Flaxseed Oil Attenuates Trimethyltin-induced Neurodegeneration via Down-regulation of Inammatory Activity of Astrocytes

Trimethyltin chloride (TMT) is an organotin neurotoxicant that selectively targets the hippocampus, and induces selective and progressive neuronal loss, gliosis, neuroinammation and cognitive decits, thus resembling critical features of Alzheimer’s diseases (AD). Flaxseed oil (FSO) is anti-inammatory agent with potent neuroprotective properties. Therefore, the presented study was designed to evaluate the protective effects of axseed oil (FSO) continuous pretreatment to alleviate TMT- (8 mg/kg) induced neurodegeneration. Ovariectomized (OVX) female rats were pretreated with FSO (1 ml/kg, orally) for two weeks. At day 14, part of animals received single dose of TMT (8 mg/kg, i.p.) and application of FSO continued for seven more days. Data have convincingly shown that FSO counteracted TMT effects. Specically, daily administration of FSO improved TMT- induced behavioral manifestations manifested as hyper-excitability, and hyper-responsiveness, reduced neuronal loss, ameliorated expression of proinammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-10, and IL-6), alleviated astrogliosis and A1-like astrocytes conversion which was related to down-regulation of aromatase/estrogen receptor α signaling, and microgliosis. Together, these ndings support benecial neuroprotective properties of FSO against TMT-induced neurotoxicity and hint at a promising preventive use of FSO in hippocampal degeneration and dysfunction.

In response to injury, astrocytes assume reactive states that may be discriminated based on the proliferation, and induction of pro-in ammatory mediators and reactive oxygen species organized to forms borders around areas of tissue damage or in ammation, and non-proliferative astrocytes that retain the basic cell structure, tissue architecture, and functional interactions established in healthy tissue (Sofroniew 2020). With the analogy to reactive states of microglia, based on their molecular signature these two broad reactive astrocyte subtypes are also classi ed as A1-like pro-in ammatory and A2-like anti-in ammatory astrocytes (Zamanian et al. 2012). We previously found that astrocytes change their morphology early after TMT-intoxication ), up-regulate transcription factors and in ammatory mediators (Dragić et al. , 2021, typical for neurotoxic A1-like astrocytes (Zamanian et al. 2012; Liddelow and Barres 2017). Speci cally, TMT induces up-regulation of NF-κB and its target proin ammatory genes, such as interleukin (IL)-1β, IL-10, IL-6, tumor necrosis factor-α (TNF-α) that was limited to activated astrocytes ( which play key role in the regulation of reactive astrogliosis (Wang et al. 2020). Collectively, astrocyte activation and neuroin ammation contribute to the pathogenesis of TMT-induced cognitive decline and neurotoxicity, and speci c prophylactic/therapeutic strategies actively targeting activated/proin ammatory astrocytes might be of great relevance.
Since their broad spectrum of pharmacological and biological activities, natural products may be promising alternatives for prevention/amelioration of neuroin ammation accompanying neurodegenerative diseases. Flaxseed oil (or linseed oil, FSO), derived from the seeds of the ax (Linum usitatissimum L., FS) is used worldwide as a functional food. It contains the highest amounts of an essential omega-6 (ω-6/n-6) and omega-3 (ω-3/n-3) polyunsaturated fatty acids (PUFAs) obtained from plants such as linoleic acid (LA) and -linoleic acid (ALA), respectively, but also lignans, proteins, and vitamin E (Parikh et al. 2019a injuries. Thus, n-3 PUFAs restored spatial memory loss and protected neurons by suppressing upregulation of pro-in ammatory cytokines, selectively targeted pro-in ammatory microglia and/or astrocytes and reduced their reactivity reverting activated cells to the physiological state (Crupi et Table 1. All animals were decapitated by a small animal guillotine (Harvard apparatus) 24 h after the last injection, and brains were isolated for tissue processing.  Table 2. Relative quanti cation was performed using the comparative 2 −ΔCt method, using peptidylprolyl isomerase A, also known as cyclophilin A (CycA), as a reference gene. Samples obtained from 5 animals for each experimental group were run in duplicate. In each run, internal standard curves were generated by several fold dilutions of generated cDNA to check ampli cation e cacy. Melt curve analysis was performed at the end of every experiment to con rm the formation of a single PCR product.

FSO pretreatment alleviated TMT-induced behavior symptoms
The behavioral score have changed over the 7-day assessment period as early stages of TMT intoxication. Behavioral severity score exhibited a bell-shaped curve which peaked at day 4 and decreases at day 7 (Fig. 2)  . The FSO-treated animals exhibited signi cantly milder behavioral symptoms which resulted in a attened behavioral score curve (Fig. 2), and reduced aggressive behavior.

FSO pretreatment attenuated TMT-induced neurodegeneration
The pattern of TMT-induced neuronal death was consistent with our previous study . Since the differences between Ctrl (OVX) and Ctrl + FSO (OVX treated with FSO) group were insigni cant (data not shown), comparisons were made in respect to Ctrl. Dying and/or necrotic neurons, apoptotic cells bodies and signi cant gliosis were observed in the pcl of CA1, in the hilus of DG and proximal part of CA3 region (hilus/pCA3) (Fig. 3a). On the contrary, in the FSO + TMT group, regular laminar organization and the absence of necrotic cells were observed in the hippocampal CA1 and the hilus, although with some level of gliosis in the hilar/pCA3 sub eld of DG (Fig. 3a).
Further, TMT-induced neurodegeneration was analyzed by FJC staining (Fig. 3b). Ctrl and Ctrl + FSO (data not shown) sections did not show the presence of FJC-positive cells, while in the TMT group strong uorescence staining suggested signi cant neuronal degeneration and cell loss in pcl of CA1 and in the hilar/pCA3 sub eld of DG (Fig. 3b). Prolonged treatment with FSO ameliorates the effects of TMT and prevents the neuronal cell loss (Fig. 3b).
Since FSO treatment induced signi cant neuroprotective effect and prevented TMT-induced neurodegeneration, we further analyzed the cell death signaling pathway by qRT-PCR (Fig. 3c) down-regulation of Bcl-2 mRNA (p < 0.01) in respect to Ctrl (Fig. 3c), while the treatment with FSO counteracted the alterations of target genes expression relative to TMT. Namely, the abundances of Casp3-(p < 0.01) and Bax-mRNA (p < 0.001) decreased, while the Bcl-2-mRNA (p < 0.001) signi cantly increased in TMT animals treated with FSO compared to TMT animals (Fig. 3c).
Furthermore, we examined expression of astrocyte marker (GFAP) and marker of pro-in ammatory A1-like reactive astrocytes (C3) at both gene and cellular levels ( Post hoc analyses showed that TMT induced signi cant up-regulation of transcripts coding for aromatase (p < 0.01) and ERα (p < 0.01) relative to Ctrl (Fig. 5a), while treatment of TMT animals with FSO signi cantly decreased relative abundance of Arom-(p < 0.01) and ERα-mRNA (p < 0.01) compared to TMT.
To con rmed cellular localization of harmful astrocytic marker C3 as well as ERα, we performed triple immuno uorescence labeling. In Ctrl group, GFAP + astrocytes were labeled throughout the entire structure, displaying normal, stellate morphology with numerous ne-branched processes radiating in all directions (Fig. 5b, c). No GFAP + /C3 + cells were observed in the hippocampus of OVX rats. C3-ir was present in the perikaryal membrane (Fig. 5b, c) in accordance with literature data (Kumar and Dhar 2020). As already reported in our previous work ), seven days after TMT intoxication two distinct astrocyte morphotypes were observed; hypertrophied astrocytes in CA1 region (Fig. 5b), and atrophied-like astrocytes in hilar/pCA3 sub eld (Fig. 5c). The response of astrocytes to TMT resulted in an increase of GFAP + /C3 + cells in CA1 (Fig. 5b) and hilar/pCA3 sub elds (Fig. 5c). After FSO pretreatment, GFAP + /C3 + cells were no longer observed in investigated hippocampal regions (Fig. 5b, c).

The effects of FSO pretreatment on TMT-induced microgliosis
Given that astrocytes activate microglial cells and precedes their activation (Röhl and Sievers 2005; Dragić et al. 2020), we examined IBA-1 imunoreactivity as an indicator of microgliosis, and weather FSO pretreatment prevents activation of microglia. A homogeneous distribution of IBA-1-immunoreactive (ir) microglial cells throughout the hippocampus of Ctrl group was noticed (Fig. 6). Higher magni cation image of CA1 (Fig. 6) and hilar/pCA3 sub eld of DG (Fig. 6) showed that IBA-1 + microglia cells had a typical rami ed morphology with small cell bodies, highly branched and elongated processes. Seven days post-TMT intoxication, total IBA-1-ir intensity increased delineating mostly rod-shaped, bushy and amoeboid microglial cells in CA1 (Fig. 6). Also, microglial cells of mixed morphology gradually populated DG (Fig. 6). Repeated FSO pretreatment prevented overall increase in IBA-1-ir cells observed after TMT (Fig. 6). Although occasional microglia with enlarged cell bodies in CA1 sub eld (Fig. 6) as well as sporadic clusters of these cells in the hilar/pCA3 sub eld of DG might be observed (Fig. 6). FSO noticeably attenuated microglia activation.

Discussion
Flaxseed oil is abundant in many nutrients, such as polyunsaturated fatty acid, protein, vitamins, and lignans, and it is eminent by α-linolenic acid in high content, which recently has been found as chie y vital for human organism (Parikh et al. 2019a). To our knowledge, this is the rst study that investigates dietary axseed oil in the animal model of TMT-induced neurodegeneration and neuroin ammation. The data demonstrate that dietary axseed oil given to animals before and over the course of TMT-induced neuropathology (a) reduced hippocampal neuronal loss, (b) exerted an anti-in ammatory effect by preventing the activation of astrocytes and microglia and thus (c) prevented the occurrence of TMTinduced behavioral signs manifested as hyper-excitability, and hyper-responsiveness. Since Flaxseed is the richest plant source of n-3 and n-6 PUFAs, especially ALA and LA, respectively (Parikh et al. 2019a). Therefore, the mechanism responsible for the healing effects of dietary axseed on the TMTinduced hippocampal neuronal loss, likely involves, directly or indirectly, this rich content of PUFAs. Our study complemented the knowledge of the anti-apoptotic actions of FSO by demonstrating that the oil pretreatment reduced the early TMT-induced cell death in the hippocampus, most probably by turning down caspase-3 signaling pathway, reducing expression of pro-apoptotic factor Bax, and enhancing expression of Bcl-2, suggesting that the oil preserved mitochondrial integrity and function.
Neuroin ammation after TMT intoxication is mainly characterized by activation of astrocytes and increased production of pro-in ammatory cytokines (Haga et  The present study indicates that FSO could induce the bene cial inhibition of neuroin ammation and could provide another path for the study of the anti-neuroin ammatory actions of FSO. In response to injury, aromatase is primarily expressed in astrocytes and locally synthesized E2 regulates/modulates astrocyte activation via ERα (Arimoto et al. 2013; Duncan and Saldanha 2019; Wang et al. 2020). Up-regulation of aromatase and ERα in activated astrocytes as well as GFAP expression observed after TMT intoxication, may be a generalized response to injury, since this phenomenon is observed in different types of CNS pathologies, like AD, penetrating brain injury, but also kainic acid model of temporal lobe epilepsy (Blurton-jones and Tuszynski 2001; Dubal et al. 2001;Lu et al. 2003). Thus, by preventing aromatase/ERα signaling, FSO might prevent astrocyte activation and its conversion towards pro-in ammatory A1-like phenotype, probably through inactivation of ERα which acts as a transcription factor over GFAP and signi cantly decreased downstream in ammatory cascade, ultimately lessening the neuronal damage.
TMT directly affect astrocytes which in turn activate microglial cells (Röhl and Sievers 2005), thus we may assume that by preventing TMT-induced astrogliosis, FSO exhibits indirect effects toward activated microglial cells and prevent their activation. However, a portion of activated ERα + /GFAP + astrocytes and microglia remained in the hilar/pCA3 sub eld of DG despite FSO pretreatment. This observation may imply that FSO could not completely protect hippocampus from primary injury caused by strong neurotoxin such as TMT, but prevents from secondary injury which contributes to degeneration of CA1 sub-region (Trabucco et al. 2009).

Conclusion
In conclusion, the current ndings indicate that administration of FSO may have anti-in ammatory and neuroprotective potential as a nutrient that may prevent neuronal injury and development of neurodegenerative disorders, such as AD. Results obtained here represent a model to further investigate the e cacy of using axseed as a dietary supplement in population predisposed to neurodegenerative disorders. However, research has cautioned that the diet may depress body growth due to an imbalance between omega-3 and omega-6 fatty acid levels (Parikh et al. 2019a), thus additional experiments are in need to address the bene cial doses without side effects.

Declarations
Funding: This study was supported by Ministry of Education, Science and Technological development, Republic of Serbia.
Con icts of interest/Competing interests: The authors declare no con ict of interest.
Availability of data and material: Data are available from corresponding author upon reasonable request.
Authors' contributions: NM-conceptualization/design, methodology, investigation, formal analysis, writing -original draft preparation; MD-investigation and data collection, writing -review and editing; MZ-investigation, data collection; NN-Resources, writing -review and editing; IG-conceptualization, formal analysis, writing -review and editing; supervision. All authors read and approved the nal manuscript.
Consent to participate: not applicable Consent for publication: not applicable