The actual role of prion protein-induced glial activation and subsequent cytokine secretion during prion diseases is still incompletely understood. The overall aim of this study is to assess the effect of an anti-inflammatory treatment with dexamethasone on different cytokines released by neuroglial cells that are potentially related to neuroinflammation in natural scrapie. This study emphasizes the complex interactions existent among several pleiotropic neuromodulator peptides and provides a global approach to clarify neuroinflammatory processes in prion diseases. Additionally, an impairment of communication between microglial and astroglial populations mediated by cytokines, mainly IL-1, is suggested. The main novelty of this study is that it is the first one assessing in situ neuroinflammatory activity in relation to chronic anti-inflammatory therapy, gaining relevance because it is based on a natural model. The cytokine profile data would suggest the activation of some neurotoxicity-associated route. Consequently, targeting such a pathway might be a new approach to modify the damaging effects of neuroinflammation.
Scrapie is considered the prototype of prion diseases, which are a group of neurodegenerative disorders caused by the conversion of a cellular protein into a pathological isoform called prion.
Neuroinflammation is currently a widely accepted concept in neurodegeneration, particularly in prion diseases. The neuroinflammatory process is defined as the prolonged activation of neuroglial cells with the corresponding production of inflammatory cytokines. Consequently, there is a particular interest in investigating the roles of the innate and adaptive immune systems in several neurodegenerative disorders, with neuroglia as a key element in the neuropathological process.
A relevant number of studies have proposed a crucial role for cytokines as neuroinflammatory mediators in the cellular communication in these prion diseases. The detection of these cytokines was described coinciding with the onset of clinical signs in both a murine model and Creutzfeldt–Jakob disease (CJD). Moreover, a recent study described the presence of several genes implicated in inflammation that are upregulated in early phases of prion infection. Nevertheless, although an altered profile of inflammatory intermediaries has been evidenced in some experimental murine models, scarce studies have focused on in situ tissue expression of these proteins, and none of them on a natural model.
Previous studies developed in scrapie-affected animals have led to conclusions about the glial role in the neurodegenerative progress that was extrapolated not only to other prion but also other neurodegenerative disorders. More recently, this same in vivo model has been used to assess the changes of activation of glial cells associated with anti-inflammatory therapy. This study constituted a powerful approach to the involvement of immune response in this neurodegenerative disease, confirming the occurrence of neuroinflammation in neurodegeneration. Specifically, a potential failure of astrocytes and a stimulation of phagocytosis of prion protein deposits by microglia were evidenced after dexamethasone (DEX) treatment. To examine the interglial communication mediated by cytokines in depth constitutes a main tool for advancement of the knowledge of how these mediators are really involved in neuroinflammatory mechanisms contributing to neurodegeneration. It is indispensable to study the possible alteration of glial crosstalk that might enhance instead of prevent neuronal damage. Thus, it could be a crucial target for therapeutic approaches in prion diseases.
Overall, the actual role of prion protein-induced glial activation and subsequent cytokine expression during prion diseases is still incompletely understood. Consequently, to investigate the possible alterations of in situ cytokine expression in brain samples from animals naturally affected by scrapie and DEX treatment would be really helpful to determine whether these proteins could be significant factors in the progress of neurodegeneration in this group of diseases. Thus, the specific aim of this study is to assess the effect of the anti-inflammatory treatment on different cytokines which could be potentially related to neuroinflammation. Both immunohistochemical and expression patterns of different pro- and anti-inflammatory cytokines in several brain regions from treated and non-treated scrapie-affected sheep are compared in this study as a first step towards the ultimate goal that is to determine whether these proteins represent relevant targets in the immunopathogenesis of neurodegeneration.
All the following experimental procedures were previously approved by the Ethical Committee of University of Zaragoza (Reference number: PI41/16, 03/10/2016). All efforts were made to minimize animal suffering during the experiments and to reduce the number of animals used.
All the analyses were performed on samples coming from animals included in a previously published study where, as cited above, the glial activation response in the progress of natural scrapie after chronic DEX treatment had been assessed. All experimental details were provided in this previous manuscript, but briefly, a total of 25 sheep (age ranging from 4 to 10 years and all except for one of them with heterozygous alanine-arginine- glutamine and alanine-arginine-histidine, ARQ/ARH, presenting homozygous alanine-arginine- glutamine, ARQ/ARQ genotype) were included in this study: 10 healthy control (of which 4 treated and 6 non-treated) and 15 clinical scrapie Rasa Aragonesa ewes (10 treated plus 5 non-treated). Healthy controls were considered essential in order to specifically observe the effect of treatment (daily intramuscular, IM 0.04 mg/kg dose until euthanasia by endpoint criteria, 16 months the longest) in normal conditions in ovine species. After euthanasia with intravenous pentobarbital injection, necropsy of each sheep was performed and 80 samples were subsequently collected and distributed for different studies. One hemi-section from each sample was fixed by immersion in 4% paraformaldehyde for immunohistochemical studies and the other hemi-section was frozen at −80 °C for molecular studies (RT-qPCR).
Immunohistochemistry (IHC) was carried out in order to assess in situ neuroinflammatory profile associated with DEX treatment in all sheep. It was compared with non-treated sheep group in four encephalic areas (frontal cortex: Fc, cerebellum: Cb, obex: O and medulla oblongata: MO).
Prior 4 µm sectioning, paraffin-embedding of fixed samples was developed. After specific pre-treatments for antigen retrieval, specific immunohistochemical protocols by using specific primary antibodies against those cytokines or their receptors mainly studied in literature related (to our knowledge, IL-1α, IL-1R, IL-2R, IL-6, IL-10R, TNFR and IFNγR) were applied. EnVision system (DAKO, Glostrup, Denmark) and diaminobenzidine (DAB; DAKO, Glostrup, Denmark) were used as the visualization system and chromogen, respectively. Hematoxylin counterstaining and mounting in DPX was finally performed on all sections.
All slides were analysed by two independent observers scoring the intensity of immunostaining from 0 (absence) to 4 (maximum presence) by counting positive cells in 5 microscopic fields in each brain region examined. Moreover, provided that cerebellum has been proposed as a pseudo reference region to detect neuroinflammation, close attention was paid to different profiles of cytokine distribution in this brain region. The focus was on Purkinje cells based on previous results referring this neuronal type as the most damaged while they are the most protected neurons in this area. Table 1 summarizes all the primary antibodies and the protocols applied as following described.
A pre-treatment consisting of hydrated heating at 121 °C in citrate buffer 10% for 20 min preceded the endogenous peroxidase blocking (DAKO, Glostrup, Denmark) for 5 min and incubation overnight 4 °C with different primary antibodies: polyclonal IL-1α (1:100; ThermoFisher Scientific, Waltham, MA, USA), polyclonal IL-1RN (1:100, Sigma, St. Louis, MO, USA), monoclonal 8H12 (1:40; ThermoFisher Scientific, Waltham, MA, USA) or polyclonal IFNGR1 (1:200, ThermoFisher Scientific, Waltham, MA, USA).
A pre-treatment consisting of hydrated heating at 96 °C in citrate buffer 10% for 20 min preceded the endogenous peroxidase blocking (DAKO, Glostrup, Denmark) for 5 min and incubation with different primary monoclonal antibodies: IL-2R.1 (1:1000, overnight 4 °C; ThermoFisher Scientific, Waltham, MA, USA), OTI1D10 (1:250, overnight 4 °C; ThermoFisher Scientific, Waltham, MA, USA) or Ber-H2 (ready to use, 30 min RT; DAKO, Glostrup, Denmark).
Cerebellum and frontal cortex frozen tissues from treated and non-treated scrapie animals were included in the following comparative molecular analysis for some inflammatory markers.
The purification of RNA was performed following the instructions of the supplier (RNeasy Lipid Tissue Mini kit, Qiagen, GmbH, Hilden, Germany). RNA integrity and 28S/18S ratios were determined with the Agilent Bioanalyzer (Agilent Technologies Inc, Santa Clara, CA, USA). Samples were treated with DNase digestion, and RNA concentration was evaluated using a NanoDrop Spectrophotometer (Ther
