Apart from its role as a digestive and absorptive organ, the gastrointestinal (GI) tract is a vital immune organ that encompasses roughly 70 % of the total immune cells of the body. As such, the physical, chemical and nutrient composition of the diet influences overall GI function, effectively as an immune organ. With the improvement in feed technology, agro-industrial co-products that are high in fibre have been widely used as a feed ingredient in the diets of pigs and poultry. Arabinoxylan (AX) and mannan are the most abundant hemicellulosic polysaccharides present in cereal grain and co-product ingredients used in the livestock industry. When monogastric animals consume diets containing high amounts of AX and mannans, stimulation of GI immune cells may occur. This involves the activation of several cellular and molecular pathways of the immune system and requires a considerable amount of energy and nutrients to be expended by the animal, which may ultimately influence overall health and growth performance of animals. Therefore, a better understanding of the role of AX and mannan in immune modulation will be helpful in modulating untoward GI immune responses, thereby minimising nutrient and energy expenditure toward this effort. This review will summarise pertinent research on the role of oligosaccharides and polysaccharides containing AX and mannans in immune modulation in order to preserve gut integrity. Nevertheless, monogastric nutritionists are recognising that dietary fibre containing various amounts of a diversity of polysaccharides can elicit immunomodulatory effects within the gastrointestinal (GI) tract. Components of the immune system that have the ability to directly detect polysaccharides include complement proteins, monocytes, macrophages, dendritic cells (DC), neutrophils, lymphocytes and others. Binding of polysaccharides to the immune cell surface receptors triggers several cellular and molecular pathways leading to immune activation, both locally and, in some cases, systemically. Polysaccharide-induced immune stimulation subsequently leads to increased infiltration and proliferation of neutrophils and leucocytes, release of proinflammatory mediators , elicits formation of reactive oxygen and nitrogen species, and induces a robust synthesis of acute-phase proteins. The entire process of this immune stimulation utilises considerable amounts of nutrients  and energy, which otherwise would have been used for animal growth and productivity.Market availability and fluctuation in the cost of conventional feed ingredients are pushing producers to increase inclusion of low-cost, fibre-rich co-products in the diets of monogastric animals. One major issue of this approach is that digestibility of high-fibre ingredients varies widely, with α-linked starch being digestible through hydrolytic–enzymatic action, but β-linked dietary fibres only being digested by exogenously supplied enzymes or through microbial fermentation. Dietary fibre has also been found to contribute to energy utilisation, though this fraction has been long ignored as an insignificant source of usable energy in the diets of monogastric animals. Energy derived from absorption of hexose sugars, like glucose, galactose and mannose, within the small intestine is higher when compared with pentose sugars like arabinose and xylose. Polysaccharides from AX and mannan are considered anti-nutritional due to their negative impact on nutrient utilisation. However, the oligomers from AX and mannan have more functional value in improving the gut health of monogastric animals,5. Supplementation of oligosaccharides, or reduction of polysaccharides to oligosaccharides, is one strategy that is helpful in reducing immune stimulation and alleviate untoward effects on gut health caused by polymeric forms within the dietary fibre source. Moreover, the nutrient-encapsulating effects of polymers are much lower when they are reduced to shorter-length oligomers. Thus, to address these concerns and have a better understanding of the immunomodulatory potential of oligosaccharides and polysaccharides containing AX and mannan, the present review will discuss nutritional concepts and strategies involving AX and mannan when fed to monogastric animals, with specific emphasis on their role in modulating gut health. Given the complex structure of AX and mannan, the present review will discuss the integral relationship between structural heterogeneity of AX and mannan and potential for immunomodulatory effects.Arabinoxylan (AX) and mannan are, respectively, the first and second most abundant hemicellulosic polysaccharides present in most cereals and agro-industrial co-products that are used in diet formulations for pigs and poultry–8.The term ‘gut health’ has a very complex and comprehensive definition which includes five distinct domains: (1) a balanced diet that furnishes all required nutrients; (2) a healthy mucosal layer that maintains gut integrity; (3) effective immune responses; (4) neuroendocrine and motor functions of the gut; and (5) a stable microbial community that maintains a balanced, healthy gut environment. Structural and immunological barriers of the GI tract protect the gut from antigens and toxins derived from food. The physical barrier includes epithelial cells, mucus and intercellular tight junctions, whereas the immunological and chemical barrier includes gastric acid, digestive secretions  and various cytokines and chemokines that regulate chemotaxis of immune cells. Tight-junction proteins regulate intestinal permeability as they act to create a seal between two adjoining enterocytes. Disruption of barrier function, therefore, leads to a condition known as leaky gut syndrome or hyperpermeability. Mucosal immunoregulation in the gut is predominantly maintained by single-layered epithelial cells because it acts as the barrier between luminal contents (containing antigens and toxins) and the lymphocyte-rich lamina propria. Epithelial cells contain pattern recognition receptors  that recognise antigens and bacterial lipopolysaccharides, which may ultimately cause production of various cytokines and chemokines by local intestinal immune cells in a defensive reaction. When gut health is impaired by the presence of polysaccharides or other immunostimulatory luminal components, it elicits negative effects on feed efficiency and growth performance, ultimately causing economic losses. As such, it is imperative to elucidate how dietary components relate to stimulation of immune response within the gut in order to develop nutritional strategies to mitigate such health challenges.The GI tract of pigs and poultry represents the largest mass of immune tissue in the body, consisting of more than 70 % of total immune cells  it is 0⋅77, in wheat it is 0⋅53, in wheat DDGS it is 0⋅66 and in macadamia nut cake it is 0⋅88. Due to their structural differences, a larger percentage of AX in some ingredients like oats is insoluble, whereas it is soluble in others like rye. The molecular weight and A:X ratio of insoluble AX are generally higher than that of soluble AX.Arabinose and xylose occur in the form of AX in feed ingredients. The β(1–4)-linked xylan forms the linear backbone of AX and arabinose gets substituted on the C(O)-2 and C(O)-3 positions, with substitution of hydrocyannamic acid derivatives on the C(O)-5 position . Arabino. α-l-Arabinofuranosidase degrades the terminal non-reducing α-arabinofuranose from the AX substrate. The complete degradation of AX requires a combination of enzymes (Xylanases are enzymes that cleave the glycosidic linkage between xylose residues in the xylan backbone. Xylanases are categorised into six different glycosidic hydrolase (GH) families: GH5, GH7, GH8, GH10, GH11 and GH43. Each of the GH families have different substrate specificities. For example, GH10 xylanases cleave xylan molecules with higher degrees of substitution whereas GH11 xylanases cleave unsubstituted xylan. However, GH11 xylanases only cleave those xylan molecules that possess three consecutive xylose without any substitution enzymes .Fig. 2.,21. The purpose of enzyme supplementation in the feed is not to degrade AX to individual xylose and arabinose as they provide only little energy for the animal. Moreover, most of these monomers will be excreted in the urine.The overall objective of enzyme supplementation via feed is to minimise poor gut health induced by AX and to liberate other nutrients that are entrapped in the fibre, protein and starch matrix. In addition, the liberated shorter fragments of xylo-oligosaccharides (XOS) or arabinoxylo-oligosaccharides may act as prebiotic substrates for microbial fermentation in the large intestine. The backbone of all mannan polysaccharides contains β(1–4)-linked mannose or a combination of mannose and glucose residues, which can be substituted with α(1–6)-linked galactose. Linear mannan is a homo-polysaccharide containing less than 5 % of galactose substitution. Galactomannans consists of β(1–4)-linked mannose units with a single α(1–6)-linked galactose attached to the chain; this form is commonly found within the endosperm of legumes. Glucomannan consists of randomly arranged mannan and glucose residues both in the backbone as well as side chains in the ratio of 3:1 with a degree of polymerisation higher than 200. GGM consists of α(1–6)-linked galactose residues attached to glucose or mannose residues as a terminal branch. Partial substitution of O-acetyl groups occurs in mannosyl residues of GGM. In general, GGM consists of galactose on its side chain, which forms strong hydrogen bonds and prevents other macromolecules from aligning to each other, increasing the solubility of GGM.Glucomannan, galactomannan, galactoglucomannan (GGM) and linear mannans are the four subfamilies of the mannan family  from the mannan backbone and acetyl groups in the GGM are removed by acetyl mannan esterases.There are mainly three mannan-degrading enzymes: β-mannanases, β-mannosidases and β-glucosidases . Additio compared with mannotetraose and mannopentaose substrates, signifying greater efficacy of β-mannanase if the mannan backbone consists of more than three contiguous mannose residues.Digestion and solubility of mannans are affected by the α(1–6) side chain on the β(1–4) mannan backbone. Most of the feedstuffs used in monogastric nutrition are rich in linear mannans or galactomannans, hence β-mannanase and α-galactosidase are the most commonly used mannan-cleaving enzymes. Activity of β-mannanase on a mannotriose substrate was found to occur at a much lower rate. Immune activation causes the shift of energy utilisation from growth to support immune defence pathways, ultimately inhibiting the productive performance of the animal. However, the exact mechanisms by which dietary fibrous polysaccharides influence the immune response and inflammation are not fully understood. Three possible pathways highlight the immunomodulatory potential of dietary fibrous polysaccharides fed to monogastric animals: (1) direct interaction with epithelial cells or leucocytes; (2) uptake by M-cells in the Peyer's patches; and (3) uptake by macrophages and DC. Polysaccharide uptake by M-cells results in transcytosis to immune cells and subsequent cytokine production, whereas uptake by macrophages results in transportation of the polysaccharides to immune tissues including lymph nodes, spleen and bone marrow. Several studies conducted on monoculture cell lines with polysaccharides report that the cell wall polysaccharides may directly stimulate epithelial cells and monocytes, subsequently causing immunological outcomes,28,29.Many researchers have investigated the immunomodulatory effect of dietary polysaccharides, though most of them have focused on bacterial lipopolysaccharides which contain lipid A. However, the polysaccharides present in feed ingredients provided to monogastric animals are of plant origin that do not contain lipid A but still possess immunomodulatory properties. Currently, ten TLR have been identified in monogastric animals and each accept distinct pathogen-associated molecular patterns (PAMP) derived from various pathogens. The PAMP represent unique molecular signatures that are only present on microbes and not on host cells. Certain TLR  recognise lipoproteins , whereas other TLR  recognise nucleic acids of microbial origin (either single- or double-stranded RNA). Besides bacteria, viruses, lipids, protein and nucleic acids, TLR are also able to recognise carbohydrate moieties. For example, inulin-type fructans modulate TLR2 to enhance barrier function and prevent entry of pathogens, yet C-type lectin receptors exclusively bind to NSP  and tend to cause immunostimulation,31.Larger polysaccharides are recognised by pattern recognition receptors (PRR) found on immune cells that survey and guard peripheral tissues; these cells stimulate the production of cytokines and proliferation of lymphocytes. The PRR are sensors of the immune system and are present on and in immune and intestinal epithelial cells. The PRR involved in the recognition of NSP are TLR and C-type lectin receptors,19. The structure and molecular weight of AX derived from maize are similar to those of bacterial lipoproteins. Both AX and bacterial lipoproteins have C-3-branched polysaccharides and an outer core of hexoses such as glucose and galactose, which means that both are recognised by TLR4. Additionally, AX has been proven to elicit an immune response in chickens experiencing an Eimeria parasitic infection. The AX derived from wheat bran was found to have higher immunostimulatory activity than AX derived from maize husk, supporting the notion that AX structure is related to the ability for recognition by host immune cells. Maize has more heavily substituted arabinose units as compared with wheat,15, and reflected in a higher A:X ratio in maize (0⋅83) v. wheat (0⋅56), suggesting arabinose enrichment may be partly associated with higher immunostimulatory activity.Receptors involved in the recognition of AX have not been identified, though it has been suggested that AX may act like PAMP; or (2) via mannose-binding lectins (MBL) that prompt inflammatory response through cytokine expression. Because the location of host receptors may relate to immunostimulatory action, it is important to note that the MR is membrane bound whereas MBL are secreted by the liver,2.Two mechanisms have been proposed by which β-mannan modulates immune responses through direct interaction: (1) via the cell surface mannose receptor (MR) that can identify both host glycoproteins and microbial glycans,38. The MR, Endo 180 and phospholipase A2 contain eight carbohydrate recognition domains whereas DEC 205 contains ten. Out of the eight CLTD in MR and Endo 180, only the 4th and 5th CLTD in MR and the 2nd CLTD in Endo 180 can directly bind mannose in a Ca-dependent manner. The MR is an endocytic receptor. Binding of mannan to MR induces endocytosis followed by its delivery through the endosomal pathway (i.e. uptake of mannan and presentation to T cells), which requires coordination with other PRR like TLR4,37.The MR is a member of the MR family which consists of four members: (1) MR proper; (2) M-type phospholipase A2 receptor (A2); (3) Endo 180 (endocytic glycoprotein); and (4) DEC 205 (cell surface protein primarily expressed by DC). The general structure of all the members of the MR family is similar in that they consist of an extracellular region containing a cysteine-rich domain, a repeating fibronectin type-2 domain, multiple C-type lectin-like carbohydrate recognition domains (CLTD), a transmembrane domain and a cytoplasmic taill-fucose, d-mannose and N-acetylglucosamine) on the microbial surface. Unlike other acute-phase proteins  that increase 10- to 1000-fold in the serum, MBL fluctuates only 2- to 3-fold during an inflammatory reaction. The repeating linked array of mannans located within plant-based β-mannan polymers provides potential targets for MBL binding,41. As such, MBL activates the lectin pathway of the complement system by binding to specific carbohydrates with the help of the mannan-binding, lectin-associated serine protease. The complement system is activated by three pathways  and is activated by microbes and MBL, which involve similar pathways.MBL is a soluble C-type lectin receptor synthesised in the liver as part of the host acute-phase response and binds to neutral carbohydrates ; hence they are able to bind TLR4 and induce an immune response. Moreover, the β-mannan in Aloe vera and soyabean have similar chemical structures and are found to activate macrophages and induce NO synthesis. NO acts as cytostatic factor that inhibits proliferation of viruses and a wide range of invading pathogens. Exogenous mannanase supplementation has been found to decrease haptoglobin production (i.e. down-regulate the acute-phase protein response), thereby suggesting decreased immune stimulation may occur with lower-molecular-weight mannans.Mannans do not get hydrolysed by endogenous host enzymes in monogastric species, therefore they are available for binding to the carbohydrate recognition domains of innate immune cells, including macrophages, to elicit an immune response. As a result, these polysaccharides can form different types of three-dimensional shapes, which permits a vast array of functional groups. The presence of functional groups like acetate and/or sulfate on the cell surface is known to affect the charge and solubility of polysaccharides, with sulfate groups providing negative charges and acetyl groups forming hydrophobic pockets.All of the NSP present in the cell wall of cereal grains are composed of monosaccharides and each monosaccharide can exist in two ring forms, namely pyranose and furanose linked by glycosidic bonds in either the α or β orientationper se, acetylation, sulfation and degree of branching of fibrous polysaccharides also influence gut condition, microbial ecology and immunostimulatory potential in the host consuming those polysaccharides via the diet. A higher degree of acetylation has been found to lower the immunostimulatory effect of mannan. However, partially O-acetylated AX induces proliferation of thymocytes, indicating increased immunostimulatory activity. Sulfation of galactans has been reported to have immunostimulating activity as sulfated and carboxymethylated functional groups of β-glucan have been proven to improve solubility by impacting inter- and intra-molecular hydrogen bonding and strengthening electrostatic repulsion ability.In addition to influencing chemical properties of the substrates ,46. Cross-linking is also associated with the presence of hydroxycinnamic acid derivatives, which are most commonly found in cereals as ferulic, coumaric and sinapic acids. Ferulic acid is the predominant form in most cereals, with the concentration of ferulic acid being highest in maize, moderate in wheat and lower in oats. AX in the aleurone layer is mostly insoluble due to its high ferulic acid content and heavy esterification of AX of the aleurone layer. This means that more cross-linked complex polysaccharides are present in maize and its co-products than wheat and oats. Additionally, more than 90 % of ferulic acid occurs in the bound form whereas the free and soluble forms are found at lower concentrations. Thus, occurrence of ferulic acid in the insoluble fraction of fibre is 100-fold higher than in the soluble fraction. As such, ferulic acid is thought to form a bridge between polysaccharide chains, and thereby influence the physiochemical properties of fibre and effectively lower the efficiency of exogenous enzymes consumed by the host.Branching, or the degree of substitution on the backbone of fibrous polysaccharides, influences the way a dietary substrate interacts with the GI environment and ultimately affects the immune response. A higher A:X ratio leads to increased cross-linking, making it difficult for the enzymes to act upon the substrate, which may relate to the concentrations of diferulates present in the respective fibrous fractions. Moreover, the concentration of diferulates present in the insoluble fraction of wheat is 5–7 times higher than that present in the soluble fraction of wheat. Higher amounts of insoluble fibre are concurrent with higher levels of diferulates and increased cross-linking,13. Finally, solubilised AX has a larger fraction of reducing ends that makes AX more susceptible to Maillard reactions.The solubility of fibres in the gut is dependent on the type of ingredients used during the feed formulation process. Soluble fibre is better degraded by enzymes and more easily fermented by microbes in the large intestine of pigs and β-glucan is far more viscous than AX when in solution. However, β-glucan is also more sensitive to reduction of molecular weight during passage through the small intestine, while AX is more resistant to degradation under the same conditions, which may help to explain the greater extent to which AX negatively influences nutrient digestibility v. β-glucan.Fibre viscosity is another property that is positively associated with the molecular weight of polysaccharides present in feed ingredients. For example, the molecular weight of β-glucan is higher than that of AX. Insoluble linear mannans with lower galactose substitution  do not form any viscous solution in the GI tract of monogastric animals, whereas mannans with a high degree of galactose substitution  cause a stark increase in digesta viscosity,21. An increase in viscosity of luminal contents is associated with an increase in the rate of villus cell loss, thereby leading to villus atrophy, slowed gastric emptying and a reduction in the contact of nutrients with enterocytes and digestive enzymes. Collectively, these alterations elicit a negative impact on overall nutrient availability of the monogastric diet. Since soyabeans are used as a major source of protein in most of the feed fed to monogastric animals, the presence of mannan can be presumed in most of the feeds. However, soya mannan is mainly linear mannan ; they do not play a significant role in increasing intestinal viscosity.Presence of higher concentrations of soluble fibre is also associated with increasing the viscosity of intestinal content, as soluble fibres absorb water and increase their bulk. Inclusion of high amounts of soluble AX and mannan (>0⋅4 %) in a poultry diet would increase digesta viscosity, causing an increase in epithelial cell proliferation inhibiting nutrient digestibility. Crypt depth is also associated with cellular replacement rates as increased turnover requires more energy and amino acids, ultimately impacting growth of monogastric animals by shifting resources away from productive processes involving saleable products (i.e. meat and eggs). In monogastric animals, approximately 20 % of all the dietary energy is used by the GI tract to maintain structures supporting digestive and absorptive processes. Supplementation of moderate amounts of guar meal (5–8 %) causes a marked increase in digesta viscosity in broilers, which has been proved to increase proliferation of Escherichia coli and Clostridium within the GI tract. Laying hens have increased digestive capacity compared with broilers and evidence suggests that the untoward effects of dietary fibre-induced viscosity are lower in layers compared with broilers fed similar concentrations of NSP. Dietary supplementation of xylan- and mannan-degrading enzymes will serve to break down polymeric fibre structures to shorter oligomeric forms, and thereby reduce viscosity of intestinal contents, enhance gut health and improve performance of monogastric species.Long villi and shallow crypts help to increase absorption of nutrients by providing a larger surface area and lower proliferation and renewal rates of enterocytes constituting villus structures results in more efficient enterocyte maturation and enzyme production. The XOS and mannan oligosaccharides (MOS) are newly developed functional oligosaccharides produced from xylan and mannan, respectively, by enzymic hydrolysis that have beneficial immunological and health effects. Depolymerisation of XOS and MOS into oligosaccharide configurations by use of enzymes in situ  is neither instantaneous nor as efficient as supplementing dietary XOS and MOS directly,51. Supplementing XOS and MOS has beneficial effects comparable with in-feed antibiotics in reducing gut inflammation,21. These oligosaccharides are intermediate between simple sugars and polysaccharides and behave both as dietary fibres and prebiotics.Oligosaccharides are low-molecular-weight carbohydrates and the degree of polymerisation can vary from 2 to 10; however, molecules with a degree of polymerisation of less than 20 are sometimes considered as oligosaccharides due to their prebiotic potential. Prebiotics are non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already resident in the colon. Several studies have found XOS to selectively increase the proliferation of bifidobacteria to a larger extent than fructo-oligosaccharide (FOS) or any other oligosaccharides. This ‘bifidogenic effect’ of XOS has been shown in both in vitro and in vivo studies. The XOS and MOS with monomer units of 5 or fewer are getting more attention for their potent prebiotic and butyrogenic effect.XOS and MOS are linked together by the β-glycosidic bond of the linear polyxylose or polymannose chains, respectively. Manno-oligosaccharides are also heat resistant as they can tolerate a higher temperature of up to 120°C for up to 20 min. Hence, both XOS and MOS should theoretically be resistant to destructive processes involved in various feed milling operations, including pelleting and some forms of extrusion. As such, XOS and MOS not only resist salivary hydrolysis or hydrolysis by gastric and pancreatic secretions, they are also not absorbed within the small intestine, hence providing a substrate for microbial fermentation in the colon of pigs and caecum of poultry. The difference between oligosaccharide and polysaccharides of xylan and mannan is that XOS and MOS cannot form cross-linkages and are not able to stimulate immune receptors. Thus, XOS and MOS may help to reduce immune stimulation in monogastric animals and all the associated metabolic costs involved to maintain the immune response.XOS are thermostable during pasteurisation and can be autoclaved at a lower pH as compared with fructo-oligosaccharide (FOS), which is more vulnerable to decomposition at lower pH and higher temperature,60, production of antimicrobial factors,61, exclusion of specific enteric pathogens and changes in gut histomorphological structure. Bacteroidetes and Firmicutes are the polysaccharide-degrading phyla possessing the largest set of glycoside hydrolase-encoding genes,63. On the other hand, oligosaccharides are a class of compounds that help the proliferation of beneficial microbes like bifidobacteria and Lactobacillus to improve the health of animals. The role of bifidobacteria in the utilisation of oligosaccharides is gaining more attention because of their stringent selectivity of carbohydrate substrates based on the degree of polymerisation. While the majority of other enteric bacteria predominantly ferment monosaccharides, bifidobacteria preferentially utilise disaccharides and other oligomeric forms over monomeric and polymeric forms.Host and gut microbes interact with each other to influence various physiological functions of the host. Functional oligosaccharides from xylan and mannan have been used in many studies to manipulate the gut microbial ecology of monogastric animals. Numerous beneficial effects on host health have been reported by oligosaccharide-mediated changes in gut microbiota including activation of the immune systemBacteroides and Roseburia, whereas the oligomeric form is only degraded by bifidobacteria and a few species of Firmicutes, including Lactobacillus brevis,64. No other Lactobacillus sp. are capable of degrading oligomeric forms of xylan as a sole carbon source. As an exception, L. brevis can only degrade unsubstituted XOS. Uptake of oligomers of AX is mediated by the ATP-binding cassette transport system, and the only known gut microbes capable of producing this transport system is bifidobacteria. Most of the Bifidobacterium sp. are shown to degrade AX, but there is variation in their degradation capabilities. Bifidobacterium adolescentis and B. vulgatus can completely degrade AX, because they are capable of degrading both substituted and unsubstituted arabinose. While B. longum and B. ovatus can partially degrade AX, B. breve and B. infantis cannot degrade XOS at all. The xylan-degrading enzymes produced by different Bifidobacterium sp. differ in their specificity and efficiency, though they have highly conserved solute-binding proteins that enable the degradation of AX oligomers. This may partly explain the variation in degradation of AX polymers exhibited by different Bifidobacterium sp.Polymeric xylans and mannans are degraded by Bacteroides can only degrade those oligomers which are larger than xylo- and manno-pentaose. Higher bifidogenic effects may be achieved when feeding XOS with higher arabinose substitution when compared with Bacteroides. Bacteroides xylanisolvens growth was hindered when xylotriose with di-substituted arabinose was used, indicating that arabinose-substituted XOS may adversely affect bacterial growth.Bacteroides, Bacillus and Clostridium are the three bacterial genera which have the best ability to produce mannanase that can degrade mannans. MOS is found to increase Bacteroidetes proliferation and many studies have reported Lactobacillus as the main genus that is influenced by MOS. However, among all different strains of Lactobacillus, L. salivarius is most effective against Salmonella colonisation and L. crispatus against both Salmonella and E. coli. Supplementation of 0⋅2 % dietary MOS to broilers significantly reduced profiles of E. coli in the caecum and ileum of birds. Bacterial diversity in the GI tract of monogastric animals is high, which assists in enabling various functions, including degradation of NSP that cannot be digested by host enzymes. Thus, XOS and MOS can be used as a dietary intervention to enhance the host's natural defence through modulation of gut microbiota. Supplementation of NSP-degrading enzymes can also have an impact on the microbial ecology of the gut that can reduce the amount of undigested substrates, degrading polymers to oligomers that potentially have functional prebiotic effect.. Mucin secretion at the brush-border membrane limits the number of bacteria that reach the epithelial layer and influence the metabolic activity of bacteria. Increased mucin production helps to improve gut barrier function as pathogenic microbes cannot penetrate the dense mucous layer. XOS have been found to increase goblet cell number and density,71, which also increases mucin secretion (i.e. glycoproteins) and protein barrier factors, thereby protecting intestinal epithelial cells. Similarly, MOS has anti-pathogenic effects, because of its ability to increase the number of sulfated goblet cells and concomitantly increase mucin secretion. Goblet cells sulfated by the use of MOS have been shown to provide greater protection for the host by enhancing pathogen degradation and reducing pathogenic microbe attachment to colonic epithelial cells. Moreover, soluble mannosyl receptors secreted in mucin competitively bind to type-1 fimbriae of pathogenic microbes like Salmonella, E. coli and other Gram-negative bacteria. Thus, increased mucin production in the gut due to the presence of MOS helps in clearing pathogens by preventing microbial attachment to the intestinal wall.The intestinal lining has a versatile function that helps to regulate nutrient and water absorption, while at the same time acting as a barrier excluding microbes and infectious agents that come into contact with the gut. The harmful effects elicited by pathogenic microbes occur after they attach to enterocytes of the intestinal wall and colonise the environment. However, current evidence suggests that dietary oligosaccharides may assist in clearing pathogenic microbes from the GI tract, partly through the production of mucins by goblet cells embedded among enterocytes. IgA is different from any other immunoglobulin as it removes antigens/pathogens without prompting inflammatory immune responses. Thus, increased secretory IgA in the small intestine promotes an efficient prevention of intestinal inflammatory conditions. As such, feeding XOS via wheat bran increased the concentration of secretory IgA, which was shown to protect mucosal epithelia by preventing the attachment of pathogenic microbes, strengthening tight junctions between epithelial cells and down-regulating the production of pro-inflammatory cytokines like TNF-α, IL-6 and interferon-γ. Among MOS, mannobiose has been found to be the most effective at preventing bacterial infections by increasing IgA production in both pigs and chickens. Supplementation of MOS increased the concentration of mucosal IgA and reduced oocyte shedding in the excreta of broilers, presumably via secretory IgA inhibiting oocyte development. Additionally, supplementation of mannobiose in cultured chicken macrophage cell lines caused increased elimination of Salmonella. This increase in phagocytic activity against Salmonella might be due to increased production of H2O2 and NO. Thus, improvement in the intestinal barrier function by the use of XOS and MOS helps prevent pathogen colonisation and enhance overall immunity and intestinal health of monogastric livestock species.Both XOS and MOS play important roles in maintaining gut integrity by increasing the secretion of IgA. Generally, mucin and secretory IgA are considered a first line of defence against the establishment of pathogenic micro-organisms in the ileum through the prevention of adhesion and subsequent epithelial invasion. Almost 98 % of all IgA-producing cells in the body reside in the small intestineAX and mannan are the major NSP present in most of the cereals and agro-industrial co-products used in the diet of monogastric animals. The immunomodulatory properties of AX and mannan are associated with their sugar composition, structure, molecular weight, solubility, presence of functional groups and degree of branching. Both AX and mannan have structural similarities with lipoproteins in terms of C3 branching and in the presence of an outer core of hexose sugars, which suggests immune responses caused by dietary AX and mannan may be due to the activation of lipoprotein receptors in the host. Reduction of high-molecular-weight polymers to lower-molecular-weight oligomers increases their solubility in water and thereby elicits gut health benefits. In addition, fermentation of AX and MOS in the distal GI tract aids in maintaining gut health by preventing pathogen attachment. Inclusion of polysaccharide-degrading enzymes or direct supplementation of extracted oligosaccharides derived from AX and mannan may be an effective strategy to mitigate the negative effects of NSP ingestion, thereby improving intestinal health and overall performance of monogastric animals.


Saccharomyces cerevisiae lysosome-like vacuole is a useful model for studying membrane fusion events and organelle maturation processes utilized by all eukaryotes. The vacuolar membrane is capable of forming micrometer and nanometer scale domains that can be visualized using microscopic techniques and segregate into regions with surprisingly distinct lipid and protein compositions. These lipid raft domains are liquid-ordered (Lo) like regions that are rich in sphingolipids, phospholipids with saturated acyl chains, and ergosterol. Recent studies have shown that these lipid rafts contain an enrichment of many different proteins that function in essential activities such as nutrient transport, organelle contact, membrane trafficking, and homotypic fusion, suggesting that they are biologically relevant regions within the vacuole membrane. Here, we discuss recent developments and the current understanding of sphingolipid and ergosterol function at the vacuole, the composition and function of lipid rafts at this organelle and how the distinct lipid and protein composition of these regions facilitates the biological processes outlined above.The  S. cerevisiae plays a number of important roles in cellular homeostasis. The importance of the eukaryotic lysosomal compartment is underscored by the fact that there are over 70 human diseases whose molecular basis is intricately tied to dysfunctions of lysosomal storage and homeostasis  and LCB-1-phosphates  into the extracellular space, which could not be rescued by overexpression of Vam3p or Ykt6p and was attributed to increased ceramide activated protein phosphatase (CAPP) activity  derived vesicles with endocytosed material from the cell’s exterior, ultimately to be degraded, recycled and/or sent to different organelles (ELO2 (formerly FEN1) and ELO3 (formerly SUR4) simultaneously , Pho8p , and Cps1p (carboxypeptidase S/CPS). The Snc family of SNARE proteins participates in exocytic, endocytic, and recycling processes, and their function is regulated by complex sphingolipids, VLCFAs, and LCB-1-phosphates  like phase consisting of unsaturated acyl-chain phospholipids and a Lo like phase composed of saturated acyl-chain phospholipids and cholesterol. Therefore, it wasn’t entirely surprising that the same phenomenon could also be observed with GUVs made from isolated yeast PtdInsPs (containing an unsaturated acyl chain), ergosterol, and inositol-phosphoceramide  , implying there may yet be a correlation between a protein’s structural biology and its residential membrane domain, but this will require in-depth studies. A screen for yeast mutants that were unable to produce these lipid raft domains in vacuoles pointed to genes in phospholipid production (NEM1 and FAB1) and endosomal maturation/multivesicular body (MVB) formation (VPS4) (o ergosterol rich domain (Ergosterol and sphingolipids in yeast are key players in the lipid raft hypothesis. Liquid phase separation of lipids into stable micrometer domains was observed in giant unilamellar vesicles (GUVs) when monitoring the temperature- and lipid composition-dependent distribution of a fluorescent lipid-based dye . These c chains) . This stchniques . This stchniques . Ivy1p wn (VPS4) . There hh domain . This st0 subunit) forms trans-complexes between vacuoles that promote homotypic fusion, and interacts with a number of fusion effectors P2), which would historically correlate well with a Ld membrane domain  electron microscopy (EM) method coupled with a highly specific PtdInsP2 binding probe to show that a large fraction of this lipid in the vacuole localizes to Ivy1p-enriched/Vph1p-depleted region following hyperosmotic stress  is required for proper acidification of the organelle, which is necessary for the activation of resident hydrolases and autophagy, and Vph1p , and this is dependent on a subset of canonical autophagy related genes (Atg)  activity at the vacuole . It was  pathway . It is nignaling . It was ignaling . When yeignaling . Wang anes (Atg) . TogetheSaccharomyces Genome Database1AUR1  displays genetic interactions with ATG1, GYP1, PEP1, SEC17, VAM7, VPH1, VPS4, VPS8, VPS9, VPS13, VPS21, VPS41, and VPS55. Future studies will require clever and well-planned in vitro and in vivo studies to understand the full contribution of sphingolipids and lipid rafts in the fusion cascade. Although complex sphingolipids are likely to be trafficked to the vacuole within the lumenal leaflet because their biosynthetic enzymes are housed in the lumen of the Golgi, ceramide may face either leaflet due to it higher rate of bilayer translocation or flip-flop, and there may be yet unknown or non-canonical transfer methods to move sphingolipids between leaflets (in vitro systems will require careful isolation and purification of yeast sphingolipids due to a lack of commercial availability, but they will allow for tight control of the system to observe the direct contribution of individual sphingolipids to membrane fusion. It is likely that both Lo and Ld like membrane domains play a role in vacuole fusion, with each being enriched in different regulatory lipids and proteins that promote fusion. This is supported by the observation that the vertex ring domains are not homogeneous in nature and show a distribution of known fusion factors (Most sphingolipid biosynthetic genes display varying levels of interactions with genes annotated as having a function in membrane trafficking and endolysosomal maturation. According to the leaflets . Alternaleaflets . It woulleaflets . Reconst factors . There i factors . Yeast w factors .in vitro and in vivo techniques to overcome limitations and obtain a complete picture detailing how the lipid and protein composition of the yeast vacuole changes following stresses and upon reaching stationary phase, and the homeostatic pathways that communicate with this dynamic organelle.While there is much left to discover, studies that focused on understanding the composition and biological relevance of lipid raft domains at the yeast vacuole have provided exciting results. Future studies should strive for technical coverage by utilizing a number of established methods in conjunction. These studies will need to seamlessly mix the cell biology and physical biochemistry of the membrane by using techniques such as mass spectrometry, Laurdan anisotropy, genetic manipulation, and advanced microscopy to name a few. While many studies referenced above use some of these techniques, further advancement in the field will require more thoughtful and advanced LH and RF conceived and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Repetitive transcranial magnetic stimulation (rTMS) has gained growing interest for the treatment of major depression (MDD) and treatment-resistant depression (TRD). Most knowledge on rTMS comes from human studies as preclinical application has been problematic. However, recent optimization of rTMS in animal models has laid the foundations for improved translational studies. Preclinical studies have the potential to help identify optimal stimulation protocols and shed light on new neurobiological-based rationales for rTMS use. To assess existing evidence regarding rTMS effects on depressive-like symptoms in rodent models, we conducted a comprehensive literature search in accordance with PRISMA guidelines (PROSPERO registration number: CRD42019157549). In addition, we conducted a meta-analysis to determine rTMS efficacy, performing subgroup analyses to examine the impact of different experimental models and neuromodulation parameters. Assessment of the depressive-like phenotype was quite homogeneous whilst rTMS parameters among the 23 included studies varied considerably. Most studies used a stress-induced model. Overall, results show a largely beneficial effect of active rTMS compared to sham stimulation, as reflected in the statistically significant recovery of both helplessness  and anhedonic  profiles. Improvement of the depressive-like phenotype was obtained in all included models and independently of rTMS frequency. Nonetheless, these results have limited predictive value for TRD patients as only antidepressant-sensitive models were used. Extending rTMS studies to other MDD models, corresponding to distinct endophenotypes, and to TRD models is therefore crucial to test rTMS efficacy and to develop cost-effective protocols, with the potential of yielding faster clinical responses in MDD and TRD. Also, relapse rates are higher than for any other medical condition2. Core symptoms of major depressive episodes include depressed mood, decreased drive, loss of interest and pleasure. Numerous accessory symptoms co-occur and illness course may vary significantly , defining different levels of depression severity. As a result, MDD is a highly heterogeneous syndrome encompassing varied symptom clusters and divergent treatment responses. 30–50% of patients do not adequately respond to first-line treatments, which generally involve a combination of antidepressant medication and cognitive-behavioral therapy3. Conventional antidepressants target the main neurotransmitter systems thought to be implicated in MDD  and are associated with considerable variations in efficacy. A recent, comprehensive and large-scale meta-analysis revealed that antidepressants are generally more effective than placebo, although the overall effect size (0.3) is modest4. Treatment resistant depression (TRD) is defined as the absence of a clinical response despite at least 2 consecutive antidepressant trials (at adequate doses for at least 4–6 weeks). TRD is a major public health concern; functional impairment is greater and suicide risk is higher5. Despite the promising recent FDA approval of esketamine for TRD, there is a clear unmet need for rapidly-acting and efficacious treatments7.Non-invasive brain stimulation (NIBS) uses magnetic pulses or weak electric currents to induce changes in cortical excitability and modulate brain networks in localized areas. Repetitive Transcranial Magnetic Stimulation (rTMS) is a neuromodulation technique that uses a magnetic field to stimulate focal cortical brain regions with electrical currents. Recently, rTMS has gained growing interest for the treatment of major depression (MDD) and treatment resistant depression (TRD). MDD is a highly prevalent psychiatric disorder that severely impairs functioning and diminishes quality of life. The global prevalence of MDD increased by almost 13% during 2007–20178. Repeated low-frequency stimulation (1 Hz) and the continuous form of theta-burst stimulation (cTBS) induce a suppression of excitatory synaptic transmission, while high-frequency stimulation (regular 5–50 Hz) and the intermittent form of theta-burst stimulation (iTBS) potentiate it10. In light of its effectiveness, rTMS is recommended by CANMAT guidelines as a first-line intervention after failure of one adequate antidepressant trial11. Evidence from meta-analyses suggests that rTMS has a comparable effect to ECT and antidepressant medication12. Recently, rTMS has been approved by the FDA for TRD. In the clinical setting, converging evidence supports a relevant role of physical therapies to treat TRD, mainly in the framework of integrated approaches, with response rates between 30 and 64% observed after rTMS interventions. Besides, rTMS is supposed to be a potential therapeutic option for substance use disorders . Usually, rTMS protocols for MDD deliver 10 Hz stimulation of the left dorsolateral prefrontal cortex (DLPFC) at an intensity of 120% of the resting motor threshold (RMT) over 4–6 weeks in once-daily stimulation sessions15. Other protocols are available  and appear to reduce depressive symptoms. Though rTMS is a promising treatment option for MDD, clinical response is partial, highlighting the need for a more thorough understanding of MDD pathophysiology and of mechanisms implicated in rTMS therapeutic action.The effects of neuromodulation produce a dynamic regulation of brain circuitry rather than directly affect neurotransmission. During rTMS, magnetic pulses are delivered by coils of different shapes  at different frequencies  and intensities to determine changes in the excitability of specific brain areas. rTMS induces lasting changes in cortical excitability16. A major setback is lack of specificity of stimulation targets. While in humans technological advances allow for a very high level of accuracy (resulting in an isolated stimulation of a specific region), difficulty in maintaining small-scale focus has slowed rTMS use in animal models17. This has hampered gaining of the necessary understanding of the neurobiological basis of rTMS to develop personalized interventions and to clarify which stimulation protocols  yield faster responses, allowing only for empirically-based treatment protocols . Recently, optimization of rTMS use in animal models  and development of smaller sized coils specifically designed for preclinical application19 have laid the foundations for improved translational studies.Most knowledge on rTMS comes from clinical studies as application to animal models has been problematic21. The chronic unpredictable mild stress  is one of the most extensively investigated models22. It involves continuous exposure over several weeks to a variety of mild manipulations acting as low-grade stressors, determining induction of depressive-like symptoms, such as anhedonia , commonly measured by the sucrose preference test (SPT). This model simulates other phenotypic alterations isomorphic to human MDD symptoms, such as increased immobility in the forced swim test (FST) and changes in sleep architecture and locomotor activity22. Preclinical studies have the potential to shed light on new neurobiological-based rationales for rTMS use and to help identify optimal stimulation protocols .Numerous preclinical approaches have been developed over the years to model aspects of MDD in rodentsIn order to assess the current status of translational application of rTMS in the preclinical field as a treatment for MDD, we systematically reviewed studies using rTMS in rodent models. We included studies applying rTMS to both animal models of depression and healthy animals that assessed changes in terms of depressive-like measures. In addition, we conducted a meta-analysis on the efficacy of rTMS treatment for recovery from the depressive phenotype, analyzing the possible impact of different experimental models and neuromodulation protocols on treatment outcome. Data are discussed to elucidate the translational relevance of preclinical findings in developing effective treatments for MDD and TRD.24. The protocol  guidelines27). The complete search strategies used in each database are presented in the supplementary material . The search strategy consisted of two main components: repetitive transcranial magnetic stimulation (rTMS) and depression, and results were limited to rats and mice studies  and the 2nd  screening phases: (1) language other than English; (2) non-original researches ; (3) no full-text articles ; (4) studies in vitro, studies in humans, studies in non-human animals other than rodents; (5) other outcome measures reported  in the absence of an assessment of the depressive-like phenotype; (6) neuromodulation interventions other than rTMS ; (7) animals not exposed to the sham rTMS intervention as comparator/control. Within each phase, two independent reviewers screened each article , with discrepancies being resolved through discussion or by consulting additional investigators .The full-text articles of studies eligible for qualitative data extraction were independently assessed by multiple reviewers , with discrepancies that could not be resolved by discussion being solved by consulting additional investigators . The data extracted included the following categories: (i) bibliographic details; (ii) animal model characteristics; (iii) study design characteristics; (iv) intervention characteristics. Detailed information on the study characteristics extracted within each category is reported in the protocol  of the anhedonic profile and of the helplessness profile. If the 24-h timepoint had not been collected, data closest to the last rTMS session were extracted. Statistical details to enable the computation of standardized effect sizes, namely number of animals, mean and standard deviation (SD), were independently extracted by multiple reviewers  from the graphs using a digital screen ruler29.Effect size calculations were based on the comparison between the group receiving active rTMS intervention and the control group (sham). The intervention effect for each individual treated-control comparison was expressed as standardized difference in means . The individual SDMs were pooled to obtain an overall SDM and 95% confidence interval . Whenever a control group served more than one experimental group, we corrected the total number of control animals in the meta-analysis by dividing the number of animals in the control group by the number of intervention groups served29), a random-effect model was used to compute both the overall effect size and the separate effect sizes for the different subgroups, in order to take into account heterogeneity that cannot be explained. In the presence of one or two studies presenting characteristics that render them different from the others, a sensitivity analysis was performed excluding those studies from the meta-analysis.Heterogeneity among results was explored by conducting subgroup analyses by rTMS intervention’s frequency and by type of animal model. Nevertheless, as animal studies are usually rather heterogeneous with respect to numerous factors , version 3.0. Statistical significance was set at p < 0.05.To assess potential publication bias, a funnel plot of study effect sizes against standard errors was visually inspected for asymmetry resulting from a relative lack of small studies with small effect sizes . Asymmetry was also statistically tested with Egger’s bias test34 by adjusting the Cochrane’s RoB tool35 for aspects of bias that play a specific role in animal studies. The RoB tool for animal studies contains 10 entries related to selection bias, performance bias, detection bias, attrition bias, reporting bias and other biases. Two independent reviewers  performed the quality assessment of each article by independently assessing the criteria.To assess the internal validity/methodological quality of the included studies, we used the SYRCLE’s Risk of Bias (RoB) tool for animal studies, developed by Hooijmans and co-authors36, was excluded as the number of animals in the control group after the required correction could not be processed by the CMA software).The comprehensive search strategy on the effects of rTMS on depressive-like symptoms in rodent models resulted in 298 bibliographic records. The study selection process is summarized in Fig. 51 investigated the helplessness profile through the Forced swim test (FST), 2 articles53 the anhedonic profile through the sucrose preference/consumption test (SPT/SCT) whilst the remaining 6 articles58 investigated both. In addition, the anxiety profile was assessed in 9 articles58 (by means of 5 different tests) and other behavioral profiles  were assessed in 12 articles58.The characteristics of the 23 included studies are described in Table 52 tests were performed only during the rTMS intervention (in this case the one closest to the end was selected). Specifically, for the short-term evaluation of rTMS efficacy , the timing of the tests relative to the neurostimulation intervention was as follows: 24 h after the last rTMS session (12 articles57); immediately after the last rTMS session (5 articles53); between 24 and 72 h after the last rTMS session (3 articles58); during the week following the end of the rTMS intervention (2 articles56); during the last week of the rTMS intervention (1 article52).In 22 out of 23 articles at least one assessment of the depressive-like phenotype was performed shortly after the end of the rTMS intervention. Only in 1 article44; during the 2nd week after a 5-weeks intervention46; during the 3rd week after a 3-weeks intervention56.A number of articles also reported ongoing and long-term evaluations of rTMS efficacy performed at various additional timepoints . Interestingly, 3 articles out of 23 extended the evaluation of rTMS efficacy to the long-term period : 1 week and 2 weeks after the last session of a 10-days intervention58) or models of other disorders with comorbid depression (3 articles51) or healthy animal models (9 articles49) receiving active rTMS intervention; control subjects were either models of depression or animals modeling other disorders or healthy animals receiving the sham rTMS intervention, respectively. Regarding the models of depression, 8 articles58 employed the chronic unpredictable mild stress (CUS) model ; 1 article50 applied a modified version of a forced swimming paradigm (10 min daily for 5 days) able to induce a depression-like state durable for 4 weeks without additional swimming; 1 article45 employed a genetic model, i.e., the Flinders sensitive line (FSL) and its control ; 1 article38 applied a lesion to obtain the olfactory bulbectomy model of agitated depression59. The models of comorbid depression were a model of anxiety from selective breeding47, a model of autism through neonatal isolation51 and a model of epilepsy by means of pentylenetetrazol administration40.Treated subjects were either models of depression . The intensity could be either expressed as Tesla (from 0.004 to 4.0T), % motor threshold  and/or % device maximum power; 2 studies38 employed more intensities in distinct groups of animals and 1 study40 did not mention this parameter. Number of pulses per single session  and total number of pulses administered during the entire intervention varied greatly, from 45 to 15,000 and from 420 to 150,000 respectively. When mentioned, the inter-train interval ranged from 2 to 120 s. The total number of sessions per intervention varied from 5 to 28 . In general, the inter-session interval was 24 h  as no accelerated protocols were applied. Whilst 5 studies57 did not mention the type of coil, the remaining studies used 3 types of coil , the remaining 21 articles employed only male subjects. Only 2 articles50 employed mice (C57 strain), the remaining 21 articles used rats (Sprague-Dawley or Wistar strains); age and/or weight were rather heterogeneous .Only 2 articles used female subjects  are illustrated in Table 38 the recovery corresponds to increased immobility duration. Overall, rTMS led to a significant recovery of the phenotype in models of disease or improvement of the behavioral profile in healthy models  was 60%.Twenty studies (29 independent comparisons) measured the short-term efficacy of rTMS on the immobility/activity duration in the FST , in other models  and in healthy models  assessed the effects of rTMS in animal models of disease: 5 studies (6 comparisons) in the chronic unpredictable stress model of depression and 6 studies (9 comparisons) in other models of depression; the latter subgroup included 3 studies (3 comparisons) in models of other disorders with comorbid depression. In the remaining 9 studies (14 comparisons) rTMS effects were evaluated in healthy models. Recovery/improvement in the helplessness profile was observed in the CUS model  and in animals treated with rTMS at low frequency  rTMS was given at high frequency (>5 Hz), in 5 studies (5 comparisons) at low frequency (<5 Hz) and in 2 studies (2 comparisons) at 5 Hz. The latter were excluded as (i) they were not enough to create an additional subgroup, (ii) they could not be attributed to either the low or the high frequency subgroups47; particularly high frequencies, i.e., 50 Hz and 30 Hz:43), confirmed the beneficial effect of rTMS on helplessness profile .A sensitivity analysis, performed excluding 3 comparisons presenting characteristics that render them different from the others (anesthesia:p = 0.001).Inspection of the funnel plot of study effect sizes (SDMs) against standard errors  was 80%.Seven studies (8 independent comparisons) measured the short-term efficacy of rTMS on the sucrose preference index/ratio in the SPT or sucrose intake in the SCT Table . Overallp = 0.001).Inspection of the funnel plot of study effect sizes (SDMs) against standard errors  on both helplessness  and anhedonia 57. Finally, although the rTMS intervention produced antidepressant effects similar to those of the antidepressant venlafaxine on both helplessness and anhedonia, the combination of the 2 interventions had no additive effect compared with either rTMS or venlafaxine alone56.As for the augmenting or antagonizing effects of concomitant pharmacological interventions, 1 study reported an additive effect of rTMS and the atypical antipsychotic quetiapine on both helplessness  and anhedonia The effects of rTMS intervention on anxiety and other profiles on all available parameters (within each test) and timepoints  are illustrated in Table 51, in 1 study out of 2 in the Open-field test (OPT), in terms of increased time spent in the center58, and in the 2 studies employing the Novelty-suppressed feeding test (NSFT), as evidenced by the decreased latency to feed56. Interestingly, the rTMS intervention did not ameliorate symptoms in the model of anxiety47, in spite of the recovery of the comorbid depressive-like profile.The effects of rTMS intervention on the anxiety profile were mixed Table ; a benef51. In 2 studies the treatment attenuated the weight reduction induced by the CUS procedure53. Finally, rTMS did not produce antinociception42 or affect the appetitive drive56.As for the effects of the neurostimulation intervention on general activity, 3 studies out of 8 reported an increase in distance traveled in the OFT (Supplementary item 61) and, consequently, studies had an overall unclear risk of bias based on SYRCLE’s RoB tool (55.3%). For 2 instances (20%), assessing reporting bias was judge as “not applicable”34. When not unclear, the risk of bias was generally low (24.2%), with the only exception of one study for the item “Attrition bias” (0.5%).The risk of bias assessment of all included studies is shown in Fig. 22). As for rTMS treatment characteristics, parameters varied considerably in terms of frequency, intensity and duration.To our knowledge, this is the first systematic review and meta-analysis aiming to evaluate rTMS efficacy in preclinical models of depression. Overall, results show a largely beneficial effect of active rTMS compared to sham stimulation, as reflected in the statistically significant decrease in depressive-like symptoms. Most studies used stress-induced depression models  were rather mixed. This differential effect suggests that a “pure” depressive phenotype may be a specific and meaningful clinical target of rTMS interventions. Present data confirm the relevance of rTMS use in MDD and give an indication as to which patient subtype may benefit the most . Preliminary suggestions on possible synergistic  and antagonizing  interactions between neuromodulation and pharmacotherapy also emerge. Translational findings from future preclinical studies should investigate other augmenting  effects of pharmacological agents and help guide integrated (rTMS plus pharmacotherapy) approaches65.Notwithstanding the paucity and heterogeneity of studies, results are generally consistent. As expected, an overt depressive-like phenotype  was more likely to be associated with a larger effect size. The positive direction of the effect was consistent across studies for both helplessness and anhedonia, though with different magnitude. Robust results were obtained both in models of depression and healthy animals. The effectiveness of rTMS in healthy models compares with human studies involving non-depressed patients and healthy subjects. For example, left human DLPFC stimulation has been demonstrated to reverse hedonic tone dysfunction in addicted subjects66) and metabolic alterations , both involved in MDD pathophysiology. The fact that up to now lateralization has not been possible in animal models is a major limitation of preclinical studies that hampers accurate translation and substantiation of the right-left prefrontal imbalance pathophysiological hypothesis66. To overcome the lack of specificity of stimulation, coils optimized for precise targets—specifically designed for preclinical application—are needed19. Hopefully, technological advances and increased interest towards rTMS in animal models will allow the development of smaller sized coils for isolated stimulation of specific regions38. The availability of optimized coils will also allow to extend the investigation to smaller species (the presence of only 2 studies employing mice should be considered as a limitation).Clinical translation of results is also significant in terms of the reversal of depressive-like symptomatology regardless of certain neuromodulation parameters . Indeed, reversal of the depressive phenotype was obtained independently of rTMS frequency. This result may be of translational relevance, given that current clinical applications of rTMS include use at both high and low frequencies, though targets have a different lateralization. Accruing evidence indicates an imbalance between the left and right DLPFC in MDD, supporting the need for differentiated stimulation/inhibition lateralized protocols to counterbalance such asymmetry. Left-right DLPFC imbalance is associated with neuropsychological . Lack of studies assessing rTMS efficacy in the same type of model (the “other” subgroup comprises 6 studies in 6 different models) precluded the possibility to refine subgroup analysis. As for anhedonia, even though all studies employed the CUS model, there was still substantial evidence of high heterogeneity, which may be partly attributed to individual differences in rats’ hedonic status, as well as to differences in sucrose concentration (1% vs. 2%). It is worth noting that the duration of stress application (3 vs. 8 weeks) and the stress regime itself may also explain some of the discrepancies among studies22. A subgroup analysis could not be performed due to the paucity of studies.When helplessness studies were grouped according to frequency, moderate to high heterogeneity persisted for high frequency studies, while for low frequency studies heterogeneity was remarkably lower. Similarly, after application of model-based subgroup analysis, the heterogeneity was considerably lower for studies performed in healthy subjects, while there was still substantial evidence of high heterogeneity particularly between studies employing “other” models. This is likely due to the diversity of approaches used to induce depression . To date, only the CUS model is well-represented . Models addressing the interaction between environmental and predisposing genetic factors in the induction of depressive-like phenotypes are still rarely employed but highly relevant as they resemble real-life clinical situations . Extending rTMS studies to other models of depression that involve different pathogenic mechanisms is therefore crucial to test its effectiveness and to inform on the biological basis of treatment response to rTMS.Though rTMS appears to significantly reverse the depressive phenotype, results from the present meta-analysis do not allow us to draw conclusions on its relative efficacy on specific depression models due to the low representation of the different existing models  for patients with different symptom profiles diagnosed as having MDD77. In this context, translating preclinical results into clinical practice requires the use of other validated models of depression (corresponding to distinct endophenotypes) and, whenever possible, of additional and/or more sophisticated tests to evaluate depressive-like symptomatology78. This will help identify diagnostic biomarkers that predict response to rTMS and develop more tailored interventions79. To date, only the FST is well-represented , the FST remains one of the most widely used tests to screen antidepressant effects83.Stress models of depression, while well-validated in preclinical research, encompass a broad array of symptoms thus possibly diluting specific target treatment effects and introducing bias when examining biological changes associated with rTMS. Narrower phenotypes may capture the biological effects of rTMS and improve prediction of treatment response7 . CUS-exposed rodents that fail to respond to conventional antidepressants are considered non-responders85. Also, Wistar-Kyoto rats subjected to CUS have been recently validated as a model of TRD87. Notably, these models showed a good response to deep brain stimulation87; however, the effects of rTMS in these treatment-resistant animals have not yet been studied.Current animal models of depression, including CUS, respond to conventional antidepressants. Therefore, they can inform about the antidepressant-like efficacy of a novel intervention but have limited utility in predicting whether such intervention will also be effective in patients suffering from TRD. To overcome this issue, future studies should involve treatment-resistant animal models of depression89. The presence of only 2 studies including female subjects should therefore be considered as a limitation. Preclinical experiments are often conducted only in males , while clinical trials include both men and women91. This issue may partly explain why clinical trials repeatedly fail to confirm the expected benefits of new treatment approaches that have shown favorable profiles in preclinical studies92. To maximize translation of research findings to the clinical practice, sex should be considered as an important biological variable from basic and preclinical research91. To overcome the issue of overlooked and underreported sex and gender in research across disciplines, the SAGER guidelines (Sex And Gender Equity in Research) were published in 201693. Accordingly, numerous scientific journals are revising their editorial policies requiring clear reporting of the sex/gender of research subjects and to analyze data by sex94. Future studies should also test whether rTMS effects persist longitudinally and, if so, to what extent, as currently follow-up evaluations are rarely performed.Sex differences in depressive-like symptomatology have been evidenced in several animal models  and tests  and there are marked sex differences in the prevalence of MDD94), even after publication in 2010 of the ARRIVE guidelines , which were developed to improve the design, analysis and reporting of research using animals. Although ARRIVE guidelines are currently endorsed by numerous scientific journals and societies96, most animal research papers still fail to meet minimum reporting standards . In particular, none of the 13 articles published after 2010 declares adherence to ARRIVE guidelines. However, in the absence of mandatory reporting standards for pre-clinical animal studies, we cannot assume that the authors conducted their experiment in an inappropriate way; therefore, studies were not excluded based on a poor-quality score. Nevertheless, there is an urgent need to overcome the issue of largely unclear risk of bias in animal studies by improving compliance with the ARRIVE guidelines as (i) poor reporting of animal research hinders the quality of research and its potential to translate into the clinic; (ii) the quality of a systematic review/meta-analysis is dependent on the quality of the included studies, and not knowing the actual risk of bias hampers our ability to draw reliable conclusions. An approach that may likely mitigate the reporting bias in preclinical studies is constituted by the possibility to prioritize manuscripts that were preceded by a pre-registration procedure analogous to systematic reviews99.Studies had an overall unclear risk of bias based on SYRCLE’s RoB tool. Unfortunately, this confirms that reporting of methodological details in animal studies remains poor  maximize clinical effects and to develop cost-effective protocols, with the potential of yielding faster clinical responses . Further exploration of rTMS use in rodent models will promote data-driven identification of prognostically-informative depression endophenotypes to be used in real-world MDD treatment settings to predict which patients are more likely to respond to rTMS, thus working toward a patient-tailored intervention for MDD and TRD. This will have a major impact in terms of clinical burden reduction and decreased exposure to non-specific and ineffective treatments.Notwithstanding its limitations, this meta-analysis supports the efficacy of rTMS interventions in ameliorating phenotypic alterations isomorphic to human MDD symptoms in laboratory rodents. Predictions of the effects of rTMS, such as changes on the molecular and cellular levels up to modulations of brain networks, need further investigation in order to reconcile the varying observations that have been made so far in the rTMS fieldSupplementary captionsSupplementary item 1Supplementary item 2Supplementary item 3Supplementary item 4Supplementary item 5


Stroke is the leading cause of serious long-term disability, significantly reducing mobility in almost half of the affected patients aged 65 years and older. There are currently no proven neurorestorative treatments for chronic stroke. To address the complex problem of restoring function in ischemic brain tissue, stem cell transplantation-based therapies have emerged as potential restorative therapies. Aligning with the major cell types found within the ischemic brain, stem-cell-based clinical trials for ischemic stroke have fallen under three broad cell lineages: hematopoietic, mesenchymal, and neural. In this review article, we will discuss the scientific rationale for transplanting cells from each of these lineages and provide an overview of published and ongoing trials using this framework. Stroke is the leading cause of serious long-term disability, significantly reducing mobility in almost half of affected patients aged 65 years and older  at rest reside within the bone marrow niche but can be mobilized to the general blood circulation in response to stimulant factors such as granulocyte colony-stimulating factor (G-CSF). A majority of trials conducted using HSC lineage cells have relied on autologous bone marrow transplantation, favoring the lack of immunogenicity and ethical challenges to obtaining a reliable source of hematopoietic cells. In these studies, bone marrow was obtained from the patient and purified, either by density gradient alone or combined with immunosorting to obtain the cell population for transplantation. The cell surface marker CD34 characterizes a population enriched for HSCs, and the proportion of CD34+ cells found in the peripheral blood immediately after stroke has been found to directly correlate with functional recovery  within 7–30 days of stroke onset and also confirmed the safety and feasibility of this treatment protocol. Friedrich et al.  infusion to 20 patients within 3–10 days of stroke onset and found this method to be safe. Of note, while not known at the time, the brain biodistribution of intra-arterially and intravenously transplanted BM-MNCs was eventually determined to be comparable in a separate clinical trial completed a year later . Similarly, Banerjee et al.’s (via the MCA) 10 million autologous sorted CD34+ cells in five patients within 1–7 days of stroke onset, and found the intervention to be safe, and associated with a decrease in infarct size over time.Hypothesizing that the hematopoietic stem cell-enriched CD34+ fraction of BM-MNCs contained the functional subset of cells responsible for repair during ischemic CNS injury, Moniche et al.  conducteet al.’s  researchvia intracarotid infusion in 29 patients and compared this cohort to 19 control patients who received a sham procedure. Disappointingly, the study found no significant difference in primary or secondary efficacy measures between treatment and placebo groups.Aldehyde dehydrogenase (ALDH) was among the first markers used by immunologists to identify populations of human hematopoietic stem and progenitor cells in the 1990s. HSCs were found to express the highest levels of ALDH, while lymphocytes expressed the lowest  transplant efficacy in treating chronic ischemia in rats  are stromal cell precursors to cells of osteogenic, chondrogenic, and/or adipogenic lineages, and can be harvested and expanded from a variety of tissue types, including bone marrow , adipose tissue , and umbilical cord (UB-MSCs). This versatility, combined with low immunogenicity due to low expression of human leukocyte antigens, makes MSCs excellent candidates for allogeneic stem cell transplants  from transplanted cells  and transplanted groups experienced statistically significant improvement, there was no statistically significant difference between the groups in terms of functional outcome. Bhasin et al.  or placebo (n = 61) within 24–48 h after stroke onset. The allogeneic BM-MSC product used in this study—MultiStem—was derived from two independent donors  and for all patients treated within the originally planned time window of ≤36 h post-stroke the benefit at 1 year was even greater . This finding was encouraging enough for Athersys to initiate another Phase III prospective, randomized, placebo-controlled, double-blind trial treating patients between 18–36 h of stroke (MASTERS-2).In the largest MSC trial to date, Hess et In a separate Phase I/II trial using allogeneic single-donor adult mesenchymal BM-MSCs  are multipotent cells that can differentiate into neurons, astrocytes, and oligodendrocytes. This native capacity to repopulate and support endogenous cell types within the brain has generated much interest in NSC transplantation for stroke. The subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampus have been identified as neurogenic sites, and in murine models, NSC’s have been shown to migrate from these niches to promote neurogenesis and vascular remodeling in response to ischemic stroke  modifying a human teratoma cell line [teratomas and cancer lines were the only non-embryonic source of pluripotent human cells before the development of induced pluripotent stem cells (iPSCs)] to induce neuronal differentiation by introducing the morphogen retinoic acid; (2) using NSCs harvested from human fetal tissue and/or clonal lines derived from these cells; or (3) using cells of non-human origin .via intracranial injection between 6 months and 6 years post-stroke. These cells were derived by differentiating the NT2/D1 human cell line, originally derived from a lung metastasis of testicular embryonal carcinoma, into neurons using a 10 μM dose of retinoic acid. Before the discovery of iPSCs in 2007, NT2/D1 was among the lines widely used to represent human pluripotent cells and was shown to be capable of terminal differentiation into multiple cell types, including neurons and astrocytes  improved significantly compared with controls and with baseline scores on the same test.Given the concerns surrounding implanting cells derived from malignant human tumors, compounded with the ethical implications and difficulty of obtaining fetal tissue, Savitz et al.  attemptevia enhanced neurogenesis and angiogenesis in a dose-dependent fashion after ischemia ; (2) OECs + NPCs ; (3) OECs + NPCs  with a second dose of NPCs at a later time point ; (4) OECs + NPCs  + later doses of NPCs  and UB-MSCs ; and (5) OECs and NPCs  + later doses of SC and NPCs  and UB-MSCs . Treatment was found to be safe and feasible in all cases, however, due to the variety of cell types and routes of administration evaluated, the authors concluded that the study was not sufficiently powered to conclude that co-transplantation is safe under all studied conditions. The authors also noted a trend towards functional benefits with intracranial injections, but not with intrathecal and intravenous cell administration.Given the significant trophic support provided by mesenchymal cells and the regenerative capacity of NSCs, combined engraftment of fetal-derived neural and cord-blood-derived mesenchymal lineage cells together has been investigated as a potential therapeutic strategy to improve and support stem cell engraftment. Chen et al.  sought t6 UB-MSC cells per kg body weight or one intravenous dose of 0.5 × 106 UB-MSC cells per kg body weight followed by three intrathecal doses of 0.5 × 106 UB-MSC cells per kg body weight and 6 × 106 human fetal derived NPCs (Qiao et al., Another Phase I study conducted by Qiao et al.  involvedvia a variety of routes and in a variety of doses, to treat ischemic stroke of varying chronicity. We have discussed the history of and scientific rationale for the different cell types transplanted, their routes of administration, and associated trial outcomes, and have provided snapshots of current ongoing trials. Additional studies are necessary to strengthen our understanding of the relationship between neural cells and their surrounding stromal, endothelial, and immune landscape in both the healthy state and in pathologic conditions. As discussed in this review, specific attention should be given to the paracrine mechanisms by which transplanted cells exert their therapeutic effect, especially in light of data that suggests that these benefits persist even after the clearance of the originally transplanted cell type.The regenerative properties of stem cells have brought cell-based transplantation studies into the spotlight as appealing therapies for otherwise recalcitrant disorders such as subacute and chronic ischemic stroke. In this review article, we have detailed the clinical trials to date, which have featured transplantation of various cell types, administered Furthermore, promising preclinical studies require adequate support and prudent design to overcome the “translational roadblock,” a notable decrease in efficacy between preclinical studies and their clinical trial counterparts. The difference is thought to be due to a series of factors. First, a combination of publication bias and overstated efficacy in preclinical studies has led to overly optimistic preclinical data that fail to result in statistically meaningful clinical interventions (Dirnagl et al., ES and GS contributed to the conception of this review. JH and ES contributed to the design and organization of the material. JH and GS created and updated the tables. JH, ES, and GS wrote sections of the manuscript, contributed to manuscript revision, read, and approved the submitted version.GS is a consultant for Qool Therapeutics, Peter Lazic US, NeuroSave, SanBio, Audaxion Therapeutics, Zeiss and Surgical Theater. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The evolutionary conflict between retroviruses and their vertebrate hosts over millions of years has led to the emergence of cellular innate immune proteins termed restriction factors as well as their viral antagonists. Evidence accumulated in the last two decades has substantially increased our understanding of the elaborate mechanisms utilized by these restriction factors to inhibit retroviral replication, mechanisms that either directly block viral proteins or interfere with the cellular pathways hijacked by the viruses. Analyses of these complex interactions describe patterns of accelerated evolution for these restriction factors as well as the acquisition and evolution of their virus-encoded antagonists. Evidence is also mounting that many restriction factors identified for their inhibition of specific retroviruses have broader antiviral activity against additional retroviruses as well as against other viruses, and that exposure to these multiple virus challenges has shaped their adaptive evolution. In this review, we provide an overview of the restriction factors that interfere with different steps of the retroviral life cycle, describing their mechanisms of action, adaptive evolution, viral targets and the viral antagonists that evolved to counter these factors. Retroviruses replicate by converting their single-stranded RNA genome into double-stranded DNA through a virus-encoded reverse transcriptase. These DNA copies are integrated into host chromosomes where they persist as a permanent part of the host cell genome. When retroviruses infect germline cells, integrated copies can be passed on to the next generation. These integrated proviruses are termed endogenous retroviruses (ERVs). As the retroviral genome encodes only a limited number of genes, replication of exogenous viruses and expression of ERVs rely on the host cell machinery. These viruses can be mutagenic and pathogenic, and this puts strong selective evolutionary pressure on the host genes appropriated for virus replication. The result has been the emergence of defensive genes that can inhibit viral infection and that have been derived from cellular genes, or more rarely, from ERVs. These restriction factors comprise the innate immune system that represents the first line of defense against viral attack.While each restriction factor has followed a unique evolutionary path, some common themes have emerged . First, Here, we describe a well-characterized set of retroviral restriction factors for which there is substantial information on their restriction mechanisms as well as their evolutionary history. The majority of these factors were discovered in primates or rodents through studies on HIV-1 which causes the acquired immune deficiency syndrome (AIDS) in humans, and on mouse leukemia viruses (MLVs) which induce lymphomas, immunodeficiencies, and neurological diseases. Some of these factors are restricted to specific taxonomic lineages, while others are carried by many mammalian species, showing broad antiviral activity against multiple retroviruses and in some cases against other families of viruses . DecadesRetroviruses enter susceptible cells through their interactions with specific cell surface receptors. These interactions can be inhibited by receptor polymorphisms as well as by other transmembrane proteins that interfere with binding or membrane fusion.CD4 in humans that affect the efficiency of HIV-1 infection, CD4 is highly variable in chimpanzees, and this variation is responsible for restricted susceptibility to SIV, the progenitor of HIV-1  deletion in the second extracellular loop of the protein leading to the introduction of a premature stop codon which renders it non-functional as a co-receptor ,4,53,4,5of HIV-1 B 11,12],12CD4 in and Nef B 13,14],14CD4 inreceptor .Mus dunni CAT1 restricts infection by Moloney MLV . Co. Co75]. s titers . Fv1 ress titers ,61,77,78s titers .Fv1 is found in all but the most basal species in the Mus phylogenetic tree, and is missing in the rat genome, so it was initially thought that Fv1 was acquired shortly after the origins of the Mus genus ..BST2 revFv1 in 1976, and this set of genes will continue to expand as new screening methods reveal additional cellular proteins that have antiviral activity [Fv1, the MLV LTR embedded in mouse Apobec3, and the Env-producing Fv4, Rmcf and Rmcf2 genes are domesticated ERVs that have no known cellular purpose.There has been explosive growth in the identification of retroviral restriction factors since the discovery of activity ,350,351.For some of these factors, restriction operates through interference with cellular processes needed for virus replication, for example, depletion of dNTP pools by SAMHD1 or tRNA pools by SLFN11 ,257. OthMus-specific factor restricting MLVs but is now known to have inhibitory activity against retroviruses of different genera such as feline foamy virus and some lentiviruses [There are many restriction factors that have unexpectedly long historical records of intense diversifying selection, and this argues for broader defensive roles, roles that for many of these factors are now known to extend well beyond their initially defined retroviral targets. Thus, Fv1 was initially defined as a iviruses ,81. MoreMX1 and MX2 can also contribute to their diversification [The rapid evolutionary changes that produce more effective host defenses are not limited to the statistical increase in the rate of non-synonymous mutations, but can also be marked by more dramatic genome reconfigurations including gene duplications and diversifications within specific lineages as seen for TRIM5 which has one copy in human and one to eight copies in rodents, or in APOBEC3 with one gene in mice and seven paralogs in humans, each having different restriction profiles and tissue specific expression patterns ,116,172.fication .While the level of restriction imposed by these factors varies from total blocks to smaller several fold decreases, even marginal inhibitions are amplified during the spreading infections that precede disease. The knowledge gained by studying the interaction between these host restriction factors and their viral targets helps us understand the barriers to cross-species viral transmission, and should also contribute to the development of novel therapeutics that either exploit the inhibitory impact of these restriction factors or target their viral antagonists.


Composite materials have become widely used in engineering applications, in order to reduce the overall weight of structures while retaining their required strength. In this work, a composite material consisting of discontinuous glass fibers in a polypropylene matrix is studied at the microstructural level through coupled experiments and simulations, in order to uncover the mechanisms that cause damage to initiate in the microstructure under macroscopic tension. Specifically, we show how hydrostatic stresses in the matrix can be used as a metric to explain and predict the exact location of microvoid nucleation that occurs during damage initiation within the composite’s microstructure. Furthermore, this work provides evidence that hydrostatic stresses in the matrix can lead to coupled microvoid nucleation and early fiber breakage, and that small fragments of fibers can play an important role in the process of microvoid nucleation. These results significantly improve our understanding of the mechanics that drive the initiation of damage in the complex microstructures of discontinuous fiber reinforced thermoplastics, while also allowing scientists and engineers to predict the microstructural damage behavior of these composites at sub-fiber resolution and with high accuracy. Specifically, polymer matrix composites have allowed for major weight savings and higher performance aircraft and vehicles. Despite their high rate of implementation, scientists and engineers have faced challenges in predicting their mechanical behavior and performance, especially past the small strain regime and into the damage initiation and damage propagation regimes, because there exist a number of damage mechanisms which are often coupled and are active throughout the life of a component. Compared to continuous fiber composites, discontinuous fiber reinforced polymers exhibit vastly heterogeneous microstructures that can vary significantly depending on the component geometry, making mechanical behavior predictions even more complicated5. Efforts in understanding and predicting the behavior of fiber composites past the elastic regime have typically focused on attempting to replicate the macroscopic bulk stress-strain behavior of a specimen through phenomenological damage parameters6. In recent years, some microstructural approaches have been used to explore the damage mechanisms in certain composites using homogenization approaches7 as well as unit cell methods8. Experimentally, one in-situ study of a thermoset polymer, reinforced with discontinuous carbon fibers, showed that fiber tips play an important role in microvoid nucleation due to high shear stresses, while fiber breakage plays no role9. However, for thermoplastic polymers reinforced with discontinuous fibers, researchers have faced even more difficulty because compared to thermoset polymers, thermoplastic polymers experience highly non-linear plasticity creating more complications from a microstructural point of view, in which researchers have applied orientation averaging in unit cells to model this behavior10.Until recently, most efforts in predicting the mechanical performance of discontinuous fiber composites have been focused on the elastic loading regime during which damage has not yet initiated and progressedin-situ scanning electron microscopy have shown that under tensile loads, damage initiation appears in the form of microvoid nucleation at fiber tips12. In attempting to improve predictive capabilities and include this damage initiation mechanism, researchers have modeled the macroscopic behavior of short fiber reinforced thermoplastic composites by defining fiber matrix debonding at fiber tips as the governing damage mechanism with good results at simulating the macroscopic stress-strain response13. To try and explain this phenomena, researchers have hypothesized that the underlying mechanism of microvoid nucleation in a thermoplastic matrix is similar to that of ductile metals and is related to high tensile hydrostatic stresses or high stress triaxiality16. However, the appropriate treatment of microvoid nucleation in composites still remains uncertain, mainly because of its stochastic nature and the challenges in computing the local stress states in complex composite microstructures17.Efforts in experimentally examining the damage mechanisms of short fiber reinforced thermoplastics through 22, sometimes in-situ, in order to observe the evolution of the microstructure24. In this work, high resolution X-ray in-situ  was created of this sub-region, in which 368 fibers were instantiated. The results showed that the 5 experimentally observed locations of microvoid nucleation corresponded with the 5 highest regions of hydrostatic stress, which substantiates the use of the hydrostatic stress metric to explain and predict microvoid nucleation in the microstructure of fiber reinforced thermoplastic composites, proving a long standing hypothesis of the role of stress triaxiality in damage initiation for thermoplastic matrix composites. Lastly, this work showed that although small fragments of fibers, which are almost negligible in size compared to the average fiber length, can be ignored during elastic mechanical property predictions, they must be included in the damage initiation and strength predictions of these composites.31. The specimen was studied in-situ by applying tensile load  were detected using a combination of Weka machine learning segmentation34 and manual correction using ModLayer35. The specimen’s free surface was detected through 2D image processing of each tomography slice using an in-house MATLAB algorithm that utilized the intensity gradient at the free surface, which can be seen in Fig. Fibers were detected using an iterative and supervised 2D and 3D combined algorithm11. The meshed fiber elements were assigned linear elastic properties with an elastic modulus of 72.4 36. The meshed matrix elements were simulated to include nonlinear plasticity through a multilinear isotropic hardening model37. The positive X, negative Y, and negative Z surfaces were constrained using roller boundary conditions, and the positive Z surface was displaced in the positive Z direction by 7.8 The voxels (which were 1.3