Neuroinflammation in Primary Cultures of the Rat Spinal Dorsal Horn Is Attenuated in the Presence of Adipose Tissue–Derived Medicinal Signalling Cells (AdMSCs) in a Co-cultivation Model

Abstract

Neuroinflammation within the superficial dorsal horn (SDH) of the spinal cord induces inflammatory pain with symptoms of hyperalgesia and allodynia. Glial activation and production of inflammatory mediators (e.g. cytokines) is associated with modulation of nociceptive signalling. In this context, medicinal signalling cells, e.g. obtained from adipose tissue (AdMSCs), gained attention due to their capacity to modulate the inflammatory response in several diseases, e.g. spinal cord injury. We applied the recently established mixed neuroglial primary cell culture of the rat SDH to investigate effects of AdMSCs on the inflammatory response of SDH cells. Following establishment of a co-cultivation system, we performed specific bioassays for tumour necrosis factor alpha (TNFα) and interleukin (IL)-6, RT-qPCR and immunocytochemistry to detect changes in cytokine production and glial activation upon inflammatory stimulation with lipopolysaccharide (LPS). LPS-induced expression and release of pro-inflammatory cytokines (TNFα, IL-6) by SDH cells was significantly attenuated in the presence of AdMSCs. Further evidence for anti-inflammatory capacities of AdMSCs derived from a blunted LPS-induced TNFα/IL-10 expression ratio and suppressed nuclear translocation of the inflammatory transcription factor nuclear factor kappa B (NFκB) in SDH microglial cells. Expression of IL-10, transforming growth factor beta (TGF-β) and TNFα-stimulated gene-6 (TSG-6) was detected in AdMSCs, which are putative candidates for anti-inflammatory capacities of these cells. We present a novel co-cultivation system of AdMSCs with neuroglial primary cultures of the SDH to investigate immunomodulatory effects of AdMSCs at a cellular level.

Optimizing the Adipogenic Induction Protocol Using Rosiglitazone Improves the Physiological Parameters and Differentiation Capacity of Adipose Tissue-Derived Mesenchymal Stem Cells for Horses, Sheep, Dogs, Murines, and Humans

“…However, the discrepancies between the induction protocols and the absence of common evaluation criteria could impact the differentiation capacity of the stem cell pool, which requires special attention. ASCs' isolation and characterization protocols were previously reported from our group for horse [23], dog [24], and rat cells [25].…”

“…The list of primary and secondary antibodies used in the current study to detect ASC markers, their target species, and their cross reactivity between species are listed in (Table S1). The protocol for ASCs' multipotency was previously described in detail by our group in horse [23], dog [24], and rat cells [25]. Cells of passages P1-P4 from all animal groups and humans were expanded and then used for all experimental procedures.…”

Gabapentinoids Suppress Lipopolysaccharide-Induced Interleukin-6 Production in Primary Cell Cultures of the Rat Spinal Dorsal Horn

“…Moreover, neuronal Ca 2+ responses upon application of excitatory neurotransmitters (glutamate, substance P) were modulated by LPS stimulation. Based on these results, we showed that this in vitro tool can further be applied to study pathophysiological mechanisms including LPS tolerance [34], as well as immunomodulatory capacities of novel treatments such as co-cultivation with adipose tissue-derived medicinal signaling cells [27].…”

“…We have recently shown that neuro-glial primary cell cultures of the rat SDH are a valuable tool to study effects of inflammatory stimulation on neuronal responses, production of inflammatory mediators, and activation of inflammatory transcription factors in vitro [26]. Thus, this approach is also useful to study putative anti-inflammatory or anti-nociceptive capacities of novel therapeutic interventions, such as cell-based therapy [27].…”

Isolation and cultivation as well as in situ identification of MSCs from equine dental pulp and periodontal ligament

“…Hence, CD11a/18, CD45, CD44, CD90, CD105, and MHCII were applied as markers. The FACS analysis was performed as described before by Leisengang et al (26). We also tested the proportion of live cells by, staining with 7amino-actinomycin D (7-AAD, Becton Dickinson, Heidelberg, Germany).…”