The Developmental Neurotoxicity of Paracetamol – Evaluation of markers involved in brain development in mice

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Dnr HT2020

The Developmental Neurotoxicity of

Paracetamol – Evaluation of markers

involved in brain development in mice

Armine Yakub

Degree Project in Pharmaceutical pharmacology, 30 hp,

Autumn semester 2020

Examiner: Alfhild Grönblad

Department of Pharmaceutical Biosciences Division for Pharmacology

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Abstract

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Introduction

Paracetamol, a non-prescription analgesic and antipyretic, is one of the most frequently used drugs in the world (Bertolini et al., 2006; de Fays et al., 2015). Paracetamol and another drug called phenacetin are both derivatives of acetanilide, a drug that was found to possess analgesic and antipyretic properties but also showed toxic effects. Paracetamol was synthesized in 1878 and was used clinically for the first time in 1887. However, the use of paracetamol was restricted because phenacetin was considered to be less toxic (Bertolini et al., 2006). Paracetamol was brought to light again in the 1950's after the studies of Brodie and Axelrod (Brodie and Axelrod, 1948). The studies showed that paracetamol was the metabolite giving the analgesic action of acetanilide and that another metabolite is responsible for its toxic effects, methemoglobinemia. This led to the reappearance of paracetamol and its marketing. Later, paracetamol gained popularity and became one of the most largely used analgesic and antipyretic drugs worldwide (Bertolini et al., 2006).

Today, paracetamol is considered the first-choice treatment regarding pain and fever during pregnancy (de Fays et al., 2015; Werler et al., 2005) and is also commonly used in children (Hawkins and Golding, 1995; Walsh et al., 2007). Pregnant women are usually not included in clinical trials. Therefore, the majority of medications are being marketed without adequate studying of the safety profile in human pregnancy and the risks that the fetus can be subjected to (de Fays et al., 2015). In spite of this, the use of medication among pregnant women is common. For example, the estimated prevalence of paracetamol use is 49% in Northern America, 51% in Western Europe and 61% in Northern Europe (Lupattelli et al., 2014). After oral administration of paracetamol, the drug is found in the breast milk of mothers (Notarianni et al., 1987), passes the blood-brain barrier (Kumpulainen et al., 2007) and placental barrier reaching the brain (Levy et al., 1975; Nitsche et al., 2017); thus, paracetamol has the ability to reach the developing brain following maternal consumption.

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third trimester compared to exposure during the first trimester. Another epidemiological study also showed that prenatal exposure to paracetamol was linked to higher risk of developing ADHD related behavioural problems (Stergiakouli et al., 2016). Moreover, in another study it was shown that paracetamol intake during pregnancy was linked to poorer communication skills and gross motor development, delay in walking and increased hyperactivity (Brandlistuen et al., 2013). Stronger effects were shown when paracetamol was used in the third trimester. However, how paracetamol exerts this neurotoxicity is still unknown. Similar findings have been observed in mice. It has been shown that a single day's exposure (30 + 30 mg/kg with 4-hour interval), during a rapid growth phase of the brain on postnatal day (PND) 10, resulted in changed spontaneous behaviour, reduced capability of habituation to a novel home cage, decreased spatial learning and working memory in radial arm maze in adulthood (Philippot et al., 2017; Viberg et al., 2014). These mice also showed decreased analgesic and anxiolytic response to paracetamol later in life (Viberg et al., 2014). Also in mice, it is unclear in what way paracetamol interferes with brain development. Therefore, it is of utmost importance to find out the mechanism behind the adverse effect of paracetamol on brain development.

Mechanism of action

Despite paracetamol has been used for a long time, its mechanism of action is still unclear and not fully known (Bauer et al., 2018; Toussaint et al., 2010). Unlike non-steroidal anti-inflammatory drugs (NSAIDs), paracetamol has largely no anti-anti-inflammatory activity (Bertolini et al., 2006). It has been found that paracetamol affects central cyclooxygenase (COX) activity, and this interaction is likely responsible for paracetamol’s antipyretic effect (Flower and Van, 1972). The inhibition of COX by paracetamol is peroxide-dependent and takes place in areas where low levels of peroxide are seen; it is therefore assumed that this is why paracetamol has anti-inflammatory effects in the brain, where peroxidase activity is low, but not in the periphery (Bertolini et al., 2006; Boutaud et al., 2002).

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increased synapse concentration of anandamide (an endogenous cannabinoid) that binds to CB1R increasing its activation (Bertolini et al., 2006). AM404 can also bind to CB1R directly. However, the binding affinity of AM404 to CB1R is quite low compared to anandamide (Zygmunt et al., 2000). Thus, the direct effect of AM404 on CB1R seems less interesting than the indirect effect (Bertolini et al., 2006).Interestingly, the analgesic effect of paracetamol has been shown to be inhibited when CB1R are blocked by antagonists further showing that paracetamol works through CB1R (Ottani et al., 2006). Furthermore, AM404 acts as a potent activator of vanilloid subtype 1 receptor which is also involved in thermoregulatory and pain pathways (Högestätt et al., 2005).

Brain Growth Spurt

The brain growth spurt (BGS) is referred to a period of brain development where the brain weight increases fast (Davison and Dobbing, 1968; Dobbing and Sands, 1979). During the BGS many developmental changes occur in terms of dendritic maturation, axonal growth, neuronal connection formation, synaptogenesis, glial multiplication and myelination (Davison and Dobbing, 1968; Dobbing and Sands, 1979). The brain is known to have increased vulnerability to toxic insults during this specific period of brain development (Eriksson, 1997, Rice and Barone, 2000).

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Figure 1. The brain growth spurt of different mammalian species (Dobbing and Sands, 1979).

Biomarkers

Synaptophysin (SYP) is a synaptic protein found in synaptic vesicles (Wiedenmann and Franke, 1985). It is commonly used as a presynaptic marker for determining synapse density (Valtorta et al., 2004). SYP is involved in regulating activity-dependent formation of synapses (Kwon and Chapman, 2011; Tarsa and Goda, 2002). Moreover, SYP is known to be involved in synaptic vesicle formation and processes that are important for synaptic vesicle exo- and endocytosis (Kwon and Chapman, 2011; Valtorta et al., 2004). In mice, SYP levels are increasing postnatally between PND 1 and 28 with the most dramatic increase between PND 7 and 14 encompassing the peak of the BGS (Viberg, 2009).

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N-methyl-D-aspartate receptor subtype 2B (NMDAR2B) is one of the subunits that make up the NMDA receptor complex which is a type of postsynaptic glutamate receptor. The NMDA receptor is a ligand gated ion channel that is permeable to Ca2+ but is blocked by Mg2+. The blockade by Mg2+ is voltage dependent where postsynaptic neuron depolarization removes Mg2+ from the channel and results in influx of Ca2+ to the cell. The influx of Ca2+ leads to long-term potentiation (LTP) induction by activating signal transduction cascades including Ca2+/calmodulin dependent protein kinase type II (CAMKII) and protein kinase C (Augustine, 2008). NMDAR2B is mostly expressed in the hippocampus and cerebral cortex and is dominant at birth and in the early postnatal brain (Magnusson, 2012). It has an important role in learning and memory (Loftis and Janowsky, 2003). Improved learning abilities has been shown in transgenic mice overexpressing NMDA2B (Tang et al., 1999). Moreover, it has been shown that LTD (long term depression) and synaptic NMDA receptor responses are absent in the hippocampus of mutant mice having defected NMDA2B (Kutsuwada et al., 1996).

CAMKII is considered as one of the most highly enriched protein kinases in the brain (Erondu and Kennedy, 1985). CAMKII is mostly expressed in the hippocampus and cerebral cortex (Sola et al., 1999). It has been shown in mice that the expression of CAMKII peaks postnatally and is increasing in the first four weeks after birth, mostly between PND 7 and 14 (Viberg et al., 2008a). CAMKII is activated by Ca2+ influx and after Ca2+/Calmodulin binding. Its activity and autophosphorylation are crucial for LTP formation in hippocampus and hippocampus-dependent spatial learning and memory (Colbran and Brown, 2004; Lisman et al., 2002). Moreover, CAMKII is involved in other significant processes such as regulating synapse formation, synaptic plasticity (Frankland et al., 2001; Kazama et al., 2007; Yamauchi, 2005), dendritic and axonal arborization (Zou and Cline, 1999).

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Growth associated protein 43 (GAP-43) is a presynaptic protein expressed highly in axonal growth cones throughout the development of the central nervous system and acts as a common biomarker for axonal sprouting and growth (Oestreicher et al., 1997). GAP-43 has a significant role in axonal pathfinding (Strittmatter et al., 1995), guiding axonal growth and establishing new axonal connections during development and axonal regeneration (Oestreicher et al., 1997). In mice, GAP-43 levels increase continuously during the first four weeks after birth and reach a peak on PND 7 in hippocampus and PND 10 in cortex. After four weeks of gradually increasing, the levels start to decrease (Viberg et al., 2008a).

Tropomyosin receptor kinase B (TRKB) is a receptor activated by its high affinity ligand the neurotrophin brain-derived neurotrophic factor (BDNF). TRKB and BDNF signalling promote neurogenesis and are vital for neuronal maturation, survival and differentiation during brain development (Huang and Reichardt, 2003; LaMantia, 2008). In mature neurons on the other hand, they play an important role in behavioural and synaptic plasticity such as hippocampal-dependent memory (Heldt et al., 2007; Huang and Reichardt, 2003). Moreover, it has been shown that TRKB knockout mice display impairments in spatial learning (Minichiello et al., 1999). Studies performed on rats show high levels of TRKB expression in the early postnatal period in the hippocampus (Freyer et al., 1996; Silhol et al., 2005).

Aim

The overall aim of this study is to investigate the biochemical changes that could possibly underlie the adult behavioural changes previously observed in mice after neonatal paracetamol exposure. The specific aim is to examine whether adult mice exposed to paracetamol during the BGS have altered expression of the biomarkers SYP, PSD-95, NMDAR2B, CAMKII, GLUR1, TRKB and GAP-43 in Cornu Ammonis subfield 3 (CA3), Cornu Ammonis subfield 1 (CA1) and dentate gyrus (DG) regions of hippocampus using an immunohistochemical method.

Materials and Methods

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Animals

Pregnant Naval Medical Research Institute (NMRI) mice (from Charles River Laboratory) were obtained from Scanbur (Sollentuna, Sweden). These mice were housed individually in macrolon cages in rooms having temperature of 22oC, 45-65% humidity and a 12-hour light and dark cycle. Standardized pellet food (Lactamin, Stockholm, Sweden) and tap water was provided to the animals. Birth was checked every day at the same time at 18:00 and the birth day was termed PND 0.

Exposure and chemicals

Paracetamol (Paracetamol Fresenius Kabi, 10mg ml/ml; Fresenius Kabi AB, Sweden; CAS no. 103-90-2) was purchased (Apoteksbolaget, Sweden) and a stock solution consisting of 6 mg paracetamol/m1 saline (0.9% sodium chloride in water) was prepared. Eight male mice pups were selected from different litters and exposed to either paracetamol (30 + 30 mg/kg, 4-hour interval) or saline (0.9% sodium chloride in water) (Apoteksbolaget, Sweden) by subcutaneous injection in their neck. The exposures had a volume of 5 ml/kg and occurred on PND 10. After becoming two months old, the adult mice were anesthetized by intraperitoneal injection of 0.01mg/g body weight sodium pentobarbital (Apoteket Farmaci, Sweden). The tissues were fixed by transcardiac perfusion with 4% formaldehyde (HistoLabs, Sweden). After that, the brains were dissected out and kept in 4% formaldehyde overnight, followed by embedding them in Killik (Bio-Optica, Italy) and freezing at -80°C until sectioning.

Cryosectioning and immunohistochemistry

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4°C overnight. Three different concentrations of primary antibodies were used for optimization to obtain a signal: 1:400, 1:200 and 1:100. The optimal concentration for each primary antibody was identified. The next day the sections were washed 3 x 10 min in PBS and were incubated for two hours at room temperature in a humidity chamber in the dark with secondary antibody diluted in Supermix. The secondary antibodies used were donkey anti-rabbit Alexa Fluor 594 (Invitrogen, A21207) diluted in 1:800 Supermix for the primary rabbit antibodies: PSD-95 (Cell Signalling technology, 2507S), NMDAR2B (Cell Signalling technology, 14544S), GLUR1 (Cell Signalling technology, 13185S), TRKB (Cell Signalling technology, 4606S) and GAP-43 (Abcam, ab16053) and donkey anti-mouse Alexa Fluor 488 (Invitrogen, A21202) diluted in 1:400 Supermix for the primary mouse antibodies SYP (Abcam, ab8049) and CAMKII (Cell Signalling technology, 50049S). The sections were then washed 3 x 5 min in PBS and each section was then mounted with 50 μl of ProLong Gold antifade reagent with 4',6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Sweden) and kept in the fridge until microscope analysis. Moreover, the autofluorescence was controlled to make sure and identify that it was the antibody that gave the signal.

Microscope, quantification and statistical analysis

Images for SYP and PSD-95 in CA3, CA1 and DG regions of hippocampus were taken using a Zeiss AxioImager M2 (Zeiss, Germany) at BioVis platform, Uppsala University. Fluorescent signals were collected at 488 nM for SYP and 594 nM for PSD-95 with an exposure time of 2.62 s for SYP and 10 s for PSD-95. Images for SYP were then handled and total immunofluorescence intensity per area unit (A.U.) was quantified in a blinded manner by manually marking the area where the signals were most visible. The quantification was done using the image processing programme, ImageJ.

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Results

In the immunohistochemical method three different concentrations of each primary antibody were used for optimization in order to obtain a signal: 1:400, 1:200 and 1:100. The optimal concentration for each primary antibody was identified which is illustrated in Table 1.

Table 1. Optimal concentrations of different primary antibodies

Primary antibody Dilution

SYP 1:100 PSD-95 1:100 NMDAR2B 1:200 CAMKII 1:200 GLUR1 1:200 GAP-43 1:200 TRKB 1:100

The biomarkers NMDAR2B, GLUR1, CAMKII, TRKB and GAP-43 were only optimized in this study where optimal concentrations resulting in signal were identified based on microscope analysis as shown in Table 1. However, the immunofluorescence intensity/A.U. of NMDAR2B, GLUR1, CAMKII, TRKB and GAP-43 was not quantified. The biomarker PSD-95 was also optimized with respect to concentration and the optimal concentration giving signal was identified as seen in Table 1. PSD-95 was double stained with SYP. However, the immunofluorescence staining of PSD-95 gave weak signal based on microscope analysis and quantifying the immunofluorescence intensity/A.U. of PSD-95 was not assessed.

Potential effects on SYP levels in CA3, CA1 and DG regions of hippocampus

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(30 + 30 mg/ kg) with 4-hour interval or saline on PND 10.An illustration of the three regions of interest, CA3, CA1 and DG in coronal sections of adult mouse hippocampus (Bregma -1.94) using nuclear staining with DAPI is shown in Figure 2.

Figure 2. Nuclear staining with 4',6-diamidino-2-phenylindole (DAPI) in coronal sections of the adult mouse hippocampus (Bregma -1.94) illustrating the Cornu Ammonis subfield 3 (CA3), Cornu Ammonis subfield 1 (CA1) and dentate gyrus (DG) subregions used in this study. Scale bar = 500 µm.

Immunofluorescence staining of SYP, nuclei staining with DAPI, SYP and DAPI merged in CA3, CA1 and DG regions in coronal sections of the adult mouse hippocampus (Bregma -1.94) is shown in Figure 3, 4 and 5 respectively. The images shown in these three figures represent the four individuals used in each exposure group. No significant difference (p-value > 0.05) in immunofluorescence intensity/A.U. of SYP was shown between paracetamol exposed mice and controls in any of CA3, CA1 and DG regions of hippocampus as shown in Figure 3, 4 and 5.

CA3

CA1

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Figure 3. Immunofluorescence staining of synaptophysin (SYP) in Cornu Ammonis subfield 3

(CA3) coronal sections of the adult mouse hippocampus (Bregma -1.94). The chosen images represent the four individuals used in each exposure group. (A). Nuclei stained with 4',6-diamidino-2-phenylindole (DAPI) are shown in blue. (B). SYP staining is shown in green. (C). SYP and DAPI merged. Scale bar = 100 µm. (D). Relative immunofluorescence intensity per area unit (A.U.) of SYP measured in male adult mice after neonatal exposure to either saline (control) or paracetamol (30 + 30 mg/ kg, 4 hours apart) on postnatal day 10 in CA3 region of hippocampus. No significant difference (p-value > 0.05) was observed between paracetamol treated mice and controls in CA3. The data were subjected to Student t-test. The height of the bars represents mean value ± standard deviation (SD). Each exposure group contained 4 mice.

CA3

A B C

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Figure 4. Immunofluorescence staining of synaptophysin (SYP) in Cornu Ammonis subfield 1

(CA1) coronal sections of the adult mouse hippocampus (Bregma -1.94). The chosen images represent the four individuals used in each exposure group. (A). Nuclei stained with 4',6-diamidino-2-phenylindole (DAPI) are shown in blue. (B). SYP staining is shown in green. (C). SYP and DAPI merged. Scale bar = 100 µm. (D). Relative immunofluorescence intensity per area unit (A.U.) of SYP measured in male adult mice after neonatal exposure to either saline (control) or paracetamol (30 + 30 mg/ kg, 4 hours apart) on postnatal day 10 in CA1 region of hippocampus. No significant difference (p-value > 0.05) was observed between paracetamol treated mice and controls in CA1. The data were subjected to Student t-test. The height of the bars represents mean value ± standard deviation (SD). Each exposure group contained 4 mice.

CA1

A

B C

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Figure 5. Immunofluorescence staining of synaptophysin (SYP) in dentate gyrus (DG) coronal

sections of the adult mouse hippocampus (Bregma -1.94). The chosen images represent the four individuals used in each exposure group. (A). Nuclei stained with 4',6-diamidino-2-phenylindole (DAPI) are shown in blue. (B). SYP staining is shown in green. (C). SYP and DAPI merged. Scale bar = 100 µm. (D). Relative immunofluorescence intensity per area unit (A.U.) of SYP measured in male adult mice after neonatal exposure to either saline (control) or paracetamol on postnatal day 10 in DG region of hippocampus. No significant difference (p-value > 0.05) was observed between paracetamol treated mice and controls in DG. The data were subjected to Student t-test. The height of the bars represents mean value ± standard deviation (SD). Each exposure group contained 4 mice.

Discussion

In this study, the synaptic density in the hippocampus, a brain region vital for memory processing and learning (Deng et al., 2010), of adult mice was examined following neonatal exposure to relevant doses of paracetamol. The dose to which the mice were exposed to has previously been shown to affect memory and learning later in life. However, in this study it was found that this exposure did not affect the synaptic marker SYP in three distinct regions of the hippocampus,suggesting that there may be other mechanisms that could underlie paracetamols developmental neurotoxicity.

Paracetamol is an interesting drug to investigate since increasing evidence from both animal and human studies over the past years have shown that neonatal exposure to paracetamol is linked with adverse behavioural outcomes later in life. As mentioned earlier, epidemiological studies suggest a link between maternal intake of paracetamol during pregnancy and higher risk of developing HKD, ADHD, autism spectrum disorder and other adverse behavioural effects

DG

A B C

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later in life (Bauer et al., 2018). In parallel, similar findings have been seen in animals (Bauer et al., 2018; de Fays et al., 2015). Animal data display that PND 10 paracetamol exposure in mice affects memory and learning in adulthood (Philippot et al., 2017; Viberg et al., 2014). The present study was therefore aimed to investigate whether neonatal exposure to paracetamol (30 +30 mg/kg, 4-hour interval) altered the expression of biomarkers important for brain development in adult mice. In this study, the effects of PND 10 paracetamol exposure were studied in adulthood. Mice exposed to PND 10 have shown to have a high sensitivity not only to paracetamol (Philippot et al., 2017; Viberg et al., 2014) but also to other xenobiotics such as propofol (Pontén et al., 2011), ketamine (Viberg et al., 2008b) and Δ (9)-tetrahydrocannabinol (THC) (Philippot et al., 2016) demonstrating adverse behavioural effects later in life.

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line with another study using the same exposure where both SYP transcript and protein levels in hippocampus were not affected 24 hours after neonatal paracetamol exposure (Philippot et al., 2018). The results shown in this study, together with the above-mentioned study, indicate that previously observed adult behavioural changes after neonatal exposure to paracetamol might probably have other origins than effects on SYP. However, a recent study on adult rats showed reduced SYP and PSD-95 protein levels in the hippocampus following long-term exposure, both 15 and 30 days, to 200 mg/kg paracetamol (Lalert et al., 2020). The alterations of protein levels were more obvious after 30 days compared to 15 days of exposure. This indicates that paracetamol has the ability to affect synaptic density when given in adulthood. The differences between the effects on SYP between the above-mentioned study and the results presented herein can be due to several reasons. First, in the study by Lalert and colleagues, higher doses were used compared to this study making it difficult to compare these studies. Second, the rats were exposed for a much longer period compared to the mice in this study. It is therefore likely that the results could have been different if the exposure had been shortened. A third reason is that they expose the adult animal, unlike the mice in this study, where the exposure occurs during their development. Although the brain is generally considered to be more sensitive during development (Rice and Barone, 2000), it cannot be ruled out that there may be exceptions.

A possible reason for the lack of difference of SYP levels between the exposure groups could be that the method used in this study may not be sensitive enough to detect actual and small differences in SYP signals. This is because there was some background fluorescence which potentially can affect signal to noise ratio making it harder to detect a potential difference. The background can theoretically be reduced by using different blocking solutions. Moreover, in the quantification software the threshold level could be optimized in order to reduce the impact of the background signals and potentially detect only signals originating from SYP. Yet another approach to reduce background fluorescence could be to use a microscope with a narrower optical filter that does not take up much of the autofluorescence and captures SYP signals only. Another reason for the lack of difference could also be the small sample size (4 individuals in each exposure group) making it statistically difficult to detect a potential difference. Therefore, to detect small potential differences larger sample size is suggested.

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noradrenergic, dopaminergic, and neurotrophic systems in adult rodents following developmental exposure to 5-15 mg/kg paracetamol (Blecharz-Klin et al. 2015, 2016, 2017). Additionally, other possible mechanistic pathways for neurotoxicity of early paracetamol exposure are proposed and include excess toxic N-acetyl-p-benzoquinone imine formation, oxidative stress, effects on prostaglandin synthesis and endocannabinoid signalling (Bauer et al., 2018).

In this study, PSD-95 was double stained with SYP. However, it was difficult to quantify the immunofluorescence intensity of PSD-95 because the signal was weak and not consistent for all microscope pictures. Thus, quantifying PSD-95 protein levels could not reliably be assessed. This is most likely because PSD-95 is at the detection limit and that the tissue quality differs slightly between different sections. Therefore, some sections may be exactly below the limit of detection and others just above the limit. This in turn, may be due to either the antibody having low affinity for its antigen or that the concentration of the antibody and/or the antigen is low. This can be improved by either increasing the concentration of PSD-95, using another PSD-95 antibody with stronger affinity or in some way affecting the binding reaction by, for example, increasing the incubation time. Therefore, further investigation on the possible effect on PSD-95 is suggested; however, this could not be done due to lack of time.

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be improved by deciding before sectioning which sections to take to not end up with too many sections since it is hard to identify the exact bregma when they are unstained. Moreover, when measuring the fluorescence intensity there is also batch-batch variation between different days of staining, which can result in different concentrations of antibody stock solutions. However, this effect was managed by having sections from each individual represented in each run. During the staining procedure, it was noted that some brain sections in almost all individuals had small holes which could be due to damages inflicted during sectioning. This could be improved by having thicker sections. In this study, only hippocampus was included. For further investigation, other brain areas could be included to obtain a general assessment on the possible effects of paracetamol developmental neurotoxicity.

Due to the growing evidence from epidemiological studies of the developmental neurotoxicity of paracetamol (Bauer et al., 2018) along with paracetamol being a non-prescription drug and easily available to the public, evaluating the possible negative effect caused by developmental paracetamol exposure is important. Therefore, further research on possible mechanisms is needed.

In conclusion, this study shows that there is no difference in SYP levels in CA3, CA1 and DG regions of hippocampus between mice neonatally exposed to paracetamol compared to controls later in life. These findings suggest that previously observed adult behavioural changes after neonatal exposure to paracetamol might probably be of other origins than effects on synaptic density in hippocampus using the synaptic marker SYP in adulthood. Moreover, further studies are required to investigate whether the expression of PSD-95, NMDAR2B, GLUR1, CAMKII, TRKB and GAP-43 in CA3, CA1 and DG regions of hippocampus is affected in adult mice neonatally exposed to paracetamol. Further research on possible mechanisms behind paracetamol-induced adverse developmental effects is however warranted to make a better and more accurate risk/benefit assessment as possible.

Svensk populärvetenskaplig sammanfattning

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rön har iakttagits hos möss. Det har visat sig att en enda dags exponering för paracetamol, under en känslig period av hjärnans utveckling så kallad “brain growth spurt” (BGS), inducerar bestående minne och inlärningsproblem hos möss. Dock är det okänt exakt på vilket sätt paracetamol påverkar hjärnan och utövar denna neurotoxicitet.

Denna studie syftar därför till att undersöka om vuxna möss som exponerats för paracetamol under hjärnans utveckling har förändrat uttryck av neuroproteiner som är viktiga för hjärnans utveckling. För att undersöka detta har möss vid ett tidigare tillfälle exponerats för antingen paracetamol eller vehikel på postnatal dag 10. Mössens hjärnor har sedan dissekerats ut när mössen var två månader gamla. I denna studie, snittades hjärnorna med fryssnittning och senare undersöktes med immunhistokemisk metod för neuroproteinerna Synaptophysin (SYP), Postsynaptic density protein 95 (PSD-95), N-methyl-D-aspartate receptor subtype 2B (NMDAR2B), Ca2+_{/calmodulin dependent protein kinase type II (CAMKII), Glutamate}

receptor 1 (GLUR1), Growth associated protein 43 (GAP-43) och Tropomyosin receptor kinase B (TRKB) i hippocampus.

Resultaten visade att det inte finns någon signifikant skillnad i proteinnivåerna av synaptiska markören SYP i tre regioner i hippocampus mellan paracetamol-behandlade möss och kontroller. Detta kan tyda på att beteendeförändringar som tidigare observerats hos möss vid vuxen ålder efter neonatal exponering för paracetamol förmodligen har ett annat ursprung och kan beror på andra mekanismer. Ytterligare studier krävs för att undersöka huruvida uttrycket av PSD-95, NMDAR2B, GLUR1, CAMKII, TRKB och GAP-43 i hippocampus är förändrat hos vuxna möss efter neonatal paracetamol exponering. Resultatet indikerar behovet av vidare forskning om möjliga mekanismer som ligger till grund för de negativa effekter som tidigare observerats i både möss och människa efter exponering för paracetamol under hjärnans utveckling.

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The Developmental Neurotoxicity of Paracetamol – Evaluation of markers involved in brain development in mice