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. 2023 Jun 2;24(11):9691.
doi: 10.3390/ijms24119691.

Oleoylethanolamide Treatment Modulates Both Neuroinflammation and Microgliosis, and Prevents Massive Leukocyte Infiltration to the Cerebellum in a Mouse Model of Neuronal Degeneration

Ester Pérez-Martín  1   2 , Laura Pérez-Revuelta  1   2 , Cristina Barahona-López  1 , David Pérez-Boyero  1   2 , José R Alonso  1   2 , David Díaz  1   2 , Eduardo Weruaga  1   2
Affiliations

Oleoylethanolamide Treatment Modulates Both Neuroinflammation and Microgliosis, and Prevents Massive Leukocyte Infiltration to the Cerebellum in a Mouse Model of Neuronal Degeneration

Ester Pérez-Martín et al. Int J Mol Sci. .

Abstract

Neurodegenerative diseases involve an exacerbated neuroinflammatory response led by microglia that triggers cytokine storm and leukocyte infiltration into the brain. PPARα agonists partially dampen this neuroinflammation in some models of brain insult, but neuronal loss was not the triggering cause in any of them. This study examines the anti-inflammatory and immunomodulatory properties of the PPARα agonist oleoylethanolamide (OEA) in the Purkinje Cell Degeneration (PCD) mouse, which exhibits striking neuroinflammation caused by aggressive loss of cerebellar Purkinje neurons. Using real-time quantitative polymerase chain reaction and immunostaining, we quantified changes in pro- and anti-inflammatory markers, microglial density and marker-based phenotype, and overall leukocyte recruitment at different time points after OEA administration. OEA was found to modulate cerebellar neuroinflammation by increasing the gene expression of proinflammatory mediators at the onset of neurodegeneration and decreasing it over time. OEA also enhanced the expression of anti-inflammatory and neuroprotective factors and the Pparα gene. Regarding microgliosis, OEA reduced microglial density-especially in regions where it is preferentially located in PCD mice-and shifted the microglial phenotype towards an anti-inflammatory state. Finally, OEA prevented massive leukocyte infiltration into the cerebellum. Overall, our findings suggest that OEA may change the environment to protect neurons from degeneration caused by exacerbated inflammation.

Keywords: PCD mouse; Purkinje cells; endocannabinoids; microglia; neurodegeneration; neuroinflammation; neurotherapeutics; oleoylethanolamide (OEA).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Experimental design and effect of OEA on mRNA levels of different inflammatory/neuroprotective factors in the short-, medium-, and long term. (a) Schematic representation of the experimental design and timeline followed in this study. (b) Dorsal view of the brain of a 3-week-old WT mouse; the dotted line indicates the specific cerebellar region employed for all mRNA experiments. (c,d) Graph showing relative mRNA levels of different inflammatory markers and neuroprotective factors 3 and 24 h after OEA treatment in 12-day-old WT healthy mice (c) and their corresponding heatmap gene expression (d). (e,f) Graph showing relative mRNA levels of different inflammatory markers and neuroprotective from WT, untreated PCD (PCD), and OEA-treated PCD mice (PCD + OEA) at P20 (e) and their corresponding heatmap gene expression (f). (g,h) Graph showing relative mRNA levels of different inflammatory markers and neuroprotective factors from the same experimental groups as in (e,f) at P30 (g) and their corresponding heatmap gene expression (h). Note that a striking increase in Tnfα gene expression was detected at the onset of the Purkinje cell degeneration process in both PCD groups, and OEA-treated mice showed an overall reduction in mRNA levels of proinflammatory markers and an increase in neuroprotective factors both in the short and the long term, even in an unharmed organism. n = six animals per experimental group (three replicates per biological sample). Kruskal–Wallis’ test for c, e, g.* p < 0.05; ** p < 0.01 (specific p-values for multiple comparisons in Table below). Scale bar: 2 mm. A, anterior; I.P., intraperitoneal; L, left; P, posterior; R, right. Adapted from [47].
Figure 2
Figure 2
Effect of OEA on microglial density in the cerebellum (total and per lobe). (a) Immunolabeling for Iba1 (magenta) showing microglial cells in lobe III (as a representation of lobes I to IX) and lobe X of the cerebellar vermis of WT, PCD, and OEA-treated PCD mice at P30. Note the difference in microglial cell morphology in the zoom inset: branched in WT mice and hypertrophied/globose in PCD mice, regardless of treatment. Solid boxes show zoom inset region and dotted boxes show the position at which micrographs of Figure 3a were taken. (b–d) Quantification of the effect of OEA on total (b) and per lobe (c,d) microglial density; note that OEA treatment decreases the density of Iba1-positive cells in PCD mice when both whole cerebellar vermis and LI to IX are analyzed. Moreover, in contrast to WT mice, an increase in the density of Iba1-positive cells is found in LI to IX compared to LX in both PCD groups. n = 5–7 animals per experimental group. One-way ANOVA followed by Bonferroni’s post hoc test for (b,d); and Student’s t test for (c). ** p < 0.01 (specific p-values for multiple comparisons in Table 2). Scale bar: 100 µm, 20 µm (zoom insets). Adapted from [47].
Figure 3
Figure 3
Effect of OEA on microglial density per cerebellar cortex layers. (a) Magnified images of the dotted boxes in Figure 2a showing Iba1-labeled microglia (magenta) in the different layers of the cerebellar cortex of lobe III (as a representation of LI-IX) and X of WT, PCD, and OEA-treated PCD mice at P30. (b–d) Quantification of microglial density in the total cerebellar cortex (b), lobe I to IX (c), and lobe X (d) for each experimental group, and comparison between layers; note that, unlike WT, the density of Iba1-positive cells is higher in the outermost layers (ML + PCL) compared with the deepest layer (GL) in both PCD groups for all comparisons. (e–g) Quantification of the effect of OEA on microglial density in the entire cerebellar cortex (e), lobe I to IX (f), and lobe X (g), per layer, and comparison among the experimental groups; note that values for OEA-treated PCD are always midway between those of WT and untreated PCD mice for most comparisons. n = 5–7 animals per experimental group. Student’s t-test for (b–d) and one-way ANOVA followed by Bonferroni’s post hoc test for (e–g). * p < 0.05; ** p < 0.01 (specific p-values for multiple comparisons in Table 2). Scale bar: 50 µm. GL, granular layer; ML, molecular layer; PCL, Purkinje cell layer. Adapted from [47].
Figure 4
Figure 4
Marker-based characterization of microglia in the cerebellum of WT, PCD, and OEA-treated PCD mice at P30. At the (top), immunolabeling for Iba1 (magenta) and CD45 (green), i.e., reactive microglial cells. In the (center), immunolabeling for Iba1 (magenta) and CD16/32 (green), i.e., proinflammatory microglial cells. At the (bottom), immunolabeling for Iba1 (magenta) and CD206 (green), i.e., neuroprotective or tissue-reparative microglial cells. Overall, note the higher expression intensity of all the markers analyzed in PCD groups, both untreated and OEA-treated, compared to that of WT mice. Quantitative changes shown in Figure 5. Images in this figure are located in lobe VIII of the cerebellar vermis and presented at the same magnification. Scale bar: 100 µm. Adapted from [47].
Figure 5
Figure 5
Quantitative analyses of microglial populations CD45, CD16/32, and CD206 markers in the cerebellum. (a) Prism representation of the percentage of CD45/Iba1, CD16/32/Iba1, and CD206/Iba1 double-positive cells in the cerebellum for each experimental group: WT, PCD, and OEA-treated PCD mice at P30. Note that WT mice show a lower percentage of all microglial populations compared to that of both PCD groups and that an increase of the anti-inflammatory CD206 marker can be observed in the OEA-treated PCD prism. (b–f) Quantification of the percentage of reactive CD45 microglia in total (b), per lobe (c,e), and per layer (d,f). (g–k) Quantification of the percentage of proinflammatory CD16/32 microglia in total (g), per lobe (h,j), and per layer (i,k). (l–p) Quantification of the percentage of anti-inflammatory CD206 microglia in total (l), per lobe (m,o), and per layer (n,p). Note that the most evident effect of OEA treatment is the increase in the percentage of the anti-inflammatory CD206 microglial marker for all the comparisons analyzed. n = 5–7 animals per experimental group. One-way ANOVA followed by Bonferroni’s post hoc test for (b–d,g–i,l–n); and Student’s t-test for (e,f,j,k,o,p). * p < 0.05; ** p < 0.01 (specific p-values for multiple comparisons in Table 3). Adapted from [47].
Figure 6
Figure 6
Effect of OEA on the overall leukocyte density and their distribution per lobe in the cerebellum. (a) Micrographs of CD45high-labeled leukocytes (green) with a clear rounded or rod-like morphology, distributed in the different layers of the cerebellum of WT, PCD, and OEA-treated PCD mice at P30; nuclei are stained with DAPI (blue); white arrows point out microglia with a clear distinguishable morphology. (b–d) Quantification of overall leukocyte density in the total cerebellar vermis (b) and per lobe (c,d). Note that the increased leukocyte density observed in untreated PCD mice was prevented in those animals treated with OEA, showing similar values to those of WT. n = 5–7 animals per experimental group. One-way ANOVA followed by Bonferroni’s post hoc test for (b,c); and Student’s t-test for (d). ** p < 0.01 (specific p-values for multiple comparisons in Table 4). Scale bar: 20 µm, 5 µm (zoom inset). GL, granular layer; ML, molecular layer; PCL, Purkinje cell layer. Adapted from [47].
Figure 7
Figure 7
OEA treatment modulates neuroinflammation in the model of selective neuronal degeneration PCD by (top to bottom) (i) decreasing microglial density and modulating its phenotype towards an anti-inflammatory/reparative state; (ii) preventing massive leukocyte infiltration into the cerebellar parenchyma; and (iii) downregulating gene expression of proinflammatory mediators and upregulating that of neuroprotective ones. All these effects together could contribute to a less neurotoxic and more neuroprotective environment that promotes the neuroprotection of Purkinje cells demonstrated in [27]. GL, granular layer; ML, molecular layer; PCL, Purkinje cell layer. Partially created with Biorender.com. Modified from [47].
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References

    1. Song W.M., Colonna M. The Identity and Function of Microglia in Neurodegeneration. Nat. Immunol. 2018;19:1048–1058. doi: 10.1038/s41590-018-0212-1. - DOI - PubMed
    1. Kwon H.S., Koh S.-H. Neuroinflammation in Neurodegenerative Disorders: The Roles of Microglia and Astrocytes. Transl. Neurodegener. 2020;9:42. doi: 10.1186/s40035-020-00221-2. - DOI - PMC - PubMed
    1. Subhramanyam C.S., Wang C., Hu Q., Dheen S.T. Microglia-Mediated Neuroinflammation in Neurodegenerative Diseases. Semin. Cell. Dev. Biol. 2019;94:112–120. doi: 10.1016/j.semcdb.2019.05.004. - DOI - PubMed
    1. Salter M.W., Stevens B. Microglia Emerge as Central Players in Brain Disease. Nat. Med. 2017;23:1018–1027. doi: 10.1038/nm.4397. - DOI - PubMed
    1. Kreutzberg G.W. Microglia: A Sensor for Pathological Events in the CNS. Trends Neurosci. 1996;19:312–318. doi: 10.1016/0166-2236(96)10049-7. - DOI - PubMed

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