Background

An excessive TLR response causes localized and sometimes disproportionate inflammation, as observed in different types of wound and biomaterial infections. These wound complications delay proper healing and increase the risk of severe infections and sepsis. Considering the latter, several experimental models of sepsis and endotoxin shock have been developed which study the development of systemic inflammation (Lewis et al., 2016). There is however a need for models that address localized inflammatory events from a mechanistic and therapeutic perspective. Activation of the transcription factor NF-κB is a key component of various inflammatory conditions, and hence, NF-κB is considered an important therapeutic target (Liu et al., 2017). Real-time, longitudinal in vivo imaging of NF-κB activation in NFκB-RE-Luc reporter mice is an important tool in studies on inflammatory disease and efficacy of drug treatments. NFκB-RE-Luc reporter mice carry a transgene containing NFκB-responsive elements from the CMVα promoter placed upstream of a basal SV40 promoter, and a modified firefly luciferase cDNA (Carlsen et al., 2002). This reporter element can be induced by LPS and TNF-α (Carlsen et al., 2002), and provides an excellent in vivo tool to monitor transcriptional responses of NF-κB. To achieve visualization of gene expression, luciferin, a substrate for luciferase, is administrated intraperitoneally to mice which in turn generate luminescent signals. Thereafter, in vivo imaging using IVIS spectrum is used for acquisition and analysis of the recorded signals.

Here, we describe a model of LPS-induced local inflammation in NFκB-RE-Luc reporter mice. LPS, mixed in a hydroxyethyl cellulose hydrogel, is injected subcutaneously on the back of mouse. Subcutaneous deposition of hydrogel provides a defined and controlled environment for studies of local NF-κB activation. In addition to LPS, therapeutic agents can also be included in the same hydrogel and their efficacy can be analyzed longitudinally in the same mouse. Moreover, drug deposition and release can also be imaged by using fluorescently labeled drugs. To evaluate local anti-inflammatory efficacy of a peptide drug and for imaging its local distribution, this in vivo imaging protocol has successfully been used by us (Puthia et al., 2020).