Grimace scale assessment during Citrobacter rodentium inflammation and colitis development in laboratory mice.

Introduction: Bacterial infections and chronic intestinal inflammations triggered by genetic susceptibility, environment or an imbalance in the intestinal microbiome are usually long-lasting and painful diseases in which the development and maintenance of these various intestinal inflammations is not yet fully understood, research is still needed. This still requires the use of animal models and is subject to the refinement principle of the 3Rs, to minimize suffering or pain perceived by the animals. With regard to this, the present study aimed at the recognition of pain using the mouse grimace scale (MGS) during chronic intestinal colitis due to dextran sodium sulfate (DSS) treatment or after infection with Citrobacter rodentium. Methods: In this study 56 animals were included which were divided into 2 experimental groups: 1. chronic intestinal inflammation (n = 9) and 2. acute intestinal inflammation (with (n = 23) and without (n = 24) C. rodentium infection). Before the induction of intestinal inflammation in one of the animal models, mice underwent an abdominal surgery and the live MGS from the cage side and a clinical score were assessed before (bsl) and after 2, 4, 6, 8, 24, and 48 hours. Results: The highest clinical score as well as the highest live MGS was detected 2 hours after surgery and almost no sign of pain or severity were detected after 24 and 48 hours. Eight weeks after abdominal surgery B6-Il4/Il10-/- mice were treated with DSS to trigger chronic intestinal colitis. During the acute phase as well as the chronic phase of the experiment, the live MGS and a clinical score were evaluated. The clinical score increased after DSS administration due to weight loss of the animals but no change of the live MGS was observed. In the second C57BL/6J mouse model, after infection with C. rodentium the clinical score increased but again, no increased score values in the live MGS was detectable. Discussion: In conclusion, the live MGS detected post-operative pain, but indicated no pain during DSS-induced colitis or C. rodentium infection. In contrast, clinical scoring and here especially the weight loss revealed a decreased wellbeing due to surgery and intestinal inflammation.

Vascular Network Formation on Macroporous Polydioxanone Scaffolds.

In this study, microvascular network structures for tissue engineering were generated on newly developed macroporous polydioxanone (PDO) scaffolds. PDO represents a polymer biodegradable within months and offers optimal material properties such as elasticity and nontoxic degradation products. PDO scaffolds prepared by porogen leaching and cryo-dried to achieve pore sizes of 326 ± 149.67 µm remained stable with equivalent values for Young's modulus after 4 weeks. Scaffolds were coated with fibrin for optimal cell adherence. To exclude interindividual differences, autologous fibrin was prepared out of human plasma-derived fibrinogen and proved a comparable quality to nonautologous commercially available fibrinogen. Fibrin-coated scaffolds were seeded with recombinant human umbilical vein endothelial cells expressing GFP (GFP-HUVECs) in coculture with adipose tissue-derived mesenchymal stem cells (AD-hMSCs) to form vascular networks. The growth factor content in culture media was optimized according its effect on network formation, quantified and assessed by AngioTool®. A ratio of 2:3 GFP-HUVECs/AD-hMSCs in medium enriched with 20 ng/mL vascular endothelial growth factor, basic fibroblast growth factor, and hydrocortisone was found to be optimal. Network structures appeared after 2 days of cultivation and stabilized until day 7. The resulting networks were lumenized that could be verified by dextran staining. This new approach might be suitable for microvascular tissue patches as a useful template to be used in diverse vascularized tissue constructs. Impact statement We consider this work as important for the current research in the field of tissue engineering and the development of new and functional tissue. The approach for the production of vascularized tissue patches, consisting of the biodegradable synthetic polymer polydioxanone and of the physiological, autologous, and patient-specific polymer fibrin, and seeded with endothelial cells and mesenchymal stem cells, displayed within this work, could be useful for the sustaining development of diverse and more complex tissue constructs. Therefore, these scaffolds could be used as a cornerstone for future tissue engineering approaches.

Lymph Node Stromal Cells From Different Draining Areas Distinctly Regulate the Development of Chronic Intestinal Inflammation.

The balance between the responsiveness of the intestinal immune system and the gut environment is fundamental for the maintenance of intestinal homeostasis, which is required for an adequate recognition of entering antigens. The disruption of this homeostasis by exaggerated immune response to harmless antigens can lead to the development of intestinal disorders such as inflammatory bowel disease. Stromal cells are sessile non-hematopoietic cells that build the backbone of the lymph node, an important site for the immune response induction, but also contribute to immune response and tolerance induction. However, the knowledge about the role of stromal cells in the regulation of inflammatory responses is still limited. Therefore, in this study we analyzed the influence of stromal cells on the development of chronic intestinal inflammation. Here, we show that intestinal inflammation alters the immune activation of the mesenteric lymph node-derived stromal cells. Podoplanin+ and CD21/35+ stromal cells showed increased expression of MHC class II molecules, but CD106 expression on CD21/35+ cells was reduced. Stromal cells secreted cytokines and chemokines such as CCL7 and CXCL16 influenced the gut-homing phenotype and proliferation of CD4+ and CD8+ T cells. Furthermore, stromal cells of peripheral lymph nodes transplanted into the mesentery attenuated colitis severity in B6-Il10-/- mice. The reduced colitis severity in these mice was associated with increased expression of IL4 and distinct activation pattern of stromal cells derived from transplanted peripheral lymph nodes. Altogether, our results demonstrate that lymph node stromal cells impact development of chronic colitis via T cell induction. Moreover, lymph node stromal cells from different draining area due to neonatally imprinted processes distinctly regulate the induction of immune responses.