Sex and Age-Related Differences in Neuroinflammation and Apoptosis in Balb/c Mice Retina Involve Resolvin D1

(1) Background: The pro-resolving lipid mediator Resolvin D1 (RvD1) has already shown protective effects in animal models of diabetic retinopathy. This study aimed to investigate the retinal levels of RvD1 in aged (24 months) and younger (3 months) Balb/c mice, along with the activation of macro- and microglia, apoptosis, and neuroinflammation. (2) Methods: Retinas from male and female mice were used for immunohistochemistry, immunofluorescence, transmission electron microscopy, Western blotting, and enzyme-linked immunosorbent assays. (3) Results: Endogenous retinal levels of RvD1 were reduced in aged mice. While RvD1 levels were similar in younger males and females, they were markedly decreased in aged males but less reduced in aged females. Both aged males and females showed a significant increase in retinal microglia activation compared to younger mice, with a more marked reactivity in aged males than in aged females. The same trend was shown by astrocyte activation, neuroinflammation, apoptosis, and nitrosative stress, in line with the microglia and Müller cell hypertrophy evidenced in aged retinas by electron microscopy. (4) Conclusions: Aged mice had sex-related differences in neuroinflammation and apoptosis and low retinal levels of endogenous RvD1.


Introduction
The decrease in the physiological functions of the aged organs can be influenced by genetic and environmental factors [1]. Age-related immune dysfunctions leading to chronic inflammation is a major risk factor for the incidence and prevalence of age-related glia, alongside with neuronal and glial cell death [3], that leads to morphol tions and visual impairment [4].
Clinical studies have indicated that eye disorders, such as cataract, gl age-related macular degeneration (AMD), are associated with sex and have incidence with age [5][6][7].
In this context, our previous work showed that the aged retina is mor the damage in male mice than in female mice [8]. This is because, in the r mice, there is a greater dysregulation of some age-related microRNAs (miRN to oxidative stress response and neurodegeneration. In fact, the thickening and the integrity of the Bruch's membrane were correlated with the dysregul 27a-3p, miR-20a-5p, miR-20b-5p, and miR-27b-3p in physiologically aged ma pared to physiologically aged female mice [8]. Since the ocular expression of some of these miRNAs is under contro resolving lipid mediator Resolvin D1 (RvD1) in male murine models of reti tion [9][10][11][12] and RvD1 itself governs neurodegenerative disorders with protec thought about its possible involvement in the sex and age differences in aged pared to younger retinas. Therefore, apoptosis, neuroinflammation, and act and microglia together with the retinal levels of RvD1 are studied here in younger retina of male and female Balb/c mice.

Microglial Activation
Ionized calcium-binding adapter molecule 1 (Iba-1), as a marker of micr tion, was inversely correlated with the levels of RvD1 measured in the retina h

Microglial Activation
Ionized calcium-binding adapter molecule 1 (Iba-1), as a marker of microglial activation, was inversely correlated with the levels of RvD1 measured in the retina homogenates (r = −0.88; p < 0.01) (Table 1). Increased Iba-1 immunoreactivity (as a marker of microglial activation) was detected in aged retina compared to control retina. This was distributed in outer retina (starting with photoreceptor outer segment) and inner retina evenly throughout the nerve fiber layer and the ganglion cell layer (Figure 2A). The retinas of both aged male and female mice showed a significant increase in Iba-1 (AM: 79 ± 8%, p < 0.01 vs. CM; AF: 45 ± 10%, p < 0.05 vs. CF) compared to the retinas of young same-sex mice (CM: 20 ± 2%; CF: 25 ± 3%) ( Figure 2A). However, it was more markedly significant for the retinas of aged male mice than for the retinas of aged female mice (p < 0.05 vs. AM) (Figure 2A). These results were confirmed by Iba-1 protein levels' detection by Western Blotting analysis ( Figure 2B). Microglial hypertrophy was evidenced in the extended damaged inner nuclear layer (INL) areas in aged retina of both sexes ( Figure 2C). in outer retina (starting with photoreceptor outer segment) and inner retina evenly throughout the nerve fiber layer and the ganglion cell layer (Figure 2A). The retinas of both aged male and female mice showed a significant increase in Iba-1 (AM: 79 ± 8%, p < 0.01 vs. CM; AF: 45 ± 10%, p < 0.05 vs. CF) compared to the retinas of young same-sex mice (CM: 20 ± 2%; CF: 25 ± 3%) ( Figure 2A). However, it was more markedly significant for the retinas of aged male mice than for the retinas of aged female mice (p < 0.05 vs. AM) ( Figure 2A). These results were confirmed by Iba-1 protein levels' detection by Western Blotting analysis ( Figure 2B). Microglial hypertrophy was evidenced in the extended damaged inner nuclear layer (INL) areas in aged retina of both sexes ( Figure 2C).

Müller Cell Activation
The regression with the RvD1 levels showed a significant negative correlation between RvD1 and glial fibrillary acidic protein (GFAP), a marker of astrocytes activation (r

Müller Cell Activation
The regression with the RvD1 levels showed a significant negative correlation between RvD1 and glial fibrillary acidic protein (GFAP), a marker of astrocytes activation (r = −0.86; p < 0.01) (Table 1). Particularly, GFAP-positive cells were marked in aged retina (AM: 58 ± 5%, p < 0.01 vs. CM; AF: 37 ± 4%, p < 0.05 vs. CF) ( Figure 3A). These were localized in the inner and outer retinal layers and were much more intense in the retinas of male mice if compared to the retinas of female mice (p < 0.01 vs. AM) ( Figure 3A). Accordingly, GFAP protein levels detected by Western Blotting showed the same trend between the control and aged retina, particularly between aged male and female mice (p < 0.01 vs. AM) ( Figure 3B). Moreover, electron microscopy evidenced Müller cell hypertrophy and hyperplasia on aged retina of both sex ( Figure 3C). Particularly, a normal aspect of the Müller cells processes between bipolar cells was present in the inner nuclear layer (INL) of the controls, while an extended network of cytoplasmic process of Müller cells was shown by both aged retinas ( Figure 3C). (AM: 58 ± 5%, p < 0.01 vs. CM; AF: 37 ± 4%, p < 0.05 vs. CF) ( Figure 3A). These were localized in the inner and outer retinal layers and were much more intense in the retinas of male mice if compared to the retinas of female mice (p < 0.01 vs. AM) ( Figure 3A). Accordingly, GFAP protein levels detected by Western Blotting showed the same trend between the control and aged retina, particularly between aged male and female mice (p < 0.01 vs. AM) ( Figure 3B). Moreover, electron microscopy evidenced Müller cell hypertrophy and hyperplasia on aged retina of both sex ( Figure 3C). Particularly, a normal aspect of the Müller cells processes between bipolar cells was present in the inner nuclear layer (INL) of the controls, while an extended network of cytoplasmic process of Müller cells was shown by both aged retinas ( Figure 3C).

Neuroinflammation
Aged retinas showed increased markers of inflammation such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) ( Figure 4) and Tumor Necrosis Factor alpha (TNF-α) ( Figure 5) detected by immunohistochemistry compared to controls.

Nitrotyrosine Measurement
The measure of the 3-nitrotyrosine levels as a marker of peroxynitrite formation in the retina homogenates was high in the aged retina of both sexes (AM: 95 ± 8 ng/mL, p < 0.01 vs. CM; AF: 74 ±7 ng/mL, p < 0.05 vs. CF) (Figure 7). This had the maximal values in retinas extracted from eyes of male mice being significantly different from retinas of female mice aged equally (p < 0.05 vs. AM) (Figure 7). Additionally, 3-nitrotyrosine levels were negatively correlated with RvD1 levels (r = −0.87, p < 0.01) (Figure 7).

Nitrotyrosine Measurement
The measure of the 3-nitrotyrosine levels as a marker of peroxynitrite formation in the retina homogenates was high in the aged retina of both sexes (AM: 95 ± 8 ng/mL, p < 0.01 vs. CM; AF: 74 ±7 ng/mL, p < 0.05 vs. CF) (Figure 7). This had the maximal values in retinas extracted from eyes of male mice being significantly different from retinas of female mice aged equally (p < 0.05 vs. AM) (Figure 7). Additionally, 3-nitrotyrosine levels were negatively correlated with RvD1 levels (r = −0.87, p < 0.01) (Figure 7).

Nitrotyrosine Measurement
The measure of the 3-nitrotyrosine levels as a marker of peroxynitrite forma the retina homogenates was high in the aged retina of both sexes (AM: 95 ± 8 ng/m 0.01 vs. CM; AF: 74 ±7 ng/mL, p < 0.05 vs. CF) (Figure 7). This had the maximal va retinas extracted from eyes of male mice being significantly different from retina male mice aged equally (p < 0.05 vs. AM) (Figure 7). Additionally, 3-nitrotyrosine were negatively correlated with RvD1 levels (r = −0.87, p < 0.01) (Figure 7).

Discussion
RvD1 is a lipid derived from docosahexaenoic acid metabolism together with protectins and maresins [13]. It binds its own specific receptor called formyl peptide receptor 2 (FPR2) and is involved in the genesis/resolution of several inflammatory pathologies [11]. The deficiency of this lipid has been linked to the onset of degenerative diseases typical of the CNS, especially if an oxidative-inflammatory insult causes the degeneration of the cells involved [14][15][16]. Accordingly, in previous papers done by this group, RvD1 showed a protective effect when applied exogenously to murine uveitic eyes and degenerating photoreceptors in vitro [9][10][11][12]. Here, we further contribute to these data by defining for the first time a key involvement of RvD1 in apoptosis and neuroinflammation occurring in the physiologically aged retina. Two novelties are evidenced: (i) the mediator RvD1 decreased in the 24-months-old retina, and (ii) it had different levels in the retina of the male mice compared to the retina of the female mice. Phenomena associated with greater damage to specific segments of the retina in males than in females (e.g., retinal pigment epithelium and Bruch's membrane, outer and inner layers) [8]. From the mechanistic point of view, the decrement of RvD1 was paralleled by microglia activation together with gliosis and increased apoptotic and nitrosative response into the retina.
Retinal damage is due to local immune-inflammatory processes affecting the RPE, photoreceptors layer, ganglion, and nerve fiber layers and inner nuclear layer (INL) [17], along with glial cells activation [18,19]. There are two glial retinal types with specific morphology, physiology, and antigenicity: macroglia (Müller cells and astrocytes) and microglia [20]. Activated Müller cells can induce gliosis and contribute to neuron death by secretion of proinflammatory factors, i.e., TNFα, monocytic chemotactic type 1 protein (MCP1), interleukins, interferon, and nitric oxide (NO), leading to free radicals' release and protein nitrosylation, with neuronal toxicity effects [21][22][23]. In line with this, retinal reactive gliosis (astrocytosis) increased since GFAP labelling was pronounced in aged retina compared to young retina, particularly in male retina compared to female retina. This was associated with microglia activation in aged mice compared to adult mice, specifically much more intensely in the retina of aged male mice than aged female mice. This latter example underlines a lesser extent of neuroinflammatory damage. The microglia activation process is a complex phenomenon, characterized by the acquisition of different functional phenotypes, schematically represented by the M1 and M2 phenotypes, associated respectively with neuro-toxic and neuroprotective functions. Accordingly, increased Iba-1 indicated that the M1 phenotype was present in aged retina.
Activated macro-and microglia cause neuroinflammation and increase retinal apoptosis [24], the final stage of cellular damage aimed at the removal of undesired cells [25]. However, dysregulation of the apoptotic mechanisms (e.g., persistent immune-inflammation, mitochondrial damage, ROS generation, and epigenetic alterations) may be disadvantageous since it may lead to increased cell loss, tissue dysfunction, and exacerbated postmitotic cell (neurons)-associated pathological conditions [25]. Retina is one tissue that is highly exposed to cellular damage because of the prolonged exposition to damaging factors such as light, microbes, and chemicals [26,27] that may cause apoptosis and cell death in the long run, as of the example of RPE cells [28]. Here, apoptosis increased, with aged male retina showing more apoptosis than female retina. Interestingly, RvD1 was much lower in the male retina than in the female retina.
Another aspect of aged retina is the presence of nitrosative stress, which exerts damaging effects [29]. In the mammalian retina, NO has been detected in amacrine cells, bipolar, and ganglion cells in the inner retina, whereas interplexiform cells, bipolar cells, and horizontal cells are sources of NO for the outer retina [30]. However, an overproduction of NO generates NOx production, such as peroxynitrite, which deranges retinal structure by reacting with several biomolecules and potentially leads to cell death [29,31]. Accordingly, here, we recorded higher levels of nitrotyrosine (index of peroxynitrites) in aged retina with respect to younger retina. In particular, the levels of nitrotyrosine were lower in the retina of aged female mice than in the retina of aged male mice.
In conclusion, the retinal RvD1 levels were decreased in aged mice when compared to younger mice, and the decrease was markedly larger in males. Several aspects of the aged retina (the astrocyte activation, neuroinflammation, apoptosis and nitrosative stress, being in line with Müller cell hypertrophy) were paralleled by changes of RvD1 levels. Considering that retinal aging is a progressive process, more in-depth studies should progressively monitor these alterations, even in different mouse strains.

Animals
Experimental procedures were conducted according to the guidelines of the Declaration of Helsinki, in compliance with European and national guidelines for research on laboratory animals and had the ethical approval from the Vasile Goldis Western University of Arad (Approval no.135/2019).
Male and female Balb/c mice aged 3 months (control groups) and 24 months (aged groups; approximately 75-85 years for humans) [8,32], respectively (n = 10/each group/sex), were used. Young females were under physiological and regular estrus cycle, while aged females were under naturally occurring physiological decline of estrus without any manipulation [8,33]. All were housed in IVC cages, in standard temperature and humidity conditions, with ad libitum access to food and water. Lighting was regulated on a 12-h light/dark cycle. Particularly, to minimize the negative effects of standard vivarium lighting on the aged retina, an illuminance level of 39 ± 7 lux was used [8,33]. This was even lower than the room light recommended for animals susceptible to phototoxic retinopathy (between 130 and 325 lux) by the National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals [34].
Once the experimental setting has been prepared, each mouse under anaesthesia had systemic perfusion via the left ventricle with 100 mL of 0.1 M ice-cold phosphate-buffered saline (PBS) + heparin (5000 IU/mL, final concentration of 0.1% v/v) [35]. At the end of perfusion, one eye for biochemical assays was excised. In a next step, animals' perfusion was continued with 100 mL more of freshly prepared 4% paraformaldehyde (PFA) in PBS for the collection of the remaining eyes and investigations detailed below.
Novocastra Peroxidase/DAB kit (Leica Biosystems, Nussloch, Germany) was used to detect immunoreactions, according to the manufacturer's instructions. The substitution of primary antibodies with irrelevant immunoglobulins of matched isotype was used to stain negative control sections and all were analysed under bright-field microscopy.

Immunofluorescence
GFAP levels were assessed by using a rabbit polyclonal anti-GFAP antibody (ab7260; Abcam PLC., Cambridge, UK) and AlexaFluor 594 labeled goat anti-rabbit IgG secondary antibody (A 11037; Thermo Fisher Scientific Inc., Rockford, IL, USA). Goat polyclonal Iba-1 (ab-5076; Abcam PLC., Cambridge, UK) and donkey anti-goat AlexaFluor 594 (a-11058; Invitrogen, Waltham, MA, USA) were used as primary antibody and secondary antibody, respectively. Bond Dewax Solution (Leica Biosystems Inc., Buffalo Grove, IL, United States) was used to deparaffinate the eye sections. They were rehydrated in alcoholic solutions. Epitope Retrieval Solution (Leica Biosystems Inc., Buffalo Grove, IL, United States) was used for antigen retrieval at 95 • C for 10 min, followed by blocking with 2% BSA in PBS. The primary antibody was applied in a dilution of 1:1000 in primary antibody diluting buffer (Bio-Optica, Milano, Italy) for 2 h at 4 • C. The slides were washed in PBS and incubated with the secondary antibody, diluted to 1:500 in PBS, for 2 h at room temperature in the dark. After a further 3 washing steps with PBS, nucleus counterstaining was performed with 1 µg/mL DAPI (Sigma-Aldrich, St Louis, MO, USA). CC/Mount aqueous mounting medium (Sigma-Aldrich, St Louis, MO, USA) was used to mount the stained slides. They were examined with a Leica SP5 confocal laser scanning microscope.

Transmission Electron Microscopy
The eye samples were prefixed in 2.