Marc Oudart

Romain Tortuyaux

Philippe Mailly

Noémie Mazaré

Anne-Cécile Boulay

Martine Cohen-Salmon
email: martine.cohen-salmon@college-de-france.fr

AstroDot: a new method for studying the spatial distribution of mRNA in astrocytes Running title: in situ quantification of mRNA in astrocytes

Keywords: Astrocyte, Microglia, Hippocampus, mRNA, In situ hybridization, Immunofluorescence, ImageJ, Gfap , Gfap  Rpl4: APPswe/PS1dE9, Alzheimer's disease, Astrocyte reactivity, Gfap, Iba1

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Astrocytes are the most abundant glial cells in the brain. Although the astrocytes' characteristics vary from one region of the brain to another, they all have a large number of processes that ramify into branches and then secondary branchlets. Hence, protoplasmic astrocytes are large, bushy-shaped cells with diameters of ~40-60 μm and volumes of ~10 4 μm [START_REF] Chever | Astroglial connexin43 hemichannels tune Basal excitatory synaptic transmission[END_REF] . Each astrocyte covers a unique domain, and (in humans) contacts up to 2 million synapses [START_REF] Ogata | Structural and quantitative analysis of astrocytes in the mouse hippocampus[END_REF] . At the synaptic interface, perisynaptic astrocyte processes (PAPs) sense the extracellular interstitial fluid, take up neurotransmitters and ions [START_REF] Dallerac | Versatile control of synaptic circuits by astrocytes: where, when and how?[END_REF] , and release neuroactive factors [START_REF] Chever | Astroglial connexin43 hemichannels tune Basal excitatory synaptic transmission[END_REF][START_REF] Sultan | Synaptic Integration of Adult-Born Hippocampal Neurons Is Locally Controlled by Astrocytes[END_REF] . Astrocytes are also in contact with blood vessels; indeed, the latter are entirely sheathed in perivascular astrocyte processes (PvAPs) [START_REF] Mathiisen | The perivascular astroglial sheath provides a complete covering of the brain microvessels: an electron microscopic 3D reconstruction[END_REF] . The astrocytes at this interface modulate the integrity and functions of the blood-brain barrier, immunity [START_REF] Alvarez | Glial influence on the blood brain barrier[END_REF] , cerebral blood flow [START_REF] Iadecola | The Neurovascular Unit Coming of Age: A Journey through Neurovascular Coupling in Health and Disease[END_REF] , and interstitial fluid drainage [START_REF] Aspelund | A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules[END_REF] . The mechanisms underlying the astrocyte's synaptic and vascular influence are critically important, and have attracted much research interest. Indeed, dysregulation of the astrocytes' functions and interplay with neurons and the vascular system contributes to the development and progression of most neurological diseases [START_REF] Iadecola | The Neurovascular Unit Coming of Age: A Journey through Neurovascular Coupling in Health and Disease[END_REF][START_REF] Dossi | Human astrocytes in the diseased brain[END_REF][START_REF] Verkhratsky | Why are astrocytes important?[END_REF] .

Recent studies of the astrocyte's functional polarity have suggested that mRNA distribution and local translation regulates protein delivery in space and time. In a previous study, we showed that local translation is determined in PvAPs and we characterized the locally translated molecular repertoire [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF] .

Local translation has also been observed in the radial glia during brain development [START_REF] Pilaz | Dynamic mRNA Transport and Local Translation in Radial Glial Progenitors of the Developing Brain[END_REF] and in PAPs in the adult cortex [START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF] . Interestingly, these studies showed that some mRNAs were specifically present in low or high levels in the astrocyte soma or processes; hence, mRNA distribution appears to obey specific rules and meet specific needs, and may help to regulate distal perivascular and perisynaptic functions.

To further characterize the mRNA distribution in astrocytes, we developed a new three-dimensional in situ method for identifying astrocyte mRNAs colocalized on GFAP-immunolabelled processes and quantifying these levels with dedicated bioinformatics tools. More precisely, we studied the distribution of mRNAs encoding the astrocyte-specific GFAP  and  isoforms (generated by alternative splicing) in the CA1 and CA3 regions of the hippocampus in wild-type (WT) mice and in the APPswe/PS1dE9 mouse model of Alzheimer's disease (AD). We further showed that our approach can be applied to microglia (immunolabelled for ionized calcium binding adaptor molecule 1 (Iba1)) and to all types of mRNA.

Results

Gfap  and Gfap  mRNAs are distributed in PAPs

Gfap mRNAs have already been detected in distal perivascular [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF] and perisynaptic processes [START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF] of astrocytes -suggesting that local GFAP translation regulates distal intermediate filament assembly.

Although previous research focused on the canonical isoform GFAP , at least 10 GFAP isoforms (generated by alterative mRNA splicing and polyadenylation signal selection) have been described 14- 17 . GFAP  has received special interest because it is a tumour marker [START_REF] Brehar | Immunohistochemical analysis of GFAP-delta and nestin in cerebral astrocytomas[END_REF][START_REF] Choi | Enhanced glial fibrillary acidic protein-delta expression in human astrocytic tumor[END_REF][START_REF] Heo | A histopathological diagnostic marker for human spinal astrocytoma: expression of glial fibrillary acidic protein-delta[END_REF] and is associated with neurogenic niches [START_REF] Van Den Berge | Longterm quiescent cells in the aged human subventricular neurogenic system specifically express GFAP-delta[END_REF] . In fact, GFAP is encoded by the same first 7 exons as GFAP  but has a different C-terminal (Fig. 1A), allowing it to form a heterodimer with GFAP and thus promote intermediate filament aggregation [START_REF] Moeton | GFAP isoforms control intermediate filament network dynamics, cell morphology, and focal adhesions[END_REF][START_REF] Perng | Glial fibrillary acidic protein filaments can tolerate the incorporation of assembly-compromised GFAP-delta, but with consequences for filament organization and alphaB-crystallin association[END_REF] . Here, we first looked for GFAP  and GFAP mRNAs in PAPs. Polysomal mRNAs were extracted by translating ribosome affinity purification (TRAP) from adult Aldh1-RPL10a mice, which express the ribosomal protein Rpl10a specifically in astrocytes [START_REF] Heiman | Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP)[END_REF] .

Extractions were performed either from hippocampus (for whole-astrocyte polysomal mRNAs) or synaptogliosome preparations (consisting of apposed pre-and post-synaptic membranes and astrocyte PAPs), in order to extract polysomal mRNAs contained in PAPs [START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF][START_REF] Carney | Proteomic analysis of gliosomes from mouse brain: identification and investigation of glial membrane proteins[END_REF] . Quantitative qPCR amplification of Gfap  and  mRNA was performed using specific primers (Fig. 1B). Both isoforms were detected in whole astrocytes (mean ± standard error of the mean level: 8.28±1.99 arbitrary units for Gfap  and 1.38±0.20 for Gfap ) and in the perisynaptic processes (17.04±9.09 for Gfap  and 1.16±0.76 for Gfap ). For polysomal mRNAs, the Gfap α/Gfap  ratio was significantly higher in PAPs (40.09±24.27; N=3; p-value=0.05) than in whole astrocytes (5.81±0.67) -suggesting the predominance of Gfap in PAPs (Fig. 1B).

We next sought to visualize Gfap  and mRNAs in hippocampal astrocytes. Fluorescent in situ hybridization (FISH) was performed on 30 m thick free-floating adult mouse brain sections, using specific fluorophore-coupled RNAscope® probes against Gfap exon 9 and Gfap exon 7a (Fig. 1C). Next, the astrocytes' somata and processes were labelled by GFAP immunostaining (Fig. 1C).

Importantly, the co-immunofluorescent detection of proteins depends on the preservation of their epitopes during the protease digestion step preceding FISH. We observed dense, continuous, GFAPlabelled arborizations, which indicated that our protocol preserved the GFAP epitopes. In line with the qPCR results presented above, Gfap  and mRNA FISH signals were detected as discrete dots in the soma and in distal astrocyte areas; Gfap  mRNA (Fig. 1B) was more abundant than Gfap  which was mainly present in the somata (Fig. 1C).

AstroDot and AstroStat: bioinformatics tools for analyzing the mRNA distribution in astrocytes

In order to analyze the distribution of Gfap α and δ mRNAs in astrocytes, we developed AstroDot -a dedicated ImageJ plug-in. We had two main objectives: (i) to detect mRNA FISH dots that colocalized with GFAP-immunostained astrocyte processes; and (ii) to quantify these dots and analyze their distribution in the astrocytes. Confocal images of the CA1 and CA3 regions of the hippocampus were acquired and then deconvoluted, so as to eliminate the inherent fluorescence blurring (the point spread function (PSF)) and noise, and to increase the resolution (Fig. 2A). For each individual astrocyte, regions of interest (ROIs, i.e. the soma and processes) were selected manually by assessing the GFAP (intermediate filaments) and DAPI (nuclei) staining and by defining the stack of confocal planes (Fig. 2B). AstroDot opens with a dialogue box that enables the operator to attribute a specific purpose for each of the three fluorescence channels, e.g. "DAPI" for nuclear staining, "IF" for GFAP immunofluorescence, and "Dots" for FISH dot thresholding and detection (Fig. 2C). This dialogue box also contains a "Specific mRNA" option, which can be selected when the mRNAs are expressed only in the cell type of interest. In such a case, all mRNA FISH dots are considered to belong to this cell type. The first step in the analysis was calculation of the mean GFAP immunofluorescence background, i.e. the value above which the signal was considered to be positive. In the second step, each astrocyte nucleus was defined; given that astrocytes interact with other brain cell types, some ROIs can contain more than one nucleus. AstroDot was designed to optimize the recognition of astrocyte nuclei on the basis on the GFAP immunostaining. The putative astrocyte nucleus appears in green, and any other nuclei appears in red. A dialogue box allows the operator to confirm or modify AstroDot's automatic selection by clicking on the correct nucleus (Fig. 2D). AstroDot then starts to detect astrocyte mRNAs, based on their specific colocalization with GFAP. A distance map was used to calculate the diameter of each GFAP-immunolabelled process. Processes with a diameter greater than the minimum distance between two confocal planes (0.3 m, in the present case), were defined as "large", and those with a smaller diameter were defined as "fine" (Fig. 2E). The somatic domain of each astrocyte corresponded to the DAPI staining and the surrounding 2 m space. A TIF image was generated for each ROI (Fig. 2F). The mRNA FISH dots appeared in red if they were outside astrocytes, in green if they colocalized with astrocyte large processes and somata, or in yellow if they colocalized with astrocyte fine processes (Fig. 2F). All the results were automatically entered on a table with the following items for each ROI: Image name; ROI name; Background intensity; Astrocyte volume; Dot density in astrocytes; Percentage of dots not in astrocytes; Percentage of dots in astrocyte somata; Percentage of dots in astrocyte fine processes; Percentage of dots in astrocyte large processes;

Mean astrocyte process diameter. To facilitate the statistical analysis of AstroDot data, we developed an optional R package named AstroStat; it automatically calculates and compares the mean ± standard deviation values, and produces a summary report on the results.

Characterization of Gfap  and mRNAs in CA1 and CA3 hippocampal astrocytes from WT mice and the APPswe/PS1dE9 mouse model of AD.

We analyzed the density and distribution of Gfap  and Gfap  mRNAs in CA1 and CA3 hippocampal adult astrocytes in WT adult mice by using AstroDot's "Specific mRNA" option (Fig. 3 and Table S2). Comparison of the astrocytes in CA1 vs. CA3 indicated that CA1 astrocytes had a slightly greater overall volume but displayed processes with the same mean diameter (Fig. 3A). The Gfap /Gfap  mRNA ratio was the same in the two regions (Fig. 3B). Overall, and in line with our initial qPCR analysis (Fig. 1B), Gfap  was 5.2 times more abundant than Gfap  in both CA1 and CA3 (Fig. 3C).

Both mRNAs were more abundant in the processes (Gfap ± 6.7 SD in CA1 and 86.7 ± 8.1 SD in CA3 Gfap ± 11.3 SD in CA1 and 71.5 ± 16.4 SD in CA3 than in the soma. Gfap  was more abundant than Gfap in the soma and in large processes Fig. 3D). We analyzed the data without taking account of the astrocytic-specific nature of GFAP expression (Fig. 3E). In this case, both Gfap  and Gfap  mRNAs were colocalized on GFAP-labelled intermediate filaments in CA1 (mean ± SD: 59.5 ± 9.0 for Gfap  and 74.4 ± 11.0) and CA3 (62.2 ± 10.1 for Gfap  and 74.7 ± 12.4 for Gfap ) -suggesting that the majority of Gfap RNAs are associated with intermediate filaments (Fig. 3E).

Astrocytes are involved in neuroinflammation, and become reactive in virtually all pathological situations in the brain. Astrocyte reactivity is characterized by GFAP overexpression and subsequent morphological changes, such as process hypertrophy and remodelling [START_REF] Hol | Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system[END_REF] . Hence, we next sought to study Gfap  and mRNAs in reactive astrocytes. We choose the example of AD, in which astrocytes undergo drastic morphological and molecular changes that perturb their physiology [START_REF] Burda | Reactive gliosis and the multicellular response to CNS damage and disease[END_REF][START_REF] Ben Haim | Elusive roles for reactive astrocytes in neurodegenerative diseases[END_REF] . Using the method described above, Gfap  and  mRNA FISH dots were detected on GFAP-immunolabelled sections of CA1 and CA3 hippocampus from 9-month-old APPswe/PS1dE9 mice (Fig. 4). We quantified astrocytes associated with a beta-amyloid plaque (A, labelled with DAPI) or more than 30 μm from an A plaque (Fig. 5 and Table S2). As reported in the literature, CA1 and CA3 astrocytes from APP/PS1dE9 mice were larger than those from WT mice (Fig. 5A) but had a slightly smaller process diameter (Fig. 5A). In astrocytes not associated with A, the Gfap  ratio was slightly but significantly higher (by a factor of 1.3) in CA1 and CA3 (Fig. 5B), with a higher Gfap mRNA level in fine processes only (Fig. 4A,5F). In contrast, the Gfap  mRNA density was the same as in WT mice in CA1 and CA3 (Fig. 5C). However, the distribution of this mRNA within the astrocytes differed; levels in large processes were lower (relative to the WT) in CA1 and CA3 (Fig. 5E), and levels in fine processes were higher in CA3 only (Fig. 5F). The relative differences in mRNA levels were greater in A-associated astrocytes (Fig. 4B); the density of Gfap  mRNAs was significantly higher than in WT cells (5.0-fold for CA1, and 4.7-fold in CA3) or in astrocytes not associated with A (3.8-fold for CA1, and 3.7-fold in CA3) (Fig. 5C). The distribution of Gfap  mRNA also differed, with lower levels (relative to the WT) in the soma (Fig. 5D) and in large processes (only in CA1) (Fig. 5E) and higher levels in fine processes in CA1 and CA3 (Fig. 5F). The same effect was observed for the Gfap mRNA, with a greater abundance in A-associated astrocytes than in WT samples (3.6-fold for CA1, and 3.5-fold in CA3) or in astrocytes far from plaques (3.3-fold for CA1, and 3.4-fold in CA3) (Fig. 5C). The redistribution was most prominent in fine processes in CA1 and CA3 (Fig. 5F).

These results show that the density and distribution of Gfap  and Gfap  mRNAs vary markedly as a function of the astrocyte's reactivity status, the brain area, and the proximity of A deposits.

Application of AstroDot and AstroStat to the analysis of ubiquitous mRNAs in astrocytes and microglia

To further validate our approach, we studied the distribution of Rpl4 mRNA (a ubiquitously expressed mRNA encoding the large subunit ribosomal protein 4) in CA1 (Fig. 6A). Interestingly, 62.52% ± 11.77 of the Rpl4 mRNA FISH dots were localized in astrocytes (n=67). Of these, 83.33% ± 5.41 were present in fine GFAP-immunolabelled processes, with 9.59% ± 3.45 in large GFAP-immunolabelled processes, and 7.09% ± 4.14 in somata. This result was unexpected because Rpl4 integrates into the 60S ribosome subunit in the nucleus [START_REF] Huber | Molecular basis for protection of ribosomal protein L4 from cellular degradation[END_REF] but was corroborated by a qPCR analysis (performed as described above) of polysomal mRNAs extracted by TRAP from adult Aldh1L1-RPL10a mouse hippocampus or PAPs; in the latter, Rpl4 was enriched 120-fold (p=0.05, n=3) (Fig. 6B). To study the distribution of non-astrocyte Rpl4 mRNA FISH dots, we performed an additional, independent experiment by immunostaining the specific microglial marker Iba1. As with the GFAP immunofluorescence experiments, our FISH protocol was designed to protect Iba1's epitopes; the protein was detected throughout the somata and processes (Fig. 6C). Our analysis of the distribution of Rpl4 mRNA in microglia (n=28) indicated that 16.07% ± 4.47 of the Rpl4 mRNA FISH dots were localized in microglial processes. Of these, 37.72% ± 9.24 were localized in fine processes, with 27.06% ± 10.78 in large processes and 35.22% ± 10.13 in somata.

Discussion

Although local translation has been recently described in astrocyte processes, tools for studying the distribution of astrocyte mRNAs were not previously available. Accordingly, we developed a colabelling method that combined mRNA in situ hybridization, the immunofluorescence detection of GFAP-containing intermediate filaments on brain slices, confocal imaging, and a bioinformatics analysis of mRNA density and distribution in astrocytes.

A key technical obstacle to the implementation of this approach was the risk of protein epitope degradation during the protease digestion step that precedes in situ RNA hybridization. Our previous tests on transgenic hGfap-eGFP mouse brain sections (in which eGFP fills the astrocyte cytoplasm) [START_REF] Nolte | GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue[END_REF] indicated that these adaptations were not sufficient to preserve eGFP (data not shown), and thus precluded the use of this reporter mouse strain to detect astrocytes in parallel with in situ hybridization. In contrast to previous reports [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF][START_REF] Pilaz | Dynamic mRNA Transport and Local Translation in Radial Glial Progenitors of the Developing Brain[END_REF] , however, our protocol preserved GFAP and enabled us to perform parallel in situ hybridization and GFAP immunodetection. Interestingly, these conditions also allowed us to immunodetect the microglia-specific protein Iba1. It is noteworthy that GFAP is not expressed uniformly in the brain, and so GFAP immunolabelling is somewhat limited by its lack of applicability to all brain regions. Nevertheless, our optimization of the GFAP immunolabeling makes it possible to distinguish between strongly labelled astrocyte processes and their secondary extensions in regions where GFAP is highly expressed (e.g. the hippocampus, olfactory bulbs, cerebellum, and hypothalamus). Another advantage of immunolabelling GFAP and Iba1 relates to the fact that both proteins are standard markers of glial reactivity -a process initiated in response to immune attack, chronic neurodegenerative disease, or acute trauma [START_REF] Liddelow | Reactive Astrocytes: Production, Function, and Therapeutic Potential[END_REF] . Hence, GFAP and Iba1 immunolabelling could therefore be used to address possible changes in mRNA distribution in reactive astrocytes and microglia, as demonstrated here in APPswe/PS1dE9 mice.

It was previously determined that GFAP immunolabeling delineates only 15% of the total astrocyte volume [START_REF] Bushong | Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains[END_REF] . Nevertheless, we found that the majority of the Gfap  and  mRNA dots were attributed to GFAP processes. The mRNA dots not detected in GFAP intermediate filaments probably belonged to fine distal astrocyte processes devoid of GFAP (e.g. PAPs). These observations suggest that the majority of Gfap mRNAs are bound to intermediate filaments, and are consistent with previous reports of colocalization between mRNAs encoding collagens [START_REF] Challa | A novel role of vimentin filaments: binding and stabilization of collagen mRNAs[END_REF] and alkaline phosphatase [START_REF] Schmidt | Osteoblastic alkaline phosphatase mRNA is stabilized by binding to vimentin intermediary filaments[END_REF] on one hand and vimentin (another intermediate filament protein) on the other. Taken as a whole, these findings suggest that intermediate filaments may have crucial roles in the distal distribution of mRNAs. Consequently, it is conceivable that GFAP alterations, deficiency or upregulation (one or the other of which occurs in most neuropathological conditions [START_REF] Hol | Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system[END_REF] ) might greatly modified the distribution of astrocyte mRNAs and their local translation. In turn, these changes might alter the astrocytes' functions -particularly at their synaptic and vascular interfaces.

In order to demonstrate the applicability of our approach, we first focused on mRNAs encoding (i) the canonical  isoform of GFAP and (ii) the  Cter variant, the assembly of which with GFAP promotes intermediate filament aggregation and dynamic changes [START_REF] Moeton | GFAP isoforms control intermediate filament network dynamics, cell morphology, and focal adhesions[END_REF][START_REF] Perng | Glial fibrillary acidic protein filaments can tolerate the incorporation of assembly-compromised GFAP-delta, but with consequences for filament organization and alphaB-crystallin association[END_REF] . Interestingly, the results of our experiments in WT mice showed that Gfap  mRNA was more likely than Gfap  mRNA to be found in the astrocyte soma. This finding corroborated the results of a previous in vitro study in which the proportion of mRNA in primary astrocyte protrusions was higher for Gfap  than for Gfap  33 .

The high Gfap and mRNA density observed in plaque-associated astrocytes was also consistent with previous qPCR-based assays of mRNA in the cortex of APPswe/PS1dE9 mice [START_REF] Kamphuis | GFAP isoforms in adult mouse brain with a focus on neurogenic astrocytes and reactive astrogliosis in mouse models of Alzheimer disease[END_REF] . Interestingly, levels of human GFAP and isoforms are elevated in plaque-associated astrocytes in the CA 1-3 region [START_REF] Kamphuis | Glial fibrillary acidic protein isoform expression in plaque related astrogliosis in Alzheimer's disease[END_REF] . Taken together, these results and our observations of elevated mRNA density and distribution in the fine processes of plaque-associated astrocytes suggest that local translation of Gfap and mRNA might be a critical mechanism for regulating intermediate filament dynamics in distal astrocyte processes during the progression of AD. Given that the GFAP  isoform ratio is known to strongly influence astrocyte proliferation and malignancy [START_REF] Stassen | GFAPdelta/GFAPalpha ratio directs astrocytoma gene expression towards a more malignant profile[END_REF][START_REF] Van Bodegraven | Importance of GFAP isoform-specific analyses in astrocytoma[END_REF] , our approach might constitute a valuable tool for accurately assessing the differentiation state of astrocytomas in preclinical and clinical settings.

Lastly, we demonstrated that our approach is applicable to any type of mRNA and can also be used in microglia. In fact, the present study is the first to have demonstrated that mRNAs are distributed across microglial processes; this is an important observation in view of the microglia's complex morphology, motility, and roles in immune surveillance and synaptic remodelling in the brain [START_REF] Squarzoni | Microglia modulate wiring of the embryonic forebrain[END_REF] . Our results strongly suggest that mRNA distribution and local translation are of physiological significance in this important neural cell type. In conclusion, our new semi-automated in situ histological method is the first to have characterized mRNA distribution in astrocytes and microglia.

Materials and Methods

Mice

Aldh1L1-Rpl10a mice [START_REF] Heiman | Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP)[END_REF] and C57BL6 WT mice were born and then housed under pathogen-free conditions in the animal facility at the Centre Interdisciplinaire de Recherche en Biologie (CIRB, Collège de France, Paris, France). The APPswe/PS1dE9 38 mice were born and housed in the MIRCen animal facility (CEA, Fontenay-aux-Roses, France).

Ethical approval

All experiments were approved by the French Ministry of Research and Higher Education, and conducted in accordance with the host institution's ethical standards (Collège de France, Paris, France).

Aldh1L1:l10a-eGFP TRAP from whole astrocytes and PAPs, and qPCR

Two hippocampi from 5-month-old mice were used for whole-astrocyte polysome extraction.

Synaptosomes were prepared from four hippocampi, as described in [START_REF] Carney | Proteomic analysis of gliosomes from mouse brain: identification and investigation of glial membrane proteins[END_REF] for perisynaptic astrocyte extraction. Polysomes were extracted using the method described in [START_REF] Boulay | Preparing the Astrocyte Perivascular Endfeet Transcriptome to Investigate Astrocyte Molecular Regulations at the Brain-Vascular Interface[END_REF] . Three independent samples were prepared for qPCR analysis. Messenger RNAs were purified using the RNeasy Lipid tissue kit (Qiagen). cDNA was synthesized from 100 ng of whole-astrocyte RNA or the all the PAP mRNA using a Reverse Transcriptase Superscript III kit (Invitrogen) with random primers, and stored at -20°C. Next, 1 μL of cDNA suspension was pre-amplified using SoAmp reagent (BioRad), and droplet qPCR was performed using a QX200™ Droplet Digital™ PCR System (BioRad). The cDNA content was normalized against 45S RNA. TaqMan probes and primer references are listed in Table S1. The data were analyzed by applying a one-way unpaired Mann-Whitney test. The threshold for statistical significance was set to p<0.05.

Brain slice preparation

Nine-month-old mice were anesthetized with a mix of ketamine/xylazine (0.1 mL/mg) and killed by transcardiac perfusion with 1X phosphate-buffered saline (PBS)/4% paraformaldehyde (PFA) The brain was removed and immersed in 4% PFA overnight at 4°C. The PFA solution was replaced with 15% sucrose for 24 h at 4°C and, lastly, by 30% sucrose for 24 h at 4°C. The brains were cut into 30 µm-thick coronal sections using a Leitz microtome (1400). Sections were stored at -20°C in a cryoprotectant solution (30% glycerol and 30% ethylene glycol in 1X PBS).

Fluorescent in situ hybridization and immunostaining

Slice preparation: Slices were carefully washed three times with 1X PBS in a 24-well plate. For the last wash, the 1X PBS was replaced with 7 drops of RNAscope® hydrogen peroxide solution (Advanced Cell Diagnostics Inc.) for 10 min at room temperature (RT); this blocked endogenous peroxidase activity, and resulted in the formation of small bubbles. The slices were washed in Trisbuffered saline with Tween® (50 mM Tris-Cl, pH 7.6; 150 mM NaCl, 0.1% Tween® 20) at RT, and mounted on Super Frost+®-treated glass slides using a paintbrush. Slices were dried at RT for 1 hour in the dark, quickly (in less than 3 s) immersed in deionized water in a glass chamber, dried again for 1 hour at RT in the dark, incubated for 1 h at 60°C in a dry oven, and dried again at RT overnight in the dark.

The slices were rehydrated by rapid immersion (for less than 3 s) in deionized water at RT. Excess liquid was removed with an absorbent paper, and a hydrophobic barrier was drawn. A drop of pure ethanol was applied on the slice for less than 3 s and removed using an absorbing paper. The slides were incubated at 100°C in a steamer, while ensuring that condensation did not fall back on them. A drop of preheated RNAscope® 1X Target Retrieval Reagent (Advanced Cell Diagnostics Inc.) was added to the steamer, and the slides were left for 15 minutes. Next, the slides were washed three times in deionized water at RT, and excess liquid was removed with absorbent paper. A drop of 100% ethanol was applied for 3 minutes, and excess liquid was then removed. A drop of RNAscope® Protease+ solution (Advanced Cell Diagnostics Inc.) was applied and slices were incubated at 40°C in a humid box for 30 minutes. Target retrieval treatment and RNAscope® Protease+ treatment were used to unmask the mRNAs. Lastly, the slides were washed three times with deionized water at RT.

FISH and immunostaining: FISH was performed using the RNAscope® Multiplex Fluorescent

Reagent Kit v2 (Advanced Cell Diagnostics Inc.) and specific probes (Table S1), according to the manufacturer's instructions. Following the FISH procedure, slides were incubated with a blocking solution (5% normal goat serum, 0.375% Triton X-100, and 1 mg.mL -1 bovine serum albumin in 1X PBS) for 1 hour at RT, incubated with the primary antibody overnight at 4°C (Table S1), rinsed three times with 1X PBS, and incubated with the secondary antibody (Table S1) for 2h at RT. Lastly, the slides were washed three times in 1X PBS and mounted in Fluor mount and DAPI (Southern Biotech).

Imaging

Images were acquired using a Yokogawa W1 Spinning Disk confocal microscope (Zeiss) with a 63x oil objective (1.4 numerical aperture). The imaging conditions and acquisition parameters were the same for all slides. The experimental PSF was obtained using carboxylate microsphere beads (diameter: 170 nm; Invitrogen/ThermoFisher Corp.). Except for DAPI, all channels were deconvoluted with Huygens Essential software (version 19.04, Scientific Volume Imaging, The Netherlands; http://svi.nl), using the classic maximum likelihood estimation algorithm and a signal-to-noise ratio of 50 (for the immunofluorescence channel) or 20 (for the FISH channel), a quality change threshold of 0.01, and 150 iterations at most.

AstroDot and AstroStat

As shown in the Results section, AstroDot can be used to study mRNA density and distribution not only in astrocytes but also in microglia immunolabelled for Iba1. In addition to FISH signals, AstroDot can be used to quantify any type of dot-shaped fluorescence signal. AstroStat was used to analyze the AstroDot results table, using an R script. The programs can be downloaded free of charge from https://github.com/pmailly/Astrocyte_RNA_Analyze and https://github.com/rtortuyaux/astroStat, respectively.

For AstroDot, an image analysis plug-in was developed for the ImageJ/Fiji software [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF][START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF] , using Bio-Format (openmicroscopy.org), mcib3D [START_REF] Ollion | TANGO: a generic tool for highthroughput 3D image analysis for studying nuclear organization[END_REF] , GDSC (A. Herbert, https://github.com) and local thickness (B. Dougherty, https://imagej.net/Local_Thickness) libraries.

An ROI enclosing each astrocyte was drawn by hand, using the Fiji polygon tool on the Z projection of the stack. In the ROI Manager, the ROI names were coded as (roi_number-z_top-z_bottom) and saved in a zip file.

Plug-in features:

The plug-in was designed to process all images in a specific folder containing MetaMorph .nd files, and to read metadata images (channel name, z step, etc.), deconvoluted image channels (except for DAPI), and ROI zip files.

AstroDot processing:

1) AstroDot's parameters (the image folder, the channel order, the threshold method, etc.) were displayed in a dialogue box (see Fig. 2C).

2) The immunofluorescence background was estimated using a 0.5 median filter, a binary mask (using Li's threshold method), and an inversion of the binary mask [START_REF] Li | Minimum Cross Entropy Thresholding[END_REF][START_REF] Li | An Iterative Algorithm for Minimum Cross Entropy Thresholding[END_REF] .The immunofluorescence value was multiplied by the inverted mask and then divided by 255. The background value (bgThreshold) was defined as the mean intensity of all voxels other than those with a value of zero.

3) For each ROI, a substack corresponding to zTop and zBottom (defined in the ROI name) was created for all channels. 4) Semi-automatic determination of the astrocyte or microglial cell nucleus: DAPI fluorescence was processed using a difference of Gaussian filter (kernel: 28-30), a binary mask (using Otsu's threshold method) and a three-dimensional watershed, to separate nucleus clusters [START_REF] Otsu | A threshold selection method from gray-level histograms[END_REF] . An astrocyte nucleus was selected on the basis of its high GFAP immunofluorescence intensity, and was displayed in green. All other detected nuclei were displayed in red. A dialogue box enabled the user to confirm or correct the software's choice of nuclei.

5)

The GFAP immunofluorescence was processed using a 0.5 median filter and a binary mask, using Li's threshold method. The three-dimensional local thickness of the processes was used to generate a distance map and calculate the local process diameters. 6) FISH dot channel processing: a value of 500 (a manual estimation of the background after deconvolution) was subtracted from each voxel. A difference of Gaussian filter (kernel: 3-1) and a binary mask were applied, using the threshold method defined in the "parameters" dialogue box.

The mean dot size volume was computed after the exclusion of dot clusters (volume >2 µm 3 ). For dot clusters (which arise when mRNAs are strongly expressed), the dot number was calculated by dividing the cluster by the previously determined mean dot size volume. For each dot, the mean intensity in the Gfap immunofluorescence channel and the distance map value (the process diameter) was calculated. Hence, Dots 1 and 2 were inside astrocytes, and Dots 0 were outside astrocytes. 8) For each image and for each computed ROI, a .csv output table was generated with the following headers: Image name; ROI name; Background intensity; Astrocyte volume; Dot density inside astrocytes (number of dots 1 + number of dots 2) / astrocyte volume); Percentage of dots outside the astrocyte (number of dots 0 / total dot number); Percentage of dots in astrocyte somata (number of dots less than 2 µm from the boundary of the nucleus / number of dots in astrocytes); Percentage of dots in fine processes (number of dots 1 / number of dots in astrocytes); Percentage of dots in large processes (number of dots 2number of dots in somata) / number of astrocyte dots); Mean astrocyte diameter. For each image and each ROI, the selected nucleus, astrocyte channel and classified dot populations were saved as .TIF images. AstroStat was designed to (i) define the template analysis using a checkbox (working directory, conditions to be compared, paired or unpaired analysis, or data normality plot); (ii) pool data appropriately for each mouse; (iii) test the normality of the data distribution of each group (using Shapiro's test). If there were more than 30 cells in each group, the central limit theorem was applied;

(iv) test the equality of variance (using Fisher's test) for an unpaired analysis; and (v) compare the means using: an unpaired analysis:

7 )

 7 FISH dot classification into three categories: a) Dot 0 (in red) was a dot in the immunofluorescence background (without using the "Specific mRNA" option only): mean GFAP immunofluorescence intensity ≤ bgThreshold; distance to the boundary of the nucleus > 2 µm. b) Dot 1 (in yellow) was a dot in a fine process: mean GFAP immunofluorescence intensity >bgThreshold; distance to the boundary of the nucleus >2 µm; astrocyte process diameter < step in the z calibration (0.3 µm). c) Dot 2 (green) was a dot in a large process: mean GFAP immunofluorescence intensity >bgThreshold; distance to the boundary of the nucleus >2 µm; astrocyte process diameter > step in the z calibration (0.3 µm); or a dot in the soma if the distance to the boundary of the nucleus 2 µm.

  a) Student's t-test, for normally distributed data and equal variances b) Welch-Satterthwaite's test, for a normal data distribution and unequal variances c) Wilcoxon's test for non-normally distributed data or a paired analysis: a) Paired Student's t test for normally distributed data b) Wilcoxon signed rank test for non-normally distributed dataThe threshold for statistical significance was set to p<0.05.

Figure legends Fig. 1 :

 legends1 Figure legends

Fig. 2 :

 2 Fig. 2: AstroDot image processing. All images correspond to a single confocal z-stack for a CA1

Fig. 3 :

 3 Fig. 3: The distribution of Gfap and mRNAs in CA1 and CA3 hippocampal astrocytes.

Fig. 4 :

 4 Fig. 4: Detection of Gfap  and mRNAs in CA1 hippocampal APPswe/PS1dE9 astrocytes. A.

Fig. 5 :

 5 Fig. 5: Comparison of Gfap and mRNA densities and distributions in CA1 and CA3

Fig. 6 :

 6 Fig. 6: Detection and characterization of Rpl4 mRNA distribution in CA1 hippocampal

Acknowledgments

This work was funded by a grant from the Fondation pour la Recherche Médicale (AJE20171039094).

Noémie Mazaré received a grant from the Fondation pour la Recherche Médicale (FDT201904008077). Romain Tortuyaux received a grant from the "Journées de Neurologie de Langue Francaise". The creation of the Center for Interdisciplinary Research in Biology (CIRB) was funded by the "Fondation Bettencourt Schueller". We thank Carole Escartin for the gift of APPswe/PS1dE9 mouse brain slices, and all members of the Orion imaging facility for their highquality technical support.

DAPI (in blue)

. Right: AstroDot analysis: Green dots are located in the soma or in GFAPimmunolabelled large processes; yellow dots are located in GFAP-immunolabelled fine processes; red dots are not colocalized with GFAP (i.e. excluded RNAs). B. The polysomal Rpl4 RNA level in hippocampal astrocytes and PAPs, determined by qPCR and normalized against 45S RNA. Statistical significance was determined in a one-way unpaired Mann Whitney's test; *, p<0.05. C. Left: Confocal z-stack of a CA1 microglial cell with FISH detection of Rpl4 mRNA (in red) and coimmunofluorescent Iba1 (in grey). The nucleus was stained with DAPI (in blue). Right: the AstroDot analysis: Green dots are located in the soma or Iba1-immunolabelled large processes; yellow dots are located in Iba1-immunolabelled fine processes; red dots did not colocalize with Iba1 (i.e. excluded RNAs). Scale bar; 10m.

Supplementary Table S1. Antibodies, qPCR and FISH probes and reagents

Supplementary Table S2. Raw data (analyzed using AstroDot and AstroStat) for Gfap α and Gfap  mRNAs in CA1 and CA3 hippocampal astrocytes from WT and APPswe/PS1dE9 mice. SD, standard deviation; N, number of cells analyzed.


Andreas Natsch
email: andreas.natsch@givaudan.com

Marco Nägelin

Hans Leijs

Michel Van Strien

Elena Giménez-Arnau

Matthias Vey

Cecile González

Exposure source for skin sensitizing hydroperoxides of limonene and linalool remains elusive: an analytical market surveillance

Keywords: Terpene hydroperoxides, skin allergy, quantification, consumer products, market surveillance Running head: hydroperoxide quantification in consumer products

Reports about positive patch test reactions to oxidized linalool and limonene remain frequent. These terpenes are fragrance ingredients widely present in consumer products. The main sensitizing ingredients in the oxidation mixtures of these terpenes are hydroperoxides (HP). Currently, it is not clear whether fragranced consumer products are a relevant exposure source for HP. Analytical methodologies had been developed and validated in blind-coded ring-trials in multiple laboratories allowing quantification of the HP in different consumer products. The analytical approach had been successfully transferred to an independent third party laboratory and was now used in the analytical investigation of consumer products. In total, 104 products were analysed with a method based on hydroperoxide reduction followed by GC-MS. Samples included aged and new samples from the same brand, products which were suspected by patch test positive patients to elicit their symptoms and some products containing high levels of essential oils. Only four samples contained > 50 µg/g of at least one of four analysed HP by the reduction method. Confirmatory analysis by LC-MS methods directly testing for presence of the hydroperoxide indicated that levels are even below those observed by the conservative reduction method. The samples retrieved from patch-test positive samples were below detection limit for all four target analytes by GC-MS, and LC analysis with three methods confirmed this negative result. This independent market surveillance indicates that concentrations of HP in investigated consumer products and patient products are orders of magnitude below reported sensitizing or elicitating doses. No evidence for hydroperoxide accumulation in aged products or products used by patients could be found. The nature and source of the inducing agent responsible for the frequent positive patch test reactions to oxidized terpenes remains elusive.

Introduction

Linalool and/ or limonene are present in the majority of leave-on cosmetic products [START_REF] Rastogi | Fragrance chemicals in domestic and occupational products[END_REF] . Both linalool and limonene are non-sensitizing according to data from most human and guinea pig studies [START_REF] Letizia | Fragrance material review on linalool[END_REF] , and they are only very weak sensitizers according to the local lymph node assay performed in mice [START_REF] Roberts | Mechanistic applicability domain classification of a local lymph node assay dataset for skin sensitization[END_REF] . Despite their widespread use, positive patch test reactions for linalool and limonene are only rarely reported from the dermatological clinics [START_REF] Schnuch | Sensitization to 26 fragrances to be labelled according to current European regulation. Results of the IVDK and review of the literature[END_REF][START_REF] Uter | Contact allergy to fragrances: current patch test results (2005-2008) from the Information Network of Departments of Dermatology[END_REF] , confirming the low sensitization potential. However, neat linalool and limonene were shown to undergo autoxidation when agitated for prolonged periods of time in the presence of air. [START_REF] Hagvall | Lavender oil lacks natural protection against autoxidation, forming strong contact allergens on air exposure[END_REF][START_REF] Karlberg | Air oxidation of d-limonene (the citrus solvent) creates potent allergens[END_REF][START_REF] Sköld | Contact allergens formed on air exposure of linalool. Identification and quantification of primary and secondary oxidation products and the effect on skin sensitization[END_REF][START_REF] Hausen | Degradation products of monoterpenes are the sensitizing agents in tea tree oil[END_REF][START_REF] Hagvall | Fragrance compound geraniol forms contact allergens on air exposure. Identification and quantification of oxidation products and effect on skin sensitization[END_REF][START_REF] Rudback | Characterization of skin sensitizers from autoxidized citronellol -impact of the terpene structure on the autoxidation process[END_REF] The airoxidized terpenes were found to be skin sensitizing in animal tests. Within the complex autoxidation mixtures, hydroperoxides (HP) were identified as the key allergens [START_REF] Sköld | Studies on the autoxidation and sensitizing capacity of the fragrance chemical linalool, identifying a linalool hydroperoxide[END_REF][START_REF] Christensson | Linalool -a significant contact sensitizer after air exposure[END_REF][START_REF] Nilsson | Analysis of contact allergenic compounds in oxidized d-limonene[END_REF][START_REF] Karlberg | Hydroperoxides in oxidized d-limonene identified as potent contact allergens[END_REF] . Oxidized samples of both limonene and linalool are commercially available to dermatologists to perform patch tests in order to identify possible causes of allergic contact dermatitis. [START_REF] Christensson | Linalool -a significant contact sensitizer after air exposure[END_REF][START_REF] Brared Christensson | Air-oxidized linalool-a frequent cause of fragrance contact allergy[END_REF][START_REF] Audrain | Allergy to oxidized limonene and linalool is frequent throughout the UK[END_REF] So far, these autoxidation mixtures have been tested on a cumulative number of >22'000 patients 16- 22 . These studies reported high frequencies of positive reactions to the two oxidized terpene preparations. In general more frequent reactions are reported to oxidized linalool, which is tested at a higher concentration than oxidized limonene. Thus on the average 4 -6% of tested patients respond to oxidized linalool, and similar or even higher frequencies of doubtful or irritant reactions were reported in parallel [START_REF] Brared Christensson | Air-oxidized linalool-a frequent cause of fragrance contact allergy[END_REF][START_REF] Audrain | Allergy to oxidized limonene and linalool is frequent throughout the UK[END_REF][START_REF] Bennike | Non-mix fragrances are top sensitizers in consecutive dermatitis patients -a cross-sectional study of the 26 EU-labelled fragrance allergens[END_REF] . It was often inferred from these studies that the HP formed from limonene and linalool are among the most common causes of contact allergy [START_REF] Christensson | Linalool -a significant contact sensitizer after air exposure[END_REF] and a causal link between these frequent reactions and widespread exposure of consumers to parent linalool and limonene from consumer products was made [START_REF] Brared Christensson | Positive patch test reactions to oxidized limonene: exposure and relevance[END_REF] . However, in order to scientifically substantiate this link one would have to prove that the parent molecules oxidize in the consumer products or that partly oxidized raw materials are used in product manufacture resulting in sufficiently high HP levels in the products to trigger active sensitization of a large number of consumers. Thus, to establish relevance of the positive patch-test reactions reported from the dermatological clinics, quantitative determination of the HP in consumer products on the market in general and more specifically in products used by the patients would be required. Reliable analytical methods to determine HP levels in consumer products have been developed in recent years and they were compared and evaluated in detailed studies within the IDEA project (International Dialogue for the Evaluation of Allergens) involving stakeholders from academia, industry and the dermatological community [START_REF]International Dialogue for the Evaluation of Allergens[END_REF] . IDEA was launched by the fragrance industry in collaboration with the European Union to address open questions on contact allergy to fragranced products following the opinion of the European Union's Scientific Committee on Consumer Safety (SCCS) on fragrance allergens [START_REF] Sccs | Opinion on Fragrance allergens in cosmetic products[END_REF] . A simple method with a reduction step using triphenylphosphine (PPh 3 ), followed by GC-MS to detect alcohols formed by HP reduction [START_REF] Kern | Stability of limonene and monitoring of a hydroperoxide in fragranced products[END_REF] was evaluated in two blind-coded method evaluation studies on fine fragrances [START_REF] Natsch | Interlaboratory evaluation of methods to quantify skin sensitizing hydroperoxides potentially formed from linalool and limonene in perfumes[END_REF] and creams and lotions [START_REF] Natsch | Interlaboratory evaluation of methods to quantify skin-sensitizing hydroperoxides of limonene and linalool (II): Analysis in cosmetic bases[END_REF] . Accurate quantification of spiked hydroperoxides in the different products by this method was shown. This method has the drawback that it may lead to an overestimation of the true content of HP if the alcohols formed by HP reduction are already present as fragrance ingredients and it is therefore a conservative method. On the other hand it avoids any potential issues due to instability of HP during sample preparation and extraction. In parallel, these method evaluation studies also tested the same blind-coded samples with several liquid-chromatography (LC) based methods directly detecting the parent hydroperoxide. These included a HPLC-chemiluminescence (HPLC-CL) method [START_REF] Calandra | An HPLC method for hydroperoxides derived from limonene and linalool in citrus oils, using post-column luminol-mediated chemiluminescence detection[END_REF] , an LC-Q-TOF-MS method and an LC-orbitrap-MS method [START_REF] Kern | Detection of potentially skin sensitizing hydroperoxides of linalool in fragranced products[END_REF] . As a general recommendation, it was concluded to use the robust GC-MS reduction method for a screening approach and to validate the result for each sample with a standard addition experiment, while the LC-based methods could then be used for verification of presence of parent HP in positive samples [START_REF] Natsch | Interlaboratory evaluation of methods to quantify skin sensitizing hydroperoxides potentially formed from linalool and limonene in perfumes[END_REF][START_REF] Natsch | Interlaboratory evaluation of methods to quantify skin-sensitizing hydroperoxides of limonene and linalool (II): Analysis in cosmetic bases[END_REF] in specific cases. Here, we collected different used products from consumers and new products from the general market covering a wide range of product types and with linalool or limonene declared on the label. Emphasis was given to include old and fresh samples from the same brand, products from patients tested positively for the oxidation mixtures and products containing essential oils, in order to test different possible exposure sources. All samples were analysed by the GC-MS reduction method, and confirmatory analysis on a subset of samples was made with LC-MS.

Materials and Methods

Analytical reference standards. HP reference standards (linalool-7-OOH, linalool-6-OOH, limonene-1-OOH and limonene-2-OOH) were prepared by GreenPharma (Orléans, France) as described before [START_REF] Natsch | Interlaboratory evaluation of methods to quantify skin sensitizing hydroperoxides potentially formed from linalool and limonene in perfumes[END_REF] . Fresh samples with a purity of > 98% were prepared for this study. These standards were also used for the standard addition experiments (see below).

Analytical methods

Reduction of terpene HP with triphenylphosphine (PPh 3 ) to form specific alcohols is possible in different consumer product matrices [START_REF] Natsch | Interlaboratory evaluation of methods to quantify skin sensitizing hydroperoxides potentially formed from linalool and limonene in perfumes[END_REF][START_REF] Natsch | Interlaboratory evaluation of methods to quantify skin-sensitizing hydroperoxides of limonene and linalool (II): Analysis in cosmetic bases[END_REF] . This transforms the hydroperoxides into an easily measurable stable analyte, which is then measured by GC-MS. Thus the four HP in oxidized linalool and limonene are linalool-7-OOH, linalool-6-OOH, limonene-1-OOH and limonene-2-OOH, and they are reduced to linalool-7-OH, linalool-6-OH, limonene-1-OH and limonene-2-OH (carveol), which are the 'target analytes' investigated here. A standard operation procedure for this method was published along with our previous publication [START_REF] Natsch | Interlaboratory evaluation of methods to quantify skin-sensitizing hydroperoxides of limonene and linalool (II): Analysis in cosmetic bases[END_REF] and was followed for analysis of all products in this study. It includes two different sample preparation methods for alcoholic fine fragrances and for complex products containing water (creams, lotions, etc.). Calibration was done based on external calibration curves with the four synthetic HP standards treated with the reduction method under identical conditions. To ensure successful detection of the HP by this method in the different product matrices, each sample was analyzed as received and as a parallel sample with a standard addition adding a final level of 50 µg/g of each of the four HP to the test sample. For this method we defined a 'reporting limit' of 50 µg/ml 28 , i.e. a level at which the alcohol from the reduced HP can be accurately measured in different product types, as it is not practicable to determine the true limit of quantification (LOQ) in each product matrix. The standard addition experiment was set to 50 µg/g to reflect this reporting limit, and it allows checking successful detection of this specified level in each different product matrix investigated. However, here also levels below this reporting limit are indicated for full transparency, but these must be viewed with some caution. LC-Chemiluminescence measurements, LC-Q-TOF-MS and LC-orbitrap-MS measurements were performed with the methods also previously described in detail [START_REF] Natsch | Interlaboratory evaluation of methods to quantify skin-sensitizing hydroperoxides of limonene and linalool (II): Analysis in cosmetic bases[END_REF] . Procedure for collection consumer products IDEA Management Team (IDEA MT) organized the collection of samples and the shipment to the analytical laboratory (Solvias, Kaiseraugst). Samples were directly collected by the team or provided by members of the IDEA hydroperoxides taskforce. In general, partly used products donated by consumers were collected and they were matched with fresh products of the same brands bought on the market to be able to determine influence of product storage. As inclusion criteria, products should have a declared linalool and/or limonene content and batch number /production code to ensure traceability. In addition, samples donated by dermatological patients were included (esp. those tested positive for the terpene oxidation mixtures) and were suspected by them to be involved in their clinical reactions (samples furnished by the Spanish Contact Dermatitis Group GEIDAC). All samples provided by the dermatological centres were tested, even if no matched fresh product could be sourced or if no linalool or limonene content was declared. Five additional fresh consumer product samples were selected, which contained essential oils as declared by the product labelling, since some samples of essential oils were reported to contain high levels of HP [START_REF] Rudbäck | Essential oils can contain allergenic hydroperoxides at eliciting levels, regardless of handling and storage[END_REF] . Tested products also included two commercial fine fragrances provided by a manufacturer with different aging: (i) as fresh samples, (ii) as longtime stored samples and (iii) as artificially aged samples. Finally, one sample of an aftershave was received from the University of Stockholm. This sample had recently been found to contain a significant level of linalool-6-OOH by a new twodimensional LC-MS method [START_REF] Ramzi | A two-dimensional non-comprehensive reversed/normal phase high-performance liquid chromatography/tandem mass spectrometry system for determination of limonene and linalool hydroperoxides[END_REF] , and it was reanalysed by the four different methods from our method evaluation and validation studies to link our investigations with those published data on the same sample. However, this sample does not form part of the 'random' sample collection, as it was selected from a larger number of random samples based on a previous analytical result. All the samples above were analysed with the GC-MS-reduction method. Five positive samples above the reporting limit defined earlier and a matched fresh sample were re-analysed by three LC-MS based methods to further evaluate the positive results in the screening. In addition, 11 samples recovered from patch test positive patients were re-analysed by LC-methods to confirm the negative results obtained. Finally, content of non-oxidized linalool and limonene was analysed semi-quantitatively based on the GC-MS results in all products. However, this was not a primary goal and the method was not optimized for the analysis of these analytes (a different dilution would be selected to quantitatively analyse these much more abundant analytes if that would be the primary goal, but primary goal was to be highly sensitive for the alcohols formed by HP reduction).

Results

All results on the individual analysed samples are provided as an excel-file in supplementary information, and data are sorted into sheets in the order they are discussed here. We collected 31 used samples from diverse product types, which we could match with samples of freshly purchased products. This set of samples forms the majority of analysed products. They all had a declared content of linalool or limonene, and with four exceptions both terpenes were labelled on these products. Only one of these 31 aged samples and none of the fresh samples contained one of the target analytes above the reporting level: 91 µg/ml of limonene-1-OH was detected in an Eau de Toilette, while 45 µg/ml of this analyte was detected in the matched fresh product. This product (Sample A in Table 1) contained > 5000 µg/ml of parent limonene according to the semi-quantitative analysis. Re-analysis by three LC-based methods overall confirmed the result of GC-MS reduction method and could identify this HP in the range of 30 -100 µg/ml in the aged and 8 -38µg/ml in the fresh sample (Table 1). Overall, analysis of this dataset indicated no widespread oxidation of the two terpenes between product manufacturing and the time the aged products were sampled. Compared to the high level of parent linalool and limonene, HP are either very minor constituents or are not detectable at all in these products. A further set of samples comprises 20 aged samples from consumers, for which no matched fresh sample could be found on the market. A body oil (sample B) contained 69 µg/ml of limonene-2-OH, while semiquantitative analysis found 710 µg/ml of parent limonene. LCanalysis by three different methods could not detect the parent HP limonene-2-OOH, while the standard addition indicated high reliability. A set of 10 samples collected by dermatologists from patients had neither limonene nor linalool content declared. Correct declaration could be confirmed by analysis for parent linalool and limonene, which were absent in these samples, and, not surprisingly, also no indication for hydroperoxide content was found in this set of samples. From the three sample sets described above, a subset had been collected by dermatologists from their patients as these products were suspected to elicit their reactions. This subset is separately summarized in a further data sheet in the supplementary file. Eleven samples were from patients, which are positive to at least one of the oxidized linalool or limonene. For the patients donating the other samples the patch test to the oxidation mixtures was either negative or not performed. In none of the 28 samples collected from patients, could we detect by the GC-MS method any of the four target analytes above 50 µg/ml (except linalool-7-OH in one sample at 38 µg/ml, all were < 22 µg/ml for all four analytes). Furthermore, absence of the hydroperoxides above reporting limit was confirmed in the 11 samples from patch test positive patients by both LC-MS methods. Also the LC-Chemiluminescence found only traces in two samples (Table 2).

As some literature data indicate rapid accumulation of hydroperoxides in essential oils [START_REF] Rudbäck | Essential oils can contain allergenic hydroperoxides at eliciting levels, regardless of handling and storage[END_REF] , five additional products were purchased which have a declared content of essential oils and declared linalool / limonene content. Two of these samples (Sample C and D in Table 1) contained at least one of the target alcohols above the reporting limit when analysed by the GC-MS-reduction method. However, re-analysis by LC-MS could not confirm the presence of parent HP. Here it has to be kept in mind that the reduction method can lead to an overestimation if the reduced alcohol is already present in the product. The shower oil sample D was thus analysed in some more detail. Based on the declaration, this sample contains a multitude of essential oils containing linalool and limonene (different citrus and orange oils, different lavender oils). Since the content of both limonene and linalool was outside of the calibration range, dilutions of the product were re-analysed. It was found that this product contains 215'000 µg/ml and 143'000 µg/ml of limonene and linalool (21.5% and 14.3% of the total product). Hence, presence of low levels of the alcohols limonene-1-OH and limonene-2-OH, which can be found in citrus oils, is not completely surprising (Table 1). Interestingly, LC-methods found no indication of significant hydroperoxide content in this product with highly exaggerated content of essential oils. We also analysed two commercial fine fragrances obtained from the manufacturer as a (i) fresh sample, (ii) an artificially aged sample (3 months at 45°C) and a (iii) sample stored at room temperature for 3 or 6 years. As indicated in Table 3, all samples were below reporting limit of 50 µg/ml for all four analytes, but above detection limit for some of the analytes. The data in Table 3 indicate no accumulation of the target analytes, nor degradation of the parent linalool or limonene under the storage conditions. We did complete this study by analysing an aftershave sample, which was received from the University of Stockholm. It had previously been analyzed by a novel heart-cut 2D LC-MS method [START_REF] Ramzi | A two-dimensional non-comprehensive reversed/normal phase high-performance liquid chromatography/tandem mass spectrometry system for determination of limonene and linalool hydroperoxides[END_REF] . This sample (labelled Sample 6 in the publication; aged > 5 years) was selected as it contained the highest HP level from a series of market products by this previous analysis and the highest ever reported terpene HP concentration in a commercial sample, i.e. it was reported to contain 420 µg/ml linalool-6-OOH and ca. 20 µg/ml linalool-7-OOH by that novel method. It was reanalysed with the toolbox of methods used here. While 81 µg/ml of the alcohol linalool-6-OH could be detected by the reduction method, LC-MS found no detectable level (<25 µg/ml, LC-Orbitrap), or 7 µg/ml (LC-Q-TOF) or 4.6 µg/ml (LC-CL) of the parent HP Linalool-6-OOH (see supporting Information). Analysis by these multiple techniques and detection modes indicates that the HP level is much lower as compared to the published value. As many negative results are reported here, especially also in the screening with the conservative reduction method, it is important that these results are validated and that potential hydroperoxides are not masked by interference with product matrix. Hence the standard addition experiments are of particular importance to prove that added hydroperoxides are successfully detected. The individual results of the standard addition experiments are all reported in the tables in the supporting information. For each of the four target hydroperoxides, the recovery in % is indicated for each product. From 4 (n HP) × 104 (n samples) = 416 datapoints, only in one case the added standard could not be detected due to interference. In 88% of the cases, the recovery was > 70%, Figure 1 shows the box plots for the recovery from all samples.

Discussion

This study analysed a broad variety of consumer products containing linalool and/or limonene. Based on the conservative GC-MS-reduction method, 99 of the 103 products (96%) contained hydroperoxides below the reporting level, while 102/103 (99%) contained less than 100 ppm. Re-analysis by LC-MS indicated that these data are still an overestimation, as only in one sample the hydroperoxide level determined by the GC-MS method could be confirmed, while esp. the essential oil containing products probably appeared not to contain the hydroperoxide but the reduced alcohol. Overall, these data indicate that hydroperoxides of linalool and limonene are not extensively present in consumer products. Analytical confidence. From an analytical standpoint the overestimation observed in few samples containing essential oils by the GC-MS-reduction method may be criticized. The possibility for some overestimation due to presence of reduced alcohols was known from the beginning and it was discussed before [START_REF] Kern | Stability of limonene and monitoring of a hydroperoxide in fragranced products[END_REF][START_REF] Natsch | Interlaboratory evaluation of methods to quantify skin sensitizing hydroperoxides potentially formed from linalool and limonene in perfumes[END_REF] , however it does not significantly affect the overall conclusion, namely the low frequency of positive samples and low levels in those samples above the reporting limit. More important is the positive predictivity, i.e. that samples containing hydroperoxides can be reliably identified. Thus the standard addition experiments indicated that positive detection of the standard addition was successful in 415 out of 416 individual HP determinations, proving successful positive detection. Moreover, in the 11 samples obtained from patients positive for the oxidized terpenes, absence of significant HP levels as determined by the GC-MS reduction method was confirmed by three independent LC-based methods. Thus all the data on lack of significant HP-presence in the investigated samples can be considered to be highly reliable based on the detailed control experiments performed here. The exact quantification of low levels of course is more challenging, as can be seen form the results of the different LC-methods with some variability between methods observed for sample A. In addition, recovery of the standard addition was below 70% in several samples in the GC-MS method. While, from an analytical standpoint, this is not completely satisfactory, confidence in negative results and quantitative accuracy in the correct order of magnitude is largely sufficient for the toxicological assessment, see below discussion on the known, toxicologically relevant doses. Comparison to previous data. Previous studies on hydroperoxide levels in consumer products focused on fine fragrances and deodorants [START_REF] Kern | Stability of limonene and monitoring of a hydroperoxide in fragranced products[END_REF][START_REF] Kern | Detection of potentially skin sensitizing hydroperoxides of linalool in fragranced products[END_REF] . Those studies were conducted before the methods were validated in the blind-coded ring trial and did not include complex products like creams and lotions. Nevertheless, the overall conclusions are congruent with the current data. In 39 aged fine fragrance samples from consumers, only 14 contained limonene-2-OOH as determined by the reduction method and levels were below 5 µg/ml [START_REF] Kern | Stability of limonene and monitoring of a hydroperoxide in fragranced products[END_REF] . In 22 of these 39 samples, linalool-OOH was detected with a geometric mean of 30 µg/ml as determined by LC-MS [START_REF] Kern | Detection of potentially skin sensitizing hydroperoxides of linalool in fragranced products[END_REF] . Only one sample contained > 100 µg/ml. In a set of 10 products recently analysed by the novel two-dimensional LC-MS method [START_REF] Ramzi | A two-dimensional non-comprehensive reversed/normal phase high-performance liquid chromatography/tandem mass spectrometry system for determination of limonene and linalool hydroperoxides[END_REF] , linalool-OOH levels were in the same range, only one sample contained significantly higher hydroperoxide level of the isomer linalool-6-OOH, which normally is the minor isomer in oxidized samples. However, this analytical result could not be reproduced with the methods used here applied to the same sample (See supplementary information for detail). Thus this broad market surveillance aligns well with smaller studies performed before. Importance of product type. In terms of product types being at highest risk to contain HP, fine fragrances would be suspected first, as they contain the highest fragrance levels. Here we could positively identify limonene-1-OOH in one eau de toilette and, as indicated above, linalool-OOH could be identified in fine fragrances before. In addition we could identify the target alcohols in three body oils and shower oils based on the reduction method, however in these three cases HP levels were not confirmed by the LC-methods and these oils probably contained low levels of the alcohols which are naturally present in essential oils used for manufacture. This explanation is particularly likely for sample C. As it contained a cumulative amount of 36'000 µg/g of linalool and limonene from natural oils, presence of a cumulative amount of 400 µg/g of the reduced alcohols (i.e. 1% compared to the linalool and limonene content) is not surprising. We could not detect HP in any of the creams and lotions tested, despite the fact that our method was specifically developed and validated for that purpose [START_REF] Natsch | Interlaboratory evaluation of methods to quantify skin-sensitizing hydroperoxides of limonene and linalool (II): Analysis in cosmetic bases[END_REF] . Effect of product age. In principle, commercial products could be clean in the beginning and then accumulate HP upon aging. Here we analysed old and new products and there was no evidence of HP formation upon product aging, this can nicely be seen in the two products shown in Table 3 but also by the overall results of the 31 products analysed fresh and aged. Only in sample A the level of limonene-1-OOH was indeed higher in the aged product, but only by a factor of two, and this could in principle also be due to a batch effect, as the aged product was produced at a different time. In theory, HP could also form over time and disappear again. However, if oxidation would progress significantly over time, we would expect lower levels of linalool and limonene in old products. Here only tentative conclusions can be made, as analysis of parent linalool and limonene was only semi-quantitative. Nevertheless, on the average, aged samples contain 95% of the limonene and 99% of the linalool content of the fresh samples. Similarly, the two fragrances with controlled aging in Table 3 contain stable levels of the parent molecules even when aged up to six years. This is in accordance with controlled stability studies over 9 months with accelerated aging of linalool in an eau de toilette matrix, where no significant degradation was observed even in half-filled, repeatedly opened bottles incubated at 45°C [START_REF] Kern | Detection of potentially skin sensitizing hydroperoxides of linalool in fragranced products[END_REF] . Products retrieved from patients. A key new aspect of our study is inclusion of samples from patients that were patch test positive to linalool ox. and / or limonenen ox. No target alcohols could be detected in any of those products (< 22 ppm for all four hydroperoxides) and 50-100% recovery of standard addition in those samples indicates that lack of HP detection cannot be attributed to analytical failure. Re-analysis by LC-MS methods confirmed these negative results. Thus, exposure to these particular product samples can neither explain the positive patch test reactions, nor an elicitation due to HP contained therein. HP content and analytical sensitivities compared to toxicological data. The analytical sensitivities and the detected levels should always be compared to the concentrations of toxicological concern based on the available data from animal and human studies. Thus the dose per area triggering an EC3 in the local lymph node in mice is at 16'000 µg/g for linalool-OOH and 3300 and 8300 µg/g for the two limonene isomers. The concentration used for induction in guinea pig tests was 15'000 µg/g for the limonene hydroperoxide fraction [START_REF] Karlberg | Hydroperoxides in oxidized d-limonene identified as potent contact allergens[END_REF] . Patch test preparations used in the clinics contain 3300 (limonene-OOH) or 10'000 (linalool-OOH) µg/g of the HP. Compared to these figures, the analytical sensitivities of the methods used here are much lower, thus we cannot attribute a widespread sensitization as stipulated by the high patch test frequencies to possible undetectable levels escaping our analytical sensitivities. A comparison of the exposure based on the analytical data with the exposure in the predictive and diagnostic human and animal tests was made in detail before for linalool hydroperoxides (Table 6 in 30 ). It was concluded that between levels in consumer products and known sensitizing doses there is a factor of at least three orders of magnitude. A similar assessment is made here in Table 4 for limonene-1-OOH, which was confirmed to be present in one fine fragrance sample in this study. Based on the results from the current study, the previous assessment remains unchanged, as the consumer exposure from the single positive sample under application conditions of fine fragrance is three orders of magnitude lower as compared to the dose used in a diagnostic study [START_REF] Brared Christensson | Limonene hydroperoxide analogues show specific patch test reactions[END_REF] . Also the defined reporting limit is 300-fold below the routine patch test concentration.

Conclusions

This study and previous studies come to congruent results: we cannot find, by analytical means, evidence of presence of hydroperoxides which can be linked to reported thresholds for sensitization in consumer products from the general market, nor in aged samples retrieved from consumers or patients. There is a wide gap between the analytical sensitivity (for negative samples) and the reported levels for the positive samples and the previously reported sensitization thresholds. Even when applying significant assessment factors to the results from animal studies, the detected hydroperoxide levels appear too low to explain active sensitization. Thus the source and nature of the inducing agent responsible for the high number of positive patch tests reported to oxidized terpene fractions remains elusive. The analytical work with GC-MS at SOLVIAS was funded by IDEA. The analysis by LC-MS was funded by the three participating laboratories (Firmenich, IFF and Givaudan). The study management was funded by IDEA. Manuscript writing was funded by Givaudan Schweiz AG. The synthesis of reference standards and sample collection was funded by the IDEA project. We thankfully acknowledge all donors of samples, in particular the Spanish dermatological network collecting samples from patients (GEIDAC, Grupo Español de Investigación en Dermatitis Alérgica de Contacto). Other members of the IDEA HP taskforce are gratefully acknowledged for fruitful discussions during various workshops. IDEA management team: Hans J. Bender; Industry scientists: Alain Chaintreau, Hugues Brévard and Neil Owen; Scientists from Academia: Jean-Marie Aubry, Anna Börje, Ann-Therese Karlberg, Ulrika Nilsson, David W. Roberts, and Véronique Rataj.

Abbreviations:

GC-MS, gas chromatography mass spectrometry; HP, hydroperoxide; HR-MS, high resolution mass spectrometry; LC-MS, liquid chromatography mass spectrometry; LC-CL, liquid chromatography coupled to chemiluminescence, LC-Q-ToF-MS; quadrupole time of flight mass spectrometer. LLNA, local lymph node assay; LOD, limit of detection; LOQ, limit of quantification 1) Mixture of isomers, not specifically 1-OOH-isomer

2) Dose used in study on specific Limonene-1-OOH isomer by Christensson 33

3) Different dose depending on product type (Cream 10 mg/cm 2 higher than fine fragrance, 2.2 mg/cm 2 )

4) Based on the typical application dose of fine fragrance per area (2.2 mg/cm 2 ) Figure 1. Recovery of standard addition (50µg/g) of four HP added to 104 products analysed by the GC-MS-reduction method.

Table 1 .

 1 Samples above reporting limit as per reduction method, re-analysed by different methods

	Limonene-1-	Limonene-	Linalool-	Linalool-
	OOH	2-OOH	7-OOH	6-OOH Limonene Linalool

Table 2 .

 2 Samples from patch test positive patients analysed by different methodsNote: This Table could also be left within supporting information

	Sample and history of		Limonene-	Limonene-	Linalool-7-	Linalool-
	donating patient	Analytical methods	1-OOH	2-OOH	OOH	6-OOH
		GC-MS red. (µg/ml)	<22	<22	<22	<22
	O12, Body cream, Positive	GC-MS red. (% recovery)	69%	70%	59%	84%
	some fragrances, Positive	LC-Orbitrap-MS (µg/ml)	NF	n.r.	NF	NF
	Limonene ox	LC-Q-ToF-MS (µg/ml)	<5	n.r.	<5	11
		LC-CL (µg/ml)	NF	NF	NF	NF
		GC-MS red. (µg/ml)	<22	<22	<22	<22
	O13, Body cream, Positive Linalool ox	GC-MS red. (% recovery) LC-Orbitrap-MS (µg/ml)	66% NF	72% n.r.	69% NF	70% NF
		LC-Q-ToF-MS (µg/ml)	<5	<5	<5	8
		LC-CL (µg/ml)	trace	trace	Trace	trace
		GC-MS red. (µg/ml)	<22	<22	<22	<22
	O14, Body wash, Positive Linalool ox	GC-MS red. (% recovery) LC-Orbitrap-MS (µg/ml)	99% NF	105% n.r.	77% NF	96% NF
		LC-Q-ToF-MS (µg/ml)	<5	<5	<5	<5
		LC-CL (µg/ml)	trace	trace	trace	trace
		GC-MS red. (µg/ml)	<22	<22	<22	<22
		GC-MS red. (% recovery)	80%	109%	118%	118%
	O15, hand cream, Positive	LC-Orbitrap-MS (µg/ml)	NF	n.r.	NF	NF
		LC-Q-ToF-MS (µg/ml)	<5	<5	<5	<5
		LC-CL (µg/ml)	NF	NF	NF	NF
		GC-MS red. (µg/ml)	<22	<22	<22	<22
		GC-MS red. (% recovery)	89%	74%	71%	70%
	O17, shampoo, Positive	LC-Orbitrap-MS (µg/ml)	NF	n.r.	NF	NF
		LC-Q-ToF-MS (µg/ml)	<5	<5	<5	<5
		LC-CL (µg/ml)	NF	NF	NF	NF
		GC-MS red. (µg/ml)	<22	<22	<22	<22
	O33, Body repair cream, Positive	GC-MS red. (% recovery) LC-Orbitrap-MS (µg/ml)	51% NF	55% n.r.	53% NF	58% NF
		LC-Q-ToF-MS (µg/ml)	<5	<5	<5	<5
		LC-CL (µg/ml)	NF	NF	NF	NF
		GC-MS red. (µg/ml)	<22	<22	<22	<22
	O34, skin repair cream, Positive	GC-MS red. (% recovery) LC-Orbitrap-MS (µg/ml)	65% NF	70% n.r.	69% NF	81% NF
		LC-Q-ToF-MS (µg/ml)	<5	<5	<5	<5
		LC-CL (µg/ml)	NF	NF	NF	NF
		GC-MS red. (µg/ml)	<22	<22	<22	<22
	O36, body milk, Positive	GC-MS red. (% recovery)	73%	76%	76%	85%
	Linalool ox, Positive	LC-Orbitrap-MS (µg/ml)	NF	n.r.	NF	NF
	Limonene ox	LC-Q-ToF-MS (µg/ml)	<5	<5	<5	<5
		LC-CL (µg/ml)	NF	NF	NF	NF

Table 3 .

 3 Two commercial fine fragrance samples with defined storage history analysed by the GC-MS-reduction

	method
		Limonene-	Limonene-	Linalool-	Linalool-
	Condition	1-OOH	2-OOH	7-OOH	6-OOH Limonene Linalool
	Perfume 1, fresh	16	33	18	<16	4100	2200
	Perfume 1, 3 years at RT	<16	<16	<16	<16	4200	2300
	Perfume 1, 3 months, 45°C	<16	18	<16	<16	4300	2300
	Perfume 2, fresh	18	18	36	<16	>5000	4200
	Perfume 2, 6 years at RT	19	<16	32	<16	>5000	4100
	Perfume 2, 3 months, 45°C	24	<16	30	<16	>5000	3900

Table 4 .

 4 Dose per area calculations for limonene-1-OOH

	Dose of
	hydroperoxide in test	Dose per area
	preparation

Acknowledgments and Funding Information

Conflict of interest disclosure

All authors except M.N. and EGA are employees of fragrance companies using linalool and limonene in their products or the International Fragrance Association funded by the fragrance industry. M.N. works for a CRO providing the methods described in this study to third parties. EGA works at the University of Strasbourg, CNRS UMR 7177, and collected the samples from patients as a member of the Spanish Contact Dermatitis Group GEIDAC.