biologically regulated marrow stimulation by blocking TGF-β1 (oral intake of losartan) provides superior repair via decreasing fibrocartilage formation and resulting in hyaline-like cartilage as compared with outcomes from BMS only.

Translational biomaterials targeted toward the regeneration of large bone defects in the mandible require a preclinical model that accurately recapitulates the regenerative challenges present in humans. Computational modeling and in vitro assays do not fully replicate the in vivo environment. Consequently, in vivo models can have specific applications such as those of the mandibular angle defect, which is used to investigate bone regeneration in a nonload-bearing area, and the inferior border mandibular defect, which is a model for composite bone and nerve regeneration, with both models avoiding involvement of soft tissue or teeth. In this protocol, we describe a reproducible load-bearing critical-size composite tissue defect comprising loss of soft tissue, bone and tooth in the mandible of a rabbit. We have previously used this procedure to investigate bone regeneration, vascularization and infection prevention in response to new biomaterial formulations for craniofacial tissue engineering applications. This surgical approach can be adapted to investigate models such as that of regeneration in the context of osteoporosis or irradiation. The procedure can be performed by researchers with basic surgical skills such as dissection and suturing. The procedure takes 1.5-2 h, with ∼2 h of immediate postoperative care, and animals should be monitored daily for the remainder of the study. For bone tissue engineering applications, tissue collection typically occurs 12 weeks after surgery. In this protocol, we will present the necessary steps to ensure reproducibility; tips to minimize complications during and after surgery; and analytical techniques for assessing soft tissue, bone and vessel regeneration by gross evaluation, microcomputed tomography (microCT) and histology.

No abstract

Managing postoperative pain in rodents is an important part of any animal care and use program, and identifying an optimal analgesic plan for a surgical procedure is critical to providing for animal welfare. Opioids and NSAID are commonly used in rodents, but few studies have evaluated their efficacy in surgical models. The current study aimed to evaluate the therapeutic efficacy of clinically relevant doses of buprenorphine (2 formulations) or meloxicam used in combination with ketamine and xylazine anesthesia in a Sprague-Dawley rat ovariohysterectomy surgical model. Rats received either subcutaneous saline once daily for 3 d, low-dose (0.05 mg/kg SC) or high-dose (0.1 mg/kg SC) buprenorphine twice daily for 3 d, a single injection of sustained-release buprenorphine (1.2 mg/kg SC), or low-dose (1 mg/kg SC) or high-dose (2 mg/kg SC) meloxicam once daily for 3 d. Clinical analgesic efficacy was assessed over 8 d according to cageside observation scoring, body weight, and behavioral testing. Ovariohysterectomy was associated with 2 d of postoperative pain, and all 3 buprenorphine dosing strategies and both doses of meloxicam demonstrated varying amounts of analgesia. Given the results of the current study, we recommend 0.05 mg/kg SC buprenorphine at least twice daily or a single dose of 1.2 mg/kg SC of sustained-release buprenorphine for rats undergoing midline laparotomy with ovariohysterectomy. Alternatively, meloxicam at 1 to 2 mg/kg SC once daily could be used for this indication.