Neuroimmune response mediated by cytokines in natural scrapie after chronic dexamethasone treatment

biomolecules Article Neuroimmune Response Mediated by Cytokines in Natural Scrapie after Chronic Dexamethasone Treatment Isabel M. Guijarro 1, Moisés Garcés1, Pol Andrés-Benito 2, Belén Marín1, Alicia Otero 1, Tomás Barrio 1, Margarita Carmona 2, Isidro Ferrer 2, Juan J. Badiola 1and Marta Monzón1,*   Citation: Guijarro, I.M.; Garcés, M.; Andrés-Benito, P.; Marín, B.; Otero, A.; Barrio, T.; Carmona, M.; Ferrer, I.; Badiola, J.J.; Monzón, M. Neuroimmune Response Mediated by Cytokines in Natural Scrapie after Chronic Dexamethasone Treatment. Biomolecules 2021,11, 204. https://doi.org/10.3390/biom11020204 Received: 14 January 2021 Accepted: 27 January 2021 Published: 2 February 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Research Centre for Encephalopathies and Transmissible Emerging Diseases, Institute for Health Research Aragón (IIS), University of Zaragoza, C/Miguel Servet 155, 50013 Zaragoza, Spain; [email protected] (I.M.G.); [email protected] (M.G.); [email protected] (B.M.); [email protected] (A.O.); [email protected] (T.B.); [email protected] (J.J.B.) 2Departamento de Patologíay Terapéutica Experimental, Universidad de Barcelona, 08907 Barcelona, Spain; [email protected] (P.A.-B.); [email protected] (M.C.); [email protected] (I.F.) *Correspondence: [email protected]; Tel.: +00-34-976-762944 Abstract: 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. Keywords: scrapie; cytokines; dexamethasone; neuroinflammation; prion diseases 1. Introduction 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 [ 1 – 4 ]. The neuroinflammatory process is defined as the prolonged activation of neuroglial cells with the corresponding production of inflammatory cytokines [ 5 ]. 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 [6–8]. A relevant number of studies have proposed a crucial role for cytokines as neuroinflammatory mediators in the cellular communication in these prion diseases [ 3 , 9 – 14 ]. The detection of these cytokines was described coinciding with the onset of clinical signs in both a murine model [ 9 ] and Creutzfeldt–Jakob disease (CJD) [ 15 ]. Moreover, a recent study described the presence of several genes implicated in inflammation that are upregulated in early phases of prion infection [ 16 ]. Nevertheless, although an altered profile of inflammatory intermediaries has been evidenced in some experimental murine models [ 9 , 14 , 15 , 17 – 21 ], scarce studies have focused on in situ tissue expression of these proteins [10,22], and none of them on a natural model. Biomolecules 2021,11, 204. https://doi.org/10.3390/biom11020204 https://www.mdpi.com/journal/biomolecules Biomolecules 2021,11, 204 2 of 18 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 [ 23 – 25 ]. More recently, this same in vivo model has been used to assess the changes of activation of glial cells associated with antiinflammatory therapy [ 26 ]. 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 [ 27 – 30 ]. 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 [31]. 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 proand 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. 2. Material and Methods 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 [ 26 ]. 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 alaninearginineglutamine and alanine-arginine-histidine, ARQ/ARH, presenting homozygous alanine-arginineglutamine, 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). 2.1. Immunohistochemical Techniques 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). Biomolecules 2021,11, 204 3 of 18 Prior 4 µ m 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-1 α , IL-1R, IL-2R, IL-6, IL-10R, TNFR and IFN γ R) 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 [ 32 ] in 5 microscopic fields in each brain region examined. Moreover, provided that cerebellum has been proposed as a pseudo reference region to detect neuroinflammation [ 33 ], 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 [ 23 , 26 , 34 ]. Table 1 summarizes all the primary antibodies and the protocols applied as following described. Table 1. Antibodies used for immunohistochemical techniques and retrieval method applied for each one. Antibody Antigen Type Dilution Retrieval Method Source IL-1 alpha IL-1 Polyclonal 1:100 Autoclave 121 ◦C (citrate buffer 10%) ThermoFisher Anti-IL-1RN IL-1R Polyclonal 1:100 Autoclave 121 ◦C (citrate buffer 10%) Sigma IL-2R.1 IL-2R Monoclonal 1:1000 PTLink 96 ◦C ThermoFisher 8H12 IL-6 Monoclonal 1:40 Autoclave 121 ◦C (citrate buffer 10%) ThermoFisher OTI1D10 IL-10R Monoclonal 1:250 PTLink 96 ◦C ThermoFisher Ber-H2 TNFR Monoclonal Ready to use PTLink 96 ◦C Dako IFNGR1 IFNγR Polyclonal 1:200 Autoclave 121 ◦C (citrate buffer 10%) ThermoFisher 2.1.1. IL-1, IL-1R, IL-6 and IFNγR Detection 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-1 α (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). 2.1.2. IL-2R, IL-10R and TNFR Detection 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). 2.2. RT-qPCR 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. Biomolecules 2021,11, 204 4 of 18 2.2.1. RNA Purification 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 (ThermoFisher Scientific, Waltham, MA, USA). RNA samples with OD 260/280 ratios close to 5.0 were selected for reverse transcription. Finally, a total of 5 treated and 4 non-treated clinical sheep were included in this molecular analysis. 2.2.2. Retrotranscription Retrotranscription of RNA into cDNA was performed according to the manufacturer’s instructions (High-Capacity CDNA Reverse Transcription Kit, Applied Bio systems, Foster City, CA, USA). 2.2.3. RT-qPCR Gene expression of IL-1, IL-6, IL-10Ra, IL-10Rb and IFN γ in both clinical non-treated and treated sheep was assessed. The parameters of the reactions were 50 ◦ C for 2 min, 95 ◦C for 10 min, and 40 cycles of 95 ◦C for 15 sec and 60 ◦C for 1 min. Data were assessed using the ∆∆ Ct method, using Hypoxanthine Phosphoribosyl transferase 1 (HRPT-1) and β -glucuronidase (GUSβ ) as reference genes. Table 2shows TaqMan probes used for these molecular studies. Table 2. Taqman probes used for RT-qPCR analysis. Gene Full Name Reference Source Gus-β β-glucuronidase (reference gene) Oa04828868_m1 ThermoFisher HPRT-1 Hypoxanthine Phosphoribosyltransferase 1 (reference gene) Oa04825272_gH ThermoFisher IL-1αInterleukin 1 alpha Oa04658681_m1 ThermoFisher IL-6 Interleukin 6 Oa04656315_m1 ThermoFisher IL-10Ra Interleukin 10 receptor alpha Oa04822455_m1 ThermoFisher IL-10Rb Interleukin 10 receptor beta Oa04894070_m1 ThermoFisher IFNγInterferon gamma Oa04657364_m1 ThermoFisher 2.3. Statistical Analysis For IHC results, the normality of distribution was first assessed by KolmogorovSmirnov test. The non-parametric Mann–Whitney Utest was used to assess quantitative differences between non-treated and DEX treated groups. Data of RT-qPCR were evaluated by Student’s ttest after assessing normality also by Kolmogorov–Smirnov test. SPSS software (SPSS Statistics for Windows, Version 17.0, Chicago, IL, USA) was used for these analyses and significance in all cases was taken at * p< 0.05. All graphs were performed with GraphPad Prism 6.0 (San Diego, CA, USA). Data presented in figures are expressed as means and the standard error of the mean (mean +/−SEM). 3. Results 3.1. Immunohistochemistry 3.1.1. IL-1 DEX-treated controls always displayed higher intensity for IL-1 staining compared to the untreated samples. However, clinically treated animals showed lower differences compared to their respective untreated group and were even reversed (Figure 1A). Biomolecules 2021,11, 204 5 of 18 Biomolecules 2021, 11, x FOR PEER REVIEW 5 of 18 Figure 1. IL-1 immunostaining. (A) Note the evident higher intensity of immunostaining in medulla oblongata, MO from DEX treated control sheep compared to an untreated one. Scale bars: 100 µm. (B) A significant effect of DEX was observed in MO from treated controls (* p < 0.05). However, in clinical stage, no significant changes were detected despite of the treatment. The Mann–Whitney U test revealed significant effects of DEX in treated controls compared to untreated samples, showing an increase of IL-1 immunostaining in MO (* p = 0.024). Meanwhile, no changes were detected in clinically treated scrapie sheep depending on the treatment (Figure 1B). In Cb, immunostaining for IL-1 was widespread in all layers (Figure 2A). Figure 2. Morphological findings in cerebellum immunostained with different primary antibodies. (A) Immunostaining for IL-1 was widespread in all layers. (B) IL-1R immunoreactivity was located mainly surrounding Purkinje cells, suggestFigure 1. IL-1 immunostaining. ( A ) Note the evident higher intensity of immunostaining in medulla oblongata, MO from DEX treated control sheep compared to an untreated one. Scale bars: 100 µ m. ( B ) A significant effect of DEX was observed in MO from treated controls (* p< 0.05). However, in clinical stage, no significant changes were detected despite of the treatment. The Mann–Whitney Utest revealed significant effects of DEX in treated controls compared to untreated samples, showing an increase of IL-1 immunostaining in MO ( *p= 0.024 ). Meanwhile, no changes were detected in clinically treated scrapie sheep depending on the treatment (Figure 1B). In Cb, immunostaining for IL-1 was widespread in all layers (Figure 2A). 3.1.2. IL-1R In general, immunostaining was lower in treated animals compared to non-treated ones, except for controls in Fc, where the patterns were exactly the opposite (higher). These differences evidently increased in O and Cb at clinical stage (Figure 3A). Despite the fact that there were no statistically significant changes observed for this marker, a trend to a reduction in immunostaining in Cb from animals in the clinical stage was detected (# p= 0.082) (Figure 3B). IL-1R immunoreactivity in Cb was nearly exclusively located in cells surrounding Purkinje cells, suggesting morphology consistent with specific astrocytes (Figure 2B). 3.1.3. IL-2R It is only relevant to point out that Fc showed an exacerbated increase of reactivity against this cytokine, regardless of treatment, and disease, in comparison with the other brain areas. No statistically significant differences were found regarding treatment in both the control and clinical groups (Figure 4A). Biomolecules 2021,11, 204 6 of 18 Biomolecules 2021, 11, x FOR PEER REVIEW 5 of 18 Figure 1. IL-1 immunostaining. (A) Note the evident higher intensity of immunostaining in medulla oblongata, MO from DEX treated control sheep compared to an untreated one. Scale bars: 100 µm. (B) A significant effect of DEX was observed in MO from treated controls (* p < 0.05). However, in clinical stage, no significant changes were detected despite of the treatment. The Mann–Whitney U test revealed significant effects of DEX in treated controls compared to untreated samples, showing an increase of IL-1 immunostaining in MO (* p = 0.024). Meanwhile, no changes were detected in clinically treated scrapie sheep depending on the treatment (Figure 1B). In Cb, immunostaining for IL-1 was widespread in all layers (Figure 2A). Figure 2. Morphological findings in cerebellum immunostained with different primary antibodies. (A) Immunostaining for IL-1 was widespread in all layers. (B) IL-1R immunoreactivity was located mainly surrounding Purkinje cells, suggestFigure 2. Morphological findings in cerebellum immunostained with different primary antibodies. ( A ) Immunostaining for IL-1 was widespread in all layers. ( B ) IL-1R immunoreactivity was located mainly surrounding Purkinje cells, suggesting a morphology consistent with astrocytes. ( C ) IL-2R was mainly found in cytoplasm of Purkinje cells and cells appearing astrocytes. ( D ) IL-6 immunostaining was mainly present in Purkinje cells as intracytoplasmic staining, as well as stained cells resembling glial cells in both granular and molecular layers. ( E ) IL-10R immunostaining appeared spot intracytoplasmic in Purkinje cells as well as granular cells and other cellular type with astrocytic morphology. ( F ) Purkinje cells expressed TNFR in a very lower extent than the rest of markers. ( G ) IFN γ R immunostaining pattern was mainly localized in the cytoplasm and dendritic spines of Purkinje cells. Scale bars: 50 µm. Biomolecules 2021, 11, x FOR PEER REVIEW 6 of 18 ing a morphology consistent with astrocytes. (C) IL-2R was mainly found in cytoplasm of Purkinje cells and cells appearing astrocytes. (D) IL-6 immunostaining was mainly present in Purkinje cells as intracytoplasmic staining, as well as stained cells resembling glial cells in both granular and molecular layers. (E) IL-10R immunostaining appeared spot intracytoplasmic in Purkinje cells as well as granular cells and other cellular type with astrocytic morphology. (F) Purkinje cells expressed TNFR in a very lower extent than the rest of markers. (G) IFNγR immunostaining pattern was mainly localized in the cytoplasm and dendritic spines of Purkinje cells. Scale bars: 50 µm. 3.1.2. IL-1R In general, immunostaining was lower in treated animals compared to non-treated ones, except for controls in Fc, where the patterns were exactly the opposite (higher). These differences evidently increased in O and Cb at clinical stage (Figure 3A). Despite the fact that there were no statistically significant changes observed for this marker, a trend to a reduction in immunostaining in Cb from animals in the clinical stage was detected (# p = 0.082) (Figure 3B). Figure 3. IL-1R immunostaining. (A) Differences observed after treatment in Cb at clinical stage are illustrated. Scale bars: 100 µm. (B) While a subtle reduction of IL-1R in treated control group was observed in the rest of areas examined, in Fc it increased after DEX treatment. At clinical stage, a decrease of immunostaining in O and a trend (# p = 0.082) to reduction in Cb were detected after treatment. IL-1R immunoreactivity in Cb was nearly exclusively located in cells surrounding Purkinje cells, suggesting morphology consistent with specific astrocytes (Figure 2B). 3.1.3. IL-2R It is only relevant to point out that Fc showed an exacerbated increase of reactivity against this cytokine, regardless of treatment, and disease, in comparison with the other brain areas. Figure 3. Cont. Biomolecules 2021,11, 204 7 of 18 Biomolecules 2021, 11, x FOR PEER REVIEW 6 of 18 ing a morphology consistent with astrocytes. (C) IL-2R was mainly found in cytoplasm of Purkinje cells and cells appearing astrocytes. (D) IL-6 immunostaining was mainly present in Purkinje cells as intracytoplasmic staining, as well as stained cells resembling glial cells in both granular and molecular layers. (E) IL-10R immunostaining appeared spot intracytoplasmic in Purkinje cells as well as granular cells and other cellular type with astrocytic morphology. (F) Purkinje cells expressed TNFR in a very lower extent than the rest of markers. (G) IFNγR immunostaining pattern was mainly localized in the cytoplasm and dendritic spines of Purkinje cells. Scale bars: 50 µm. 3.1.2. IL-1R In general, immunostaining was lower in treated animals compared to non-treated ones, except for controls in Fc, where the patterns were exactly the opposite (higher). These differences evidently increased in O and Cb at clinical stage (Figure 3A). Despite the fact that there were no statistically significant changes observed for this marker, a trend to a reduction in immunostaining in Cb from animals in the clinical stage was detected (# p = 0.082) (Figure 3B). Figure 3. IL-1R immunostaining. (A) Differences observed after treatment in Cb at clinical stage are illustrated. Scale bars: 100 µm. (B) While a subtle reduction of IL-1R in treated control group was observed in the rest of areas examined, in Fc it increased after DEX treatment. At clinical stage, a decrease of immunostaining in O and a trend (# p = 0.082) to reduction in Cb were detected after treatment. IL-1R immunoreactivity in Cb was nearly exclusively located in cells surrounding Purkinje cells, suggesting morphology consistent with specific astrocytes (Figure 2B). 3.1.3. IL-2R It is only relevant to point out that Fc showed an exacerbated increase of reactivity against this cytokine, regardless of treatment, and disease, in comparison with the other brain areas. Figure 3. IL-1R immunostaining. ( A ) Differences observed after treatment in Cb at clinical stage are illustrated. Scale bars: 100 µ m. ( B ) While a subtle reduction of IL-1R in treated control group was observed in the rest of areas examined, in Fc it increased after DEX treatment. At clinical stage, a decrease of immunostaining in O and a trend (# p= 0.082) to reduction in Cb were detected after treatment. Biomolecules 2021, 11, x FOR PEER REVIEW 7 of 18 No statistically significant differences were found regarding treatment in both the control and clinical groups (Figure 4A). Figure 4. No significant differences were found either in control or in clinical groups for immunostaining intensity for (A) IL-2R (B) TNFR or (C) IFNγR. A higher intensity in obex, O and medulla oblongata, MO after treatment was evidenced in controls that reversed in DEX clinical animals. In Cb, IL-2R was limited to the cytoplasm of Purkinje cells and cells with an astrocytic appearance (Figure 2C). 3.1.4. IL-6 Immunostaining for IL-6 in DEX-treated controls was higher in all brain areas compared to untreated controls, except in O, where the labeling intensity was lower. This reduction in O was more evident at clinical stages of disease (Figure 5A). The increase of immunostaining intensity reached significance in Fc of the control group when animals were DEX-treated (* p = 0.040). Meanwhile, the immunoreactivity for this cytokine showed a decreasing trend in control animals (# p = 0.071) that converted into significant in O in the clinical group (* p = 0.041) (Figure 5B). This neuromodulator peptide was mainly present in Purkinje cells as intracytoplasmic staining (Figure 2D). Moreover, the cytokine detected was present in specific cells resembling glial cells in both granular and molecular layers. Figure 4. No significant differences were found either in control or in clinical groups for immunostaining intensity for ( A ) IL-2R ( B ) TNFR or ( C ) IFN γ R. A higher intensity in obex, O and medulla oblongata, MO after treatment was evidenced in controls that reversed in DEX clinical animals. In Cb, IL-2R was limited to the cytoplasm of Purkinje cells and cells with an astrocytic appearance (Figure 2C). 3.1.4. IL-6 Immunostaining for IL-6 in DEX-treated controls was higher in all brain areas compared to untreated controls, except in O, where the labeling intensity was lower. This reduction in O was more evident at clinical stages of disease (Figure 5A). Biomolecules 2021,11, 204 8 of 18 Biomolecules 2021, 11, x FOR PEER REVIEW 8 of 18 Figure 5. IL-6 immunostaining. ( A ) Micrographs represent an increase of IL-6 immunoreactivity in frontal cortex, Fc in treated controls while slightly reduced in both treated groups in obex, O. Scale bars: 100 µ m. ( B ) DEX control group presented a significant increase in Fc (* p< 0.05) and a trend to a decrease in O (# p= 0.071). It was significantly decreased in this area when clinical group was treated (* p< 0.05). Biomolecules 2021,11, 204 9 of 18 The increase of immunostaining intensity reached significance in Fc of the control group when animals were DEX-treated (* p= 0.040). Meanwhile, the immunoreactivity for this cytokine showed a decreasing trend in control animals (# p= 0.071) that converted into significant in O in the clinical group (* p= 0.041) (Figure 5B). This neuromodulator peptide was mainly present in Purkinje cells as intracytoplasmic staining (Figure 2D). Moreover, the cytokine detected was present in specific cells resembling glial cells in both granular and molecular layers. 3.1.5. IL-10R Immunostaining for this marker was the highest in all assessed samples in both the control and clinical groups, regardless of treatment, but especially in Fc, where the highest score was reached regardless of treatment or disease. Along the same lines, although not significant, a trend toward a decrease (# p= 0.074) in Cb of clinical sheep after treatment was observed (Figure 6A). In general, statistically, immunostaining for IL-10R did not reveal any changes between groups after treatment ( p> 0.05 ). Nevertheless, a very relevant decrease in O intensity in treated clinical animals was evidenced to reach a lower intensity than untreated, while the staining intensity was higher in controls (Figure 6B). Biomolecules 2021, 11, x FOR PEER REVIEW 9 of 18 Figure 5. IL-6 immunostaining. (A) Micrographs represent an increase of IL-6 immunoreactivity in frontal cortex, Fc in treated controls while slightly reduced in both treated groups in obex, O. Scale bars: 100 µm. (B) DEX control group presented a significant increase in Fc (*p < 0.05) and a trend to a decrease in O (# p = 0.071). It was significantly decreased in this area when clinical group was treated (* p < 0.05). 3.1.5. IL-10R Immunostaining for this marker was the highest in all assessed samples in both the control and clinical groups, regardless of treatment, but especially in Fc, where the highest score was reached regardless of treatment or disease. Along the same lines, although not significant, a trend toward a decrease (# p = 0.074) in Cb of clinical sheep after treatment was observed (Figure 6A). In general, statistically, immunostaining for IL-10R did not reveal any changes between groups after treatment (p > 0.05). Nevertheless, a very relevant decrease in O intensity in treated clinical animals was evidenced to reach a lower intensity than untreated, while the staining intensity was higher in controls (Figure 6B). Figure 6. IL-10R immunostaining. (A) Decrease in intensity in Cb of treated clinical sheep is illustrated. Scale bars: 100 µm. (B) No significant changes were detected after treatment in control group, only a higher increase in O was outstanding. No significant changes were either observed in clinical stage after treatment, just a trend to lower intensity in Cb of treated clinical sheep (# p = 0.074). This marker stained Purkinje neurons consistent with a spot intracytoplasmic pattern. Additionally, the staining showed a high intensity in granular cells and some others with astrocytic morphology (Figure 2E). Figure 6. IL-10R immunostaining. ( A ) Decrease in intensity in Cb of treated clinical sheep is illustrated. Scale bars: 100 µ m. ( B ) No significant changes were detected after treatment in control group, only a higher increase in O was outstanding. No significant changes were either observed in clinical stage after treatment, just a trend to lower intensity in Cb of treated clinical sheep (# p= 0.074). 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