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Folia Neuropathologica
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vol. 55
Original paper

Early increased density of cyclooxygenase-2 (COX-2) immunoreactive neurons in Down syndrome

Maria Mulet
José Miguel Blasco-Ibáńez
Carlos Crespo
Juan Nácher
Emilio Varea

Folia Neuropathol 2017; 55 (2):154-160
Online publish date: 2017/06/30
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Down syndrome (DS) is the most common chromosomal aneuploidy [26]. Trisomy of chromosome 21 induces a phenotype with two hallmarks: intellectual disability and early development of Alzheimer’s disease (AD) [20].
Alzheimer’s disease development may be related to the presence of the amyloid precursor protein (APP) and the S100 genes on chromosome 21 [1,11]. Alzheimer’s disease has been widely associated with neuroinflammation, a process that may be respon­sible for neuronal death observed in patients. One of the key enzymes at the top of the neuroinflammatory cascade is the COX family (cyclooxygenases) which comprises two members: COX-1 that is expressed under basal conditions and COX-2, an inducible isoform, although expressed weakly under basal conditions. Several studies have analysed the expression of COX-2 in AD brains showing contradictory effects, probably due to the different disease stages in which the studies were performed [5,35].
The Ts65Dn mouse is a DS model which is segmentally trisomic for a portion of mouse chromosome 16 and orthologous to the long arm of the human chromosome 21. This segment contains approximately 140 genes, many of which are highly conserved between mice and humans [32]. Ts65Dn mice reproduce many of the alterations observed in DS, including the cholinergic degeneration present in AD.
In this study, we aim to characterize COX-2 ex­pression in the human temporal cortex in DS and in the Ts65Dn mouse model with age.

Material and methods

Experimental mice were generated by repeated backcrossing of Ts65Dn females to C57/6Ei 9 C3H/HeSnJ (B6EiC3) F1 hybrid males. The parental generation was obtained from the research colony of Jackson Laboratory. Euploid littermates of Ts65Dn mice served as controls. We used a total of 18 trisomic and 18 euploid mice in three groups: young (1 month), adult (3-4 months) and old (12-14 months). The genotypic characterization was established by qRT-PCR using SYBR Green PCR master mix (Applied Biosystems). The amount of each gene was quantified by the ABI PRISM 7700 (Applied Biosystems). The genes analysed were APP (3 copies) and Apo-B (2 copies) [13,18]. Animal experimentation was conducted in accordance with Directive 2010/63/EU of the European Parliament and Council of 22 September 2010 on the protection of animals used for scientific purposes and was approved by the Committee of Bioethics of the University of Valencia. Every effort was made to minimize the number of animals used and their suffering.
Animals were transcardially perfused under deep anaesthesia (choral hydrate 4%, 1 ml/100 gr bw) using 4% paraformaldehyde in phosphate buffer. Brains were cryoprotected using 30% sucrose. Fifty microns thick sections (6 subseries) were collected from each brain using a sliding freezing microtome.
Human samples were obtained from the BiOBANC HCB – IDIBAPS (Barcelona, Spain). Temporal cortex human brain tissue had been fixed (24 h, paraform­aldehyde 4% in buffered solution), cryoprotected (sucrose 30%), stored at –80ºC and cut (8-10 m) with a cryostat. We tested 5 controls (average age 55 years old, 31-78, PMI 11 h, 7-17 h) and 5 individuals with DS (average age 53.6 years old, 36-67, PMI 11.5, 6-18 h).

Single and double immunofluorescence

Tissue was processed “free-floating” (mouse brain sections) or on slides (human sections) for immunofluorescence as follows. Sections were incubated with citrate buffer (0.01 M, pH 6.0) for 1 minute at 100ºC. After this, sections were treated for 1 h with 5% normal donkey serum (NDS) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in PBS with 0.2% Triton-X100 (Sigma-Aldrich, St. Louis, MO, USA) and incubated overnight at room temperature either with only polyclonal goat IgG anti-COX2 (1 : 500, Santa Cruz) antibody or with a mix of COX-2 antibody and one of the following antibodies: monoclonal mouse IgG anti-NeuN (1 : 100, Chemicon), monoclonal mouse IgG anti-Iba1 (1 : 1000, Chemicon), polyclonal rabbit IgG anti-GFAP (1 : 1000, Sigma-Aldrich) or monoclonal mouse IgG anti-RIP (1 : 500, DSHB). Secondary antibodies were Alexa 488 donkey anti-goat IgG (1 : 200 Molecular Probes) and one of the following: Alexa 555 donkey anti-mouse IgG (1 : 200 Molecular Probes) or Alexa 555 donkey anti-rabbit IgG (1 : 200 Molecular Probes). Sections were mounted using Dako fluorescent medium (Dako North America, California). The sections were analysed using a confocal microscope (Leica TSC-SPE). Stacks (z-step 1.15 µm) were analysed using ImageJ software. All studied sections passed through all procedures simultaneously. All slides were coded prior to analysis until the experiment was completed.

Quantification of COX-2 expression

We analysed (1) the number of cells expressing COX-2 (high expression and low expression cells) and (2) the intensity of expression per cell in the temporal cortex of humans and mice (euploid and trisomic) of different ages. (1) For the number of cells, we counted the immunoreactive cells in 500-µm-wide strips (20 strips per group) running perpendicular to the pial surface including all layers of the temporal cortex. After measuring the area, we calculated cellular density. (2) For the intensity of expression per cell, we measured the intensity of fluorescence emission in 50 cells per individual using ImageJ software. Means were determined for each experimental group and data were statistically analysed using SPSS (version 15). The difference between groups was analysed in humans with one way ANOVA (phenotype) and in mice with two way ANOVA (age and phenotype). Parallel Nissl-stained sections were used to locate the analysed region.


COX-2-positive cells could be found in all layers of the temporal cortex, as well as in other cortical regions of the adult mouse. We observed COX-2 expression in two types of cells: small cells with high expression (arrowheads, Fig. 1A) and large cells with low expression (Fig. 1A). Expression of COX-2 in the temporal cortex of humans presented a similar pattern to that of mouse; however the population of small intensely stained cells was absent (Fig. 1B).
Phenotypic characterization of COX-2-expressing cells in the mouse temporal cortex reflected that the large COX-2-positive cells corresponded to neurons (NeuN positive, Fig. 1C) while the small COX-2 positive cells corresponded to microglia (Iba-1 positive, Fig. 1D). COX-2 was absent in astrocytes (GFAP, Fig. 1E) and oligodendrocytes (RIP, Fig. 1F).
Next, we studied COX-2 expression in DS individuals (5 controls and 5 individuals with DS) and found more COX-2-positive cells in individuals with DS (428.0 ± 29.4 cells/mm2) than in controls (331.0 ± 25.4 cells/mm2, p < 0.05) (Fig. 2A). Analysis of the intensity of COX-2 expression in both, control and DS individuals, showed that it was similar in the positive cells in the two groups (Fig. 2B).
In the second part of the study we set out to study whether the increase in COX-2-positive cell number observed in humans was present in the temporal cortex of the mouse model for DS Ts65Dn and if so, to determine the time point of onset of the overexpression of this molecule. As shown above, there are two populations of cells in mice (Fig. 1B): small, high COX-2-expressing cells (microglia) and large, low COX-2-expressing cells (neurons) in young (1 month), adult (4 months) and old (12-14 months) mice. Analysis of the microglial cells expressing COX-2 (Fig. 3A) revealed a phenotype-dependent decrease (p < 0.05). Moreover, the number of microglial cells expressing COX-2 was reduced with age (young-adult p < 0.001, and adult-old p < 0.05). Young: control 122.0 ± 11.2 vs. trisomic 94.4 ± 4.1 cells/mm2; adults: control 70.3 ± 3.0 vs. trisomic 62.5 ± 7.9 cells/mm2; old: control 48.9 ± 10.6 vs. trisomic 38.4 ± 10.8 cells/mm2. Analysis of the number of neurons expressing COX-2 in mice (Fig. 3B) (similar population as observed in humans) showed that the COX-2 neuronal density was not altered between age groups, but similar to humans, we found more COX-2-expressing neurons in trisomic mice (p < 0.001; Fig. 3B). Control: young 696.9 ± 20.1; adult 680.3 ± 25.2; old 669.7 ± 22.4 cells/mm2; trisomic: young 843.9 ± 25.7; adult 836.4 ± 30.5; old 824.2 ± 58.2 cells/mm2. Finally we analysed the intensity of COX-2 expression in the cytoplasm of neurons (Fig. 3C) in the different groups of mice. We observed a decrease in the intensity related to age, however, similar to our findings in humans, there was no difference in the intensity of expression of COX-2 between control and trisomic mice at any age studied.


In conclusion, we have found an increased number of COX-2 immunoreactive cells in the human DS temporal cortex as well as in the brain of the mouse model for DS (Ts65Dn) at any age examined. Our findings in humans confirm previous studies where an increased expression of COX-2 has been observed in other brain regions [24].
In mice it has been shown previously that COX-2 is present in neurons [16,35], although some studies found it in microglia [4,34], and even astrocytes [14]. Using an antibody previously tested in humans, we and others have shown that in humans COX-2-positive cells are always neurons [22]. However, in the mouse temporal cortex we found, in addition to large COX-2-expressing NeuN-positive cells (neurons, Fig. 1C), small COX-2-expressing cells which were Iba-1-positive and therefore microglia (Fig. 1D). COX-2 was absent in astrocytes (GFAP, Fig. 1E) and oligodendrocytes (RIP, Fig. 1F). Here we show that in the Ts65Dn mouse model, the increased number of COX-2-expressing neurons in DS is independent of age. The principal observation of our study is that this alteration starts early in DS, even in young animals (1 month old). This fact opens the possibility that, given that the alteration in COX-2 is previous to any deleterious observation, perhaps the degeneration observed in DS could be related to the alterations observed in markers such as COX-2.
The mechanism underlying the overexpression of COX-2 in DS has not been elucidated. One possibility is that the higher expression of COX-2 may be related to an extra copy of S100B present in individuals with DS [4]. S100B induces the expression of NF-kB, which is responsible, in turn, for COX-2 transcription.
Overexpression of COX-2 seems to be beneficial in the short run [9]. However, a chronic expression may be deleterious for the brain. In our animals, the density of COX-2 neurons remains unaltered with age, although some studies in old animals reported an increase with age [17]. Perhaps the reduced expression observed in the cytoplasm of old neurons in control and Ts65Dn mice, could lead to an underestimation of the number of positive cells in old animals.
Control of COX-2 expression is fundamental because of the reaction products of COX-2, pro-inflam­matory prostanoids and reactive oxygen species, may be cytotoxic and cause CNS injury [29]. However the clear role of COX-2 in the neuroinflammatory process is still a matter of controversy (for a review, see [31]). COX-2 shows a low expression under basal conditions, but is increased during inflammatory responses and can be induced by cytokines and tumour necrosis factor [33]. Pathologies are AD [23] or epilepsy [27] course with an increment in COX-2. Both pathologies present a high prevalence in DS individuals [20,30]. Moreover, DS is a syndrome that induces a premature ageing process. Human fibroblasts of individuals with DS show an excess of COX-2, and, when submitted to pro-inflammatory treatment, respond strongly by increasing COX-2 expression [24]. The elevated expression of COX-2 in DS could underlie, at least in part, the observed neurodegeneration in this syndrome and could explain the early onset of AD.
COX-2 has been related to adult neurogenesis and neuronal function. The KO mice for COX-2 display a reduction in hippocampal neurogenesis [21], moreover the treatment with COX-2 blockers reduces neurogenesis [8] and impairs the formation of LTP [6]. Even animal models for diabetes display parallel alterations in COX-2 expression and neurogenesis [15]. However, other studies have observed that the increased expression of COX-2 reduced the survival of the newborn cells. [2]. However, the model for DS Ts65Dn, showing an increased expression of COX-2 display a reduction in neurogenesis in the hippocampus [7,19].
This apparent discrepancy could be explained attending to three main facts: (a) the analysis of the increased expression of COX-2 observed in the hippocampus of some animal models of insult, such ischemia, which induces an increase in the expression of COX-2 and neurogenesis reflects that the phenotype of the newly generated cells are mainly glia cells [28]; (b) the second possibility is related to the excess of cellular activity observed in DS [10] as well in the Ts65Dn model (Carbonell in preparation), this overactivation could be the basis of the increased expression of COX-2 and (c) the dysregulation observed in the balance between excitation and inhibition observed in DS [3,12,25]. This dysregulation leads to an increase in some inhibitory neurons, including the so-called IS cells [12,13], which inhibits other inhibitory neurons generating an over-activation that is related with the high prevalence of epilepsy in individuals with DS [30] and could be related to the increased expression of COX-2 observed in neurons in the Ts65Dn model.


This study was supported by the Jerome Lejeune Foundation.


Authors report no conflict of interest.


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Copyright: © 2017 Mossakowski Medical Research Centre Polish Academy of Sciences and the Polish Association of Neuropathologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
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