The Neuroprotective Effects of Spray-Dried Porcine Plasma Supplementation Involve the Microbiota−Gut−Brain Axis

Dietary supplementation with spray-dried porcine plasma (SDP) reduces the Alzheimer’s disease (AD) hallmarks in SAMP8 mice. Since gut microbiota can play a critical role in the AD progression, we have studied if the neuroprotective effects of SDP involve the microbiota−gut−brain axis. Experiments were performed on two-month-old SAMP8 mice fed a standard diet and on six-month-old SAMP8 mice fed a control diet or an 8% SDP supplemented diet for four months. Senescence impaired short- and long-term memory, reduced cortical brain-derived neurotrophic factor (BDNF) abundance, increased interleukin (Il)-1β, Il-6, and Toll-like receptor 2 (Tlr2) expression, and reduced transforming growth factor β (Tgf-β) expression and IL-10 concentration (all p < 0.05) and these effects were mitigated by SDP (all p < 0.05). Aging also increased pro-inflammatory cytokines in serum and colon (all p < 0.05). SDP attenuated both colonic and systemic inflammation in aged mice (all p < 0.05). SDP induced the proliferation of health-promoting bacteria, such as Lactobacillus and Pediococcus, while reducing the abundance of inflammation-associated bacteria, such as Johnsonella and Erysipelothrix (both q < 0.1). In conclusion, SDP has mucosal and systemic anti-inflammatory effects as well as neuroprotective properties in senescent mice; these effects are well correlated with SDP promotion of the abundance of probiotic species, which indicates that the gut–brain axis could be involved in the peripheral effects of SDP supplementation.


Introduction
Alzheimer's disease (AD) is the most common type of dementia in elderly people. It is characterized by memory loss, the extracellular deposition of β-amyloid (Aβ) peptides and the aggregation of hyperphosphorylated tau (p-tau) protein, which is the main component of neurofibrillary tangles [1]. Neuroinflammation plays a critical role in AD because it can promote the accumulation of Aβ and p-tau [2]. There is currently no cure for this disease and the etiology underlying its progression remains unclear [3]. Although aging is the major risk factor for AD [4], many factors can influence its development, such as family history, susceptibility genes, and diet [5].
The gut microbiota, which comprises bacteria, archaea, fungi, and viruses that reside in the gastrointestinal tract, plays a key role in health and disease. In a healthy state, the microbiota is involved in the maintenance of the intestinal barrier, inhibition of pathogen adhesion to the epithelial surface, production of short-chain fatty acids (SCFAs), and regulation and maturation of the mucosal immune system [6]. During aging, the microbiota composition is altered, resulting in an increase in the abundance of pathogenic bacteria

Sample Collection
The day before animals were euthanized, their feces were collected under clean conditions and immediately frozen in liquid nitrogen. At the end of the experimental period, the mice were anesthetized by an intraperitoneal injection of ketamine:xylazine (100:10 mg/kg). Blood was obtained directly from cardiac puncture and tissue samples were taken from the brain cortex and colonic mucosa. All samples were quickly frozen at −80 • C until their use.

Open Field Test
The open field test (OFT) evaluates the locomotor activity and anxiety-like behavior of mice, based on their aversion to luminous and open spaces. It was performed as described by Puigoriol-Illamola et al. [27]. Briefly, mice were placed in the center of a white wooden box (50 × 50 × 25 cm) divided into central and peripheral zones (15 cm between the central zone and the wall). Mice explored the box for 5 min and each trial was recorded using a camera placed on the top of the box. The locomotor activity of the mice calculated as the sum of total distance travelled, the number of rearings, and the border stay duration were analyzed with SMART®v3.0 software (Harvard Bioscience, Holliston, MA, USA). Between each trial, the box was carefully cleaned with 70% ethanol.

Novel Object Recognition Test
The novel object recognition test (NORT) allows the evaluation of short-and long-term memory. It was carried out as described by Garcia-Just et al. [19]. Briefly, mice were placed in a 90-degree two-arm (25-cm long, 20-cm high, 5-cm wide) black maze. Mice were habituated to the maze for 10 min on three consecutive days. On the fourth day, the animals underwent a 10 min acquisition trial, in which they were placed in the maze in the presence of two identical objects situated at the end of each arm. During this trial, side preference and explorative index were measured. The first retention trial was performed two hours later to evaluate short-term memory. During this trial, one of the objects was replaced by a new one and the behavior of the mice was recorded for 10 min. The second retention trial, which measures long-term memory, was conducted 24 h after the acquisition trial. Another new object replaced the other new object in the previous trial. The mice were recorded for 10 min. The time that mice spent exploring the new object (NO) and the time that mice spent exploring the old object (OO) were measured from video recordings of each trial. To evaluate the cognitive performance, the discrimination index (DI) was calculated, which is defined as (NO − OO)/(NO + OO). A lower DI indicates less memory capacity.

Real-Time PCR
RNA was extracted from the brain cortex and colonic mucosa as described previously [20]. Total RNA was reverse-transcribed using the High-capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). Real-time PCR was performed on a MiniOpticon Real-Time PCR System (Bio-Rad). The primers used are shown in Supplementary Table S1. TaqMan gene expression assays (Applied Biosystems, Foster City, CA, USA) were used for Occludin (Mm0128968_m1) according to the manufacturer's instructions. Product fidelity was confirmed by melting-curve analysis. Each PCR run included duplicates of reverse transcription for each sample and negative controls (reverse transcription-free samples, RNAfree sample). Target gene transcripts were quantified using Hprt1 gene expression as reference and with the 2 −∆∆CT method [29].

Quantification of Cytokines
Cytokine concentrations in serum and cortex samples were quantified using Bio-Plex Pro™ Cytokine, Chemokine and Growth Factor Assay kit (Bio-Rad), according to the manufacturer's instructions.

Lipopolysaccharide Determination
Serum and cortical lipopolysaccharide (LPS) levels were determined using the Competition ELISA kit for LPS (Antibodies-online Inc., Atlanta, GA, USA), according to the manufacturer's instructions.

Extraction and Purification of Total Genomic DNA
DNA was extracted as described previously by Moretó et al. [23]. Briefly, fecal samples were suspended in 4 M guanidine thiocyanate, 10% N-lauroyl sarcosine and 5% N-lauroyl sarcosine (all from Sigma-Aldrich). DNA was extracted by mechanical disruption of the microbial cell wall using Zirconia/silica beads (BioSpec Products, Bartlesville, OK, USA). The disruption was performed by shaking the mixture using the FastPrep®−24 instrument (MP Biomedicals, Solon, OH, USA). Then, polyvinylpolypyrrolidone was added (Sigma-Aldrich) and tubes were vortexed and centrifuged for 3 min at 12,000× g. The pellet was washed with TENP (50 mM Tris (pH 8), 20 mM EDTA (pH 8), 1% polyvinylpolypyrrolidone, 100 mM NaCl). DNA was precipitated by isopropanol and incubated with RNase (Qiagen, Venlo, The Netherlands). To precipitate nucleic acids, 3 M sodium acetate (Sigma-Aldrich) and absolute ethanol (JT Baker, Deventer, The Netherlands) were added. DNA was quantified using a NanoDrop ND-100 Spectrophotometer (Thermo Fisher Scientific).

16S rDNA Gene Analysis
Extracted genomic DNA was processed and the variable V3 and V4 regions of the 16S rRNA gene were amplified. The primers to detect 16S rRNA were: 5 -TCGTCGGCAGCGTC AGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3 (forward) and reverse 5 -GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3 (reverse). High-throughput sequencing was performed using the Illumina MiSeq platform (Illumina, San Diego, CA, USA) at the Genomics and Bioinformatics Service, Universitat Autònoma de Barcelona (Bellaterra, Spain).

Statistical Analysis
Results are presented as means ± standard error of the mean (SEM). All results were analyzed using GraphPad Prism®software v8 (GraphPad Software Inc., La Jolla, CA, USA). First, Grubb's test was performed to detect outliers, in addition to Levene's test to check the homogeneity of variance and the Shapiro-Wilk test to verify data normality for all groups. To compare groups, one-way ANOVA followed by the Fisher posthoc test was used for normally distributed data. In contrast, the Kruskal-Wallis test was used for non-normally distributed data. To analyze the evolution of body weight and feed consumption of the aged animals, the area under the curve was calculated for each animal and these values were compared using the t-student test. Differences were considered statistically significant when p < 0.05. Microbial data were analyzed with ANOVA test, followed by the Benjamini and Hochberg method that corrected p values (or q values) for the false discovery rate (FDR) for normally distributed data. In contrast, the Kruskal-Wallis test was used for non-normally distributed data. Differences were considered statistically significant when q < 0.1 [30].

Body Weight and Food Intake
Aged animals fed the SDP feed showed a higher rate of body weight gain than control (CTL) mice (p < 0.001; Figure 1A). At the end of the experimental period, body weight gain was greater in SDP mice than in CTL mice (p = 0.008; Figure 1B). Both groups of animals had similar food intake ( Figure 1C).

Statistical Analysis
Results are presented as means ± standard error of the mean (SEM). All results were analyzed using GraphPad Prism® software v8 (GraphPad Software Inc., La Jolla, CA, USA). First, Grubb's test was performed to detect outliers, in addition to Levene's test to check the homogeneity of variance and the Shapiro-Wilk test to verify data normality for all groups. To compare groups, one-way ANOVA followed by the Fisher posthoc test was used for normally distributed data. In contrast, the Kruskal-Wallis test was used for nonnormally distributed data. To analyze the evolution of body weight and feed consumption of the aged animals, the area under the curve was calculated for each animal and these values were compared using the t-student test. Differences were considered statistically significant when p < 0.05. Microbial data were analyzed with ANOVA test, followed by the Benjamini and Hochberg method that corrected p values (or q values) for the false discovery rate (FDR) for normally distributed data. In contrast, the Kruskal-Wallis test was used for non-normally distributed data. Differences were considered statistically significant when q < 0.1 [30].

Body Weight and Food Intake
Aged animals fed the SDP feed showed a higher rate of body weight gain than control (CTL) mice (p < 0.001; Figure 1A). At the end of the experimental period, body weight gain was greater in SDP mice than in CTL mice (p = 0.008; Figure 1B). Both groups of animals had similar food intake ( Figure 1C).

Behavioral and Cognitive Tests.
AD is associated with behavioral and cognitive abnormalities, so we evaluated whether SDP could mitigate these alterations in aged SAMP8 mice. Representative tracking maps of the OFT of all groups are shown in Figure 1A. Aging reduced the locomotor activity of the mice (6M-CTL vs. 2M; p < 0.001; Figure 2B) and SDP attenuated this reduction (6M-SDP vs. 6M-CTL; p = 0.027). Furthermore, we showed reduced exploratory activity (6M-CTL and 6M-SDP, p < 0.001 and p = 0.008; Figure 2C), and increased anxietylike behavior (6M-CTL and 6M-SDP, p = 0.019 and p < 0.001; Figure 2D) in both groups of aged mice, without differences due to SDP supplementation.

Behavioral and Cognitive Tests
AD is associated with behavioral and cognitive abnormalities, so we evaluated whether SDP could mitigate these alterations in aged SAMP8 mice. Representative tracking maps of the OFT of all groups are shown in Figure 1A. Aging reduced the locomotor activity of the mice (6M-CTL vs. 2M; p < 0.001; Figure 2B) and SDP attenuated this reduction (6M-SDP vs. 6M-CTL; p = 0.027). Furthermore, we showed reduced exploratory activity (6M-CTL and 6M-SDP, p < 0.001 and p = 0.008; Figure 2C), and increased anxiety-like behavior (6M-CTL and 6M-SDP, p = 0.019 and p < 0.001; Figure 2D) in both groups of aged mice, without differences due to SDP supplementation.
To assess the working memory of mice, they were evaluated using the NORT. During the familiarization phase, all groups had a similar explorative index and object preference ( Figure 3A,B, respectively). Regarding short-term memory, aged mice had lower values of DI than younger mice (6M-CTL vs. 2M; p = 0.008; Figure 3C). SDP supplementation prevented this reduction (6M-SDP vs. 6M-CTL; p = 0.039), showing similar DI values to those of young mice. Regarding long-term memory, aged mice had a lower DI than the young group (6M-CTL vs. 2M; p < 0.001; Figure 3D). SDP supplementation prevented this To assess the working memory of mice, they were evaluated using the NORT. During the familiarization phase, all groups had a similar explorative index and object preference ( Figure 3A,B, respectively). Regarding short-term memory, aged mice had lower values of DI than younger mice (6M-CTL vs. 2M; p = 0.008; Figure 3C). SDP supplementation prevented this reduction (6M-SDP vs. 6M-CTL; p = 0.039), showing similar DI values to those of young mice. Regarding long-term memory, aged mice had a lower DI than the young group (6M-CTL vs. 2M; p < 0.001; Figure 3D). SDP supplementation prevented this reduction, as these mice showed higher DI values than aged mice (6M-SDP vs. 6M-CTL; p = 0.040).

Effects on the CNS
Molecularly, it has been well-established that SAMP8 presents changes in brain-derived neurotrophic factor (BDNF) pathway. Neither aging nor SDP supplementation changed the relative abundance of the precursor form of BDNF (pro-BDNF; Figure 4A) in the cortex. However, senescence reduced the abundance of mature form of BDNF (m-  To assess the working memory of mice, they were evaluated using the NORT. During the familiarization phase, all groups had a similar explorative index and object preference ( Figure 3A,B, respectively). Regarding short-term memory, aged mice had lower values of DI than younger mice (6M-CTL vs. 2M; p = 0.008; Figure 3C). SDP supplementation prevented this reduction (6M-SDP vs. 6M-CTL; p = 0.039), showing similar DI values to those of young mice. Regarding long-term memory, aged mice had a lower DI than the young group (6M-CTL vs. 2M; p < 0.001; Figure 3D). SDP supplementation prevented this reduction, as these mice showed higher DI values than aged mice (6M-SDP vs. 6M-CTL; p = 0.040).

Effects on the CNS
Molecularly, it has been well-established that SAMP8 presents changes in brain-derived neurotrophic factor (BDNF) pathway. Neither aging nor SDP supplementation changed the relative abundance of the precursor form of BDNF (pro-BDNF; Figure 4A) in the cortex. However, senescence reduced the abundance of mature form of BDNF (m-

Gut Microbiota
The Shannon's index, used as a reflection of the diversity of species in a community, was similar in the different groups ( Figure 8A). The number of species in feces (species richness) was not modified by age or SDP supplementation ( Figure 8B). The gut microbiota of these mice predominantly comprised Bacteroidetes (42.4%-44.3%; Figure 8D-F) and Firmicutes (30.7%-38.0%), which constituted the dominant phyla, and their ratio (F/B) was not modified by age or SDP supplementation ( Figure 7C). They were followed by Proteobacteria (14.2%-15.5%), Verrucomicrobia (0.5%-6.7%), Actinobacteria (0.4%-0.8%), Tenericutes (0.6%-1.0%), Deferribacteres (0.2%-0.5%), and others. No significant differences could be attributed to age or SDP supplementation. The effects of age and SDP supplementation at the family level are shown in Table 1. Senescence reduced the abundance of the families Lactobacillaceae and Clostridiaceae, from the Firmicutes phylum; Sphingobacteriaceae, Flavobacteriaceae, and Prevotellaceae, from Bacteroidetes phylum; and Helicobacteraceae, from the Proteobacteria phylum (6M-CTL vs.

Discussion
There is consistent evidence supporting the ability of diet and dietary supplements to influence the progression of Alzheimer's disease (AD) through the microbiota−gut−brain axis [14]. Mechanisms of communication between the gut microbiota and brain primarily involve the regulation of the immune system and the activation of neural and endocrine pathways by the gut microbiota or its by-products [31]. Dietary supplementation with SDP improves brain resilience against the neuropathological hallmarks of AD [15] and prevents the cognitive decline in SAMP8 mice [19]. Previous studies have shown that SDP supplementation has anti-inflammatory properties because it can modulate the mucosal immune response. Indeed, it can mitigate the harmful effects of Staphylococcus aureus enterotoxin B challenge in rodents [22] or E. coli infection in pigs [32]. Furthermore, SDP supplementation has prebiotic effects, raising the abundance of healthpromoting bacteria in weaned mice [23], and weaned pigs [33]. Accordingly, in this study, we hypothesized that SDP promotes changes in the microbiota profile that could be involved in the neuroprotective effects of SDP.
As aforementioned, memory loss is one of the earliest reported symptoms in AD [1]. In our study, we observed age-associated short-and long-term memory deterioration, which is widely seen in SAMP8 mice [34]. We also found that SDP supplementation was protective against age-related cognitive decline [19]. The beneficial effect of SDP on cognition is related to a lower loss of synaptophysin in the brain [19], consistent with the fact that cognitive impairment can be due to synaptic dysfunction rather than neuronal loss [35]. Cognitive decline is also associated with reduced locomotor activity [36]. This agerelated motor dysfunction is typically observed in SAMP8 mice [37], as we corroborated in aged SAMP8. For the first time, we have shown that SDP attenuated motor dysfunction in aged SAMP8 mice, similar to the increase in locomotor activity found in LPS-challenged C57BL/6 mice [38].
BDNF plays an essential role in synaptic plasticity, brain neuroprotection, and memory [39]. Of note, BDNF levels decreased in AD patients because Aβ peptides inhibit its maturation [40], leading to neurodegeneration and synaptic loss [41]. In our study, aged mice showed a lower abundance of mature form of BDNF, which correlates well

Discussion
There is consistent evidence supporting the ability of diet and dietary supplements to influence the progression of Alzheimer's disease (AD) through the microbiota−gut−brain axis [14]. Mechanisms of communication between the gut microbiota and brain primarily involve the regulation of the immune system and the activation of neural and endocrine pathways by the gut microbiota or its by-products [31]. Dietary supplementation with SDP improves brain resilience against the neuropathological hallmarks of AD [15] and prevents the cognitive decline in SAMP8 mice [19]. Previous studies have shown that SDP supplementation has anti-inflammatory properties because it can modulate the mucosal immune response. Indeed, it can mitigate the harmful effects of Staphylococcus aureus enterotoxin B challenge in rodents [22] or E. coli infection in pigs [32]. Furthermore, SDP supplementation has prebiotic effects, raising the abundance of health-promoting bacteria in weaned mice [23], and weaned pigs [33]. Accordingly, in this study, we hypothesized that SDP promotes changes in the microbiota profile that could be involved in the neuroprotective effects of SDP.
As aforementioned, memory loss is one of the earliest reported symptoms in AD [1]. In our study, we observed age-associated short-and long-term memory deterioration, which is widely seen in SAMP8 mice [34]. We also found that SDP supplementation was protective against age-related cognitive decline [19]. The beneficial effect of SDP on cognition is related to a lower loss of synaptophysin in the brain [19], consistent with the fact that cognitive impairment can be due to synaptic dysfunction rather than neuronal loss [35]. Cognitive decline is also associated with reduced locomotor activity [36]. This age-related motor dysfunction is typically observed in SAMP8 mice [37], as we corroborated in aged SAMP8. For the first time, we have shown that SDP attenuated motor dysfunction in aged SAMP8 mice, similar to the increase in locomotor activity found in LPS-challenged C57BL/6 mice [38].
BDNF plays an essential role in synaptic plasticity, brain neuroprotection, and memory [39]. Of note, BDNF levels decreased in AD patients because Aβ peptides inhibit its maturation [40], leading to neurodegeneration and synaptic loss [41]. In our study, aged mice showed a lower abundance of mature form of BDNF, which correlates well with memory loss. SDP supplementation increased m-BDNF levels that could be responsible for the beneficial effect observed on memory retention. In addition, SDP supplementation reduced sAPPβ accumulation and Aβ 40 concentration in the brain. In fact, SDP supplementation also reduced the expression of Bace-1, an essential enzyme in the amyloidogenic pathway [20], correlating the reduced activation of the amyloidogenic pathway with an increased abundance of the mature form of this neurotrophin. These results also agree with studies showing that dietary supplements and prebiotics can increase BDNF abundance in the brain and improve memory retention in adult rats [42].
Neuroinflammation and oxidative stress can worsen neurodegeneration and is considered a pathological hallmark of AD [2]. We have shown that SDP supplementation prevented neuroinflammation by diminishing the gene expression of the pro-inflammatory cytokines Tnf-α and Il-6 and increasing the levels of the anti-inflammatory cytokines Tgf-β and IL-10 in the cortex of aged mice. Moreover, in previous work, SDP supplementation also decreased the concentration of hydrogen peroxide in the cortex, reducing oxidative stress [19]. In addition, in this study, SDP supplementation also reduced gene expression of Tlr2 as well as NF-κB activation. The expression of Tlr2 is upregulated in brains of AD patients and in a mouse model of neurodegeneration [43]. Activation of TLR is not only essential for generating neuroinflammation, but this pathway also plays an essential role in promoting the amyloidogenic processing of Aβ [44]. Specifically, TLR2 and TLR4 are binding sites for Aβ, and the signaling of both receptors can activate microglia and induce the release of proinflammatory mediators [45]. The pathway activated through TLRs can be either Myd88-dependent or -independent pathways, both of which induce inflammatory cascades via activation of the transcription factor NF-κB [46]. Here, we observed that TLRs were activated in a Myd88-independent pathway because the Trif adaptor was increased in aged mice, while Myd88 was not modified. Activation of NF-κB is associated with inflammatory responses and neurodegenerative diseases, such as in AD patients, where it induces the amyloidogenic pathway and tau phosphorylation [47]. The reduction in this signaling pathway observed in SDP-supplemented mice could be associated with the ability of SDP to decrease the amyloidogenic pathway and tau phosphorylation, as well as microglial activation [20].
Increasing evidence confirms the relationship between the microbiota and AD. The microbiota can modulate microglia maturation, regulate blood−brain barrier permeability and synaptic plasticity, or induce the formation of Aβ and neurofibrillary tangles [48]. Moreover, during aging, there is an alteration of the microbiota composition that contributes to the increase in the low-intensity inflammatory state present during senescence known as inflammaging. Dysbiosis also promotes LPS release and reduces the amount of beneficial bacterial metabolites such as SCFA [49]. In this study, the senescent mice exhibited a reduced abundance of beneficial species and an increase in pro-inflammatory bacteria. For instance, aging increased the abundance of the Erysipelotrichaceae family, and of the Johnsonella and Erysipelothrix genera, which are involved in inflammatory processes [50]. These pathogenic bacteria are more abundant in an AD mouse model [51] and may contribute to the cognitive decline in AD [52].
The SDP supplement mainly comprises peptides and proteins, which differentiates it from other conventional prebiotics [23]. Some of its compounds can resist digestion in the small intestine and reach the colonic lumen [53], where they can modify the colonic microbiota. SDP supplementation enhanced the abundance of probiotic genera with wellknown anti-inflammatory properties at mucosal interfaces and reduced the abundance of pathogenic bacteria, such as the Johnsonella and Erysipelothrix genera. More specifically, at the species level, SDP prevented the effects of aging on the abundance of Erysipelhotrix muris and Prevotella dentasini. Furthermore, an SDP diet also enhanced the abundance of Eubacteriaceae family, which has health-promoting effects for the host, and is negatively correlated with dementia [54].
SDP supplementation promotes the abundance of the Lactobacillaceae family, as well as different health-promoting genera within it, such as Lactobacillus and Pediococcus. These effects of SDP on Lactobacillus have been also observed in other mice strains [23] and in pigs [33]. Furthermore, oral administration of Lactobacillus bacteria prevents cognitive decline in both humans and rats [55] and improves memory retention in aged SAMP8 mice [56], in addition to increasing BDNF levels in the rodent brain [57]. These observations are consistent with our findings regarding augmented BDNF abundance and prevention of cognitive decline. Furthermore, Lactobacillus exerts anti-inflammatory effects by increasing plasma IL-10 levels [57], which is also increased in SDP-supplemented senescent mice.
SDP supplementation also augmented the abundance of other bacterial genera of the Firmicutes phylum, such as Acetobacterium, which is an acetyl-CoA producer [58]. Increased acetyl-CoA levels improve cognitive function during aging [59] and acetyl-CoA is the main precursor of butyrate [60]. SCFAs, especially butyric acid, can inhibit the expression of the cytokines Il-6, Il-1β, and Tnf-α, thereby exerting anti-inflammatory effects [61]. SCFAs can mediate cellular functions by activating cell surface G protein-coupled receptors (GPCR), such as Ffar2 and Ffar3, which are expressed on epithelial and immune cells [31]. Here, SDP supplementation increased the gene expression of Ffar2 and Ffar3, which would suggest that some of the anti-inflammatory and neuroprotective effects of SDP may be mediated through SCFA. These byproducts produced by the microbiota may circulate to the brain, modulating the degree of immune system activation and regulating microglia maturation [31], and could therefore be a link between the gut microbiota and the reduction in neuroinflammation.
Aging-related dysbiosis promotes the production of LPS and the increase of the intestinal barrier permeability, which allows the translocation of bacteria and their by-products into the bloodstream [62]. Recent studies have revealed that increased intestinal permeability induces systemic inflammation, impairing the blood−brain barrier function, triggering neuroinflammation and cognitive dysfunction [8,63]. In the present study, it was observed that aged mice showed a lower expression of genes related to the production and maintenance of the mucus layer (Tff3 and Muc2) and to the epithelial barrier tightness (Occludin) and, as consequence of the impaired intestinal barrier, the serum LPS concentration was elevated in aged mice. Indeed, AD patients show higher plasma LPS concentration, which may promote the development of the amyloid pathology [64]. In contrast, dietary supplementation with SDP prevented the age-associated changes in the gene expression of Occludin, Muc2 and Tff3. These effects of SDP may be related to the effects observed on the microbiota, since Lactobacillus bacteria enhance the intestinal barrier, and reduce colon permeability [65], promoting mucus secretion, providing antimicrobial protection and preventing the translocation of pathogenic bacteria [66]. Indeed, this ability of SDP to improve intestinal barrier tightness has been observed in different experimental models of intestinal inflammation, both acute [67] and chronic [21] inflammation.
These effects of aging on intestinal barrier function are accompanied by colonic mucosal inflammation, and a recruitment of activated T cells (Cd25) and macrophages (F4/80). Bacteria can activate immune cells through TLR receptors. This is consistent with increased expression of Tlr2 and Tlr4 in the colonic mucosa. Activation of the immune response results in systemic inflammation which is also common along aging [68]. In this work, the systemic concentration of pro-inflammatory cytokines (e.g., IL-1β and TNF-α), increased with aging. Patients with AD show elevated concentrations of circulating IL-1β and TNFα [69], which are considered as peripheral biomarkers for AD [70]. Indeed, chronic systemic inflammation induces AD-like neuropathology in mice [63]. Various factors of the inflammatory cascade can circulate to the brain, cross the altered blood−brain barrier [10], and activate microglia, thereby altering cognitive function [48]. Dietary supplementation with SDP showed immunomodulatory effects by reducing the intensity of the immune response in the colon. The SDP diet decreased the gene expression of the innate receptors Tlr2 and Tlr4 and the recruitment of activated T cells and macrophages, as well as the expression of the pro-inflammatory cytokine Tnf-α and Il-6. This effect of SDP is relevant because immune activation and the release of pro-inflammatory cytokines can disassemble tight-junction proteins increasing epithelial permeability [71] and promoting and perpetuating intestinal inflammation [72]. SDP supplementation not only attenuated the immune response in the colon, but also systemically, by reducing the concentration of pro-inflammatory cytokines (TNF-α and IL-1β) in the serum as well as by increasing the concentration of the anti-inflammatory cytokine IL-10. The SDP induced reduction in systemic inflammation is consistent with previous results in aged mice [73].

Conclusions
In conclusion, dietary supplementation with SDP increases the abundance of probiotic species in the gut and reduces the local and systemic inflammatory responses. This attenuation of the immune response results in reduced neuroinflammation, improving cognitive performance in senescent animals ( Figure 14). Taken together, this study shows that SDP supplementation has prebiotic effects and suggests that the microbiota−gut−brain axis could play a role in the neuroprotective effects of SDP.
moting and perpetuating intestinal inflammation [72]. SDP supplementation not only attenuated the immune response in the colon, but also systemically, by reducing the concentration of pro-inflammatory cytokines (TNF-α and IL-1β) in the serum as well as by increasing the concentration of the anti-inflammatory cytokine IL-10. The SDP induced reduction in systemic inflammation is consistent with previous results in aged mice [73].

Conclusions
In conclusion, dietary supplementation with SDP increases the abundance of probiotic species in the gut and reduces the local and systemic inflammatory responses. This attenuation of the immune response results in reduced neuroinflammation, improving cognitive performance in senescent animals ( Figure 14). Taken together, this study shows that SDP supplementation has prebiotic effects and suggests that the microbiota−gut−brain axis could play a role in the neuroprotective effects of SDP. Figure 14. Schematic integrative representation of the effects of SDP supplementation on the gut−brain axis in an AD mouse model. Dietary supplementation with SDP promotes the growth of probiotic genera and reduces the levels of pathogenic genera. This is accompanied by a reduction in the intestinal permeability and local and systemic immune and inflammatory responses. These effects culminate in a reduction in neuroinflammation and an amelioration of the cognitive decline. AD, Alzheimer's disease; GPCR, G protein-coupled receptors; SDP, spray-dried porcine plasma; TLR, toll-like receptor. Figure 14. Schematic integrative representation of the effects of SDP supplementation on the gut−brain axis in an AD mouse model. Dietary supplementation with SDP promotes the growth of probiotic genera and reduces the levels of pathogenic genera. This is accompanied by a reduction in the intestinal permeability and local and systemic immune and inflammatory responses. These effects culminate in a reduction in neuroinflammation and an amelioration of the cognitive decline. AD, Alzheimer's disease; GPCR, G protein-coupled receptors; SDP, spray-dried porcine plasma; TLR, toll-like receptor.