Gut-Microbiota-Brain Axis
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What is gut microbiota? What is the influence of diet on the proper functioning of our gut microbiota? How does the gut-brain axis (GBA) influence the emotional and cognitive centers of the brain? Tune into this webinar to find out more about this timely topic. Learning Objectives: List the neurological and physiological connections that enable the bidirectional communication between the gut and the brain Identify lifestyle, dietary, and microbial influences on the flow and function of signaling molecules along the gut-microbiota-brain axis Implement dietary regimens that target the gut and gastrointestinal microbiota to improve or maintain optimal physical and mental health RDNs earn 1.0 CEU
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Gut-Microbiota-Brain Axis
- 1. NW SMS icons 1 learn.extension.org/events/3055 The Gut-Microbiota-Brain Axis
- 2. Connecting military family service providers and Cooperative Extension professionals to research and to each other through engaging online learning opportunities militaryfamilies.extension.org MFLN Intro Sign up for webinar email notifications at militaryfamilies.extension.org/webinars
- 3. Dr. Hannah Holscher, PhD, RD Assistant Professor in the Department of Food Science and Human Nutrition at the University of Illinois. The long-range goal of her research is to develop targeted dietary interventions for disease prevention and treatment by identifying key foods and/or nutrients that can be utilized for modulation of the human gastrointestinal microbiome for health benefit. Current clinical research in her laboratory, the Nutrition and Human Microbiome Laboratory, includes controlled feeding studies investigating the impact of specific whole foods and beverages, including, avocados, broccoli, walnuts, almonds, whole grains, and kefir on the human gastrointestinal microbiome, metabolic health, and the gut-microbiota-brain axis. Today’s Presenter 3
- 4. The Gut-Microbiota-Brain Axis Hannah D. Holscher, PhD, RD Assistant Professor Department of Food Science and Human Nutrition Division of Nutritional Sciences University of Illinois
- 5. List the neurological and physiological connections that enable the bidirectional communication between the gut and the brain Identify lifestyle, dietary, and microbial influences on the flow and function of signaling molecules along the gut- microbiota-brain axis Implement dietary regimens that target the gut and gastrointestinal microbiota to improve or maintain optimal physical and mental health Learning Objectives 5
- 6. Microbiome - a collection of microbial genomes Microbiota – a collection of microbes As many bacteria as host cells in human body 150x more bacterial genes than our human genome Microbiome Disease Health 6
- 7. Colon 1010 – 1012 CFU/mL Bacteroides Prevotella Facaelbacterium Ruminococcus Roseburia Clostridium Bifidobacteria Collinsella Desulfovibrio Bilophila Akkermansia Methanobrevibacter Jejunum & Ileum 104 – 108 CFU/mL Bacteroides Streptococcus Lactobacillius Bifidobacteria Fusobacteria Stomach & Duodenum 101 – 102 CFU/mL Helicobacter Streptococcus GI Microbiota 7
- 8. Protective functions Structural functions Metabolic functions Fermenting dietary fiber into short-chain fatty acids Synthesizing vitamins Grenham S, Front Physiol, 2011 Microbiota Functions 8
- 9. Flint, H. J. et al. (2012) Nat. Rev. Gastroenterol. Hepatol. Microbiota Functions 9
- 10. Gut-Microbiota- Brain Communication Cryan, John F., and Timothy G. Dinan. "Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour." Nature reviews neuroscience 13.10 (2012): 701-712. 10
- 11. Bidirectional communication Central nervous system (brain and spinal cord) Autonomic nervous system (sympathetic and parasympathetic) Enteric nervous system (intrinsic nervous system of GI tract) Hypothalamic pituitary adrenal axis (HPA) Microbiome (collection of microorganisms and their genomes in the gut) Clarkea, Gerard, Ted Dinanb, and John Cryanc. "Microbiome–Gut–Brain Axis." (2013). Gut-Microbiota-Brain Communication 11
- 12. Major nerve of the parasympathetic division of the autonomic nervous system Important pathway for bidirectional communication between the gut microbes and the brain Preclinical/animal studies demonstrate that probiotic effects on brain are dependent on vagal afferent signals Lactobacillus rhamnosus directly activates vagal neurons Induces region-dependent alterations in GABA receptor expression in the brain and reduced stress-induced corticosterone and anxiety- and depression-like symptoms via vagus nerve signaling in mice Vagotomized mice do not exhibit this effect Bravo, Javier A., et al. Proceedings of the National Academy of Sciences 108.38 (2011): 16050-16055. Vagus Nerve 12
- 13. Acetylcholine Noradrenaline Adrenaline Gamma-amino butyric acid (GABA) Serotonin Neurotransmitters 13Burokas et al., Advances in Applied Microbiology. 91 (2015): 1-62
- 14. Neurotransmitter Released By Function GABA Central Nervous System (CNS) Relaxes lower esophageal sphincter Norepinephrine CNS, spinal cord, sympathetic nerves Decreases motility, increased contraction of sphincters, inhibits secretions Acetylcholine CNS, autonomic system, other tissues Increases motility, relaxes sphincters, stimulates secretion Serotonin GI tract, spinal cord Facilitates secretion and peristalsis Neurotransmitters & GI Function 14
- 15. Neurotransmitter Genus GABA Lactobacillus, Bifidobacterium Norepinephrine Escherichia, Bacillus, Saccharomyces Acetylcholine Lactobacillus Serotonin Candida, Streptococccus, Escherichia, Enterococcus Lyte, Mark. Bioessays 33.8 (2011): 574-581. Bacteria & Neurotransmitters 15
- 16. Biogenic amine that functions as a neurotransmitter Tryptophan is precursor Involved in GI secretion Gut motility Pain perception Maintenance of mood and cognition 95% of serotonin is contained in the gut in the mucosa and nerve terminals of the enteric nervous system Alterations in serotonin transmission may underlie pathological symptoms Selective serotonin reuptake inhibitors are known to modulate psychiatric and GI disorders (e.g., IBS) O’Mahony, S. M., et al. Behavioural brain research 277 (2015): 32-48. Serotonin 16
- 17. Holzer, Peter, Florian Reichmann, and Aitak Farzi. "Neuropeptide Y, peptide YY and pancreatic polypeptide in the gut–brain axis." Neuropeptides 46.6 (2012): 261-274. Gut Hormones and Neuropeptides 17
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- 20. Gut can communicate with the brain via hormonal signaling: gut peptides from enteroendocrine cells can act directly on the brain. Ghrelin Gastrin Orexin Cholecystokinin Leptin Neuropeptide Y Feeding Behavior Energy Homeostasis Circadian Rhythm Arousal Anxiety Forsythe, Paul, and Wolfgang A. Kunze. Cellular and molecular life sciences 70.1 (2013): 55-69. Cameron, Jameason, and Eric Doucet. Applied Physiology, Nutrition, and Metabolism32.2 (2007): 177-189. Schéle, Erik, et al. Endocrinology 154.10 (2013): 3643-3651. Germ-free studies suggest that the gut microbiota mediates and regulates the release of gut peptides Gut Hormones and Neuropeptides 20
- 21. Under the anaerobic conditions of the large intestine, undigested carbohydrates are fermented mainly to SCFAs Acetate Propionate Butyrate Gases (H2, CO2, CH4, and H2S). SCFAs have multiple effects on the host Energy sources for the host Butyrate being consumed mainly by the colonic epithelium Acetate becomes available systemically Circulating SCFAs can be carried by across the blood–brain barrier Short-Chain Fatty Acids (SCFAs) Pomare, E. W., W. J. Branch, and J. H. Cummings. Journal of Clinical Investigation 75.5 (1985): 1448. Maurer, Martin H., et al. Neuroscience letters 355.1 (2004): 105-108. Microbiota-Derived Signaling 21
- 22. Evans, James M., Laura S. Morris, and Julian R. Marchesi. Journal of Endocrinology 218.3 (2013): R37-R47. Anti-CancerEffects Peptide YY GLP-1 Enteroendocrine Serotonin secretion SCFAs Appetite Control Inflammatory GutMotility Energy Expenditure Anti-Cancer Microbiota-Derived Signaling 22
- 23. Intricate neurological and physiological factors enable the bidirectional communication between the gut and the brain Bidirectional communication with afferent and efferent signals Vagus nerve Neuropeptides Microbiota can influence behavior, neurophysiology, and neurochemistry SCFAs represent set of bacterial metabolites with potentially widespread health benefits Importance of dietary fiber intake Microbial-based therapeutics remain topic of further investigation Summary 23
- 24. List the neurological and physiological connections that enable the bidirectional communication between the gut and the brain Objective 1 Review 24
- 25. List the neurological and physiological connections that enable the bidirectional communication between the gut and the brain Central nervous system: brain and spinal cord Autonomic nervous system: vagal nerve Enteric nervous system: GI nerves Microbes and their metabolites SCFAs: acetate, propionate, & butyrate Neurotransmitters: serotonin and GABA Gut Hormones: PYY Neuropeptides: NPY Objective 1: Answers 25
- 27. Fecal communities clustered into enterotypes distinguished by levels of Bacteroides and Prevotella. Enterotypes were associated with long-term diets: Bacteroides: protein and animal fat Prevotella: carbohydrates Wu et al., 2011; Science Bacte ro ide s Pre vo te lla Enterotypes associated with habitual diet 27
- 28. Fiber: Plant Based: 25 g per 1000 kcal Animal Based: 0 g per 1000 kcal Fat: Plant: 20 % kcal Animal: 70% kcal Protein: Plant: 10% Animal: 30% David et al. Nature 2014 Diet can rapidly impact microbiotaDiet can rapidly impact microbiota 6 male + 4 females; 21-33 years of age BMI 19-32 kg/m2 Cross-over design: ad libitum 5-day consumption of diets composed entirely of animal or plant products Diet Composition 28
- 29. Diets Snacks Fresh banana Fresh mangoes Fresh papayas Banana chips David et al. Nature 2014 29
- 30. Plant Based Diet Animal Based Diet David et al. Nature 2014 Diet can rapidly impact microbiota 30
- 31. • Plant-based: increased short-chain fatty acids, acetate and butyrate • Animal-based: increased branch- chain fatty acids, isovalerate and isobutyrate David et al. Nature 2014 Bile acids tended to increase on animal-based diet P<0.1 P>0.1 Diet can rapidly impact microbiota 31
- 32. Fiber: “Non-digestible soluble and insoluble carbohydrates ( > 3 monomeric units), and lignin that are intrinsic and intact in plants; isolated or synthetic non-digestible carbohydrates (> 3 or more monomeric units) determined by FDA to have physiological effects that are beneficial to human health.” Different types of fibers in different types of plants Supplemental fibers in a range of foods Different botanical origins and chemical structure Cellulose B-glucans Inulin Polydextrose Soluble Corn Fiber Dietary Fiber Food and Drug Administration 2016 32
- 33. Prebiotic – a substrate that is selectively utilized by host microorganisms conferring a health benefit1 Probiotic– live microorganisms that when administered in adequate amounts confer a benefit to the host2 Synbiotic: Product that contains both probiotics and prebiotics Pro-, Pre- & Syn-biotics 1. Gibson GR et al (2017) Nature Reviews Gastro Hept; 2. World Health Organization (2010) 33
- 34. Linus Pauling Institute Image: http://lpi.oregonstate.edu/mic/other-nutrients/fiber Microbes ferment dietary fiber 34
- 35. Randomized, double-blind, placebo-controlled crossover trial with three 21-day treatment periods Healthy adult men (n = 21) Daily food and GI tolerance records 3 fecal specimens were collected on days 16 - 21 Holscher et al. Am J Clin Nutr 2015 Study Design: PDX & SCF 35
- 36. a b b a ab p<0.01 Holscher et al. Am J Clin Nutr 2015 PDX & SCF shifted Bacteroidetes:Firmicutes Ratio 36
- 37. PDX NFC SCF PC2 (9%) PC3 (5%) PC1 (10%) p<0.001 Holscher et al. Am J Clin Nutr 2015 Microbiome changes with daily PDX and SCF 37
- 38. Randomized, double-blind, placebo-controlled crossover trial with three 21-day treatment periods Healthy adults (n=30) Daily food and GI tolerance records 3 fecal specimens were collected on days 16 - 20 Holscher, H.D. et al., Food & Function, 2014 Design: Agave Inulin 38
- 39. Holscher, H.D. et al., J. Nutr 2015 Treatment (Agave inulin g/day) b a c a a b Increased Bifidobacterium & decreased Desulfovibrio 39
- 40. Holscher, H.D. et al., J. Nutr 2015 Agave inulin: phenotypic responses 40
- 41. Identify dietary and microbial influences on the the gut-microbiota-brain axis True or False? Habitual diet and rapid changes in diet can influence microbial composition and microbial metabolites Objective 2: Review 41
- 42. Identify dietary and microbial influences on the the gut-microbiota-brain axis True Both habitual diet and rapid changes in diet have been shown to influence microbial composition and microbial metabolites in adults Objective 2: Answer 42
- 43. Identify dietary and microbial influences on the the gut-microbiota-brain axis Matching 1)Prebiotic 2)Probiotic 3)Synbiotic Objective 2: Review A. Microorganisms + substrates B. Energy substrate for microbes C. Microorganisms 43
- 44. Identify dietary and microbial influences on the the gut-microbiota-brain axis 1. Prebiotic (B)– a substrate that is selectively utilized by host microorganisms conferring a health benefit 2. Probiotic (C)– live microorganisms that when administered in adequate amounts confer a benefit to the host 3. Synbiotic (A): product that contains both probiotics and prebiotics Objective 2: Answers 44
- 45. Identify dietary and microbial influences on the the gut-microbiota-brain axis True or False? All fibers and prebiotics change the composition of the microbiome in the same way Objective 2: Review 45
- 46. Identify dietary and microbial influences on the the gut-microbiota-brain axis False Fibers and prebiotics differentially change the composition of the microbiome. For example, SCF & PDX increased the abundances Bacteroidetes, while agave inulin increased Bifidobacterium Objective 2: Answer 46
- 47. What about diet? Implement dietary regimens that target the gut and gastrointestinal microbiota 47
- 48. Fruits Vegetables Whole Grains Nuts Legumes Food sources of dietary fiber 48
- 49. Foods Oatmeal Barley Onions Greens Berries Bananas Legumes Sources of Prebiotics Supplements Tablets Powders Prebiotic – a substrate that is selectively utilized by host microorganisms conferring a health benefit 49
- 50. Supplements Capsule Powder Tablets Foods Yogurt Fermented milk drinks Kefir Infant formula Cheese Cereal Sources of Probiotics Probiotic– live microorganisms that when administered in adequate amounts confer a benefit to the host 50
- 51. Galactooligosaccharies (GOS) 5.5 g/day Reduced waking salivary cortisol Suppression of the neuroendocrine stress response Increased attentional vigilance in the processing of positive versus negative interpreted as showing an early anxiolytic-like profile, where threatening stimuli are less likely to be attended to Reduction of anxiety like behavior and dampening of fight-or-flight response Schmidt, K et al., Psychopharmacology (2015) 232:1793–1801 Gut-Brain Axis: Prebiotics 51
- 52. 4-week intake of a 250 g of fermented milk by healthy women affected activity of brain regions that control central processing of emotion and sensation Bifidobacterium animalis subsp. lactis (2.5 x 1010 CFU/day) Streptococcus thermophiles (2.4 x 109 CFU/day) Lactobacillus bulgaricus (2.4 x 109 CFU/day) Lactococcus lactis subsp. lactis Beneficial effect on general signs of anxiety and depression, reduced cortisol L. helveticus and B. longum (3 x 109 CFU/day) Tillisch et al., Gastroenterology 2013;144:1394 –1401 Messaoudi et al., British Journal of Nutrition (2011);105: 755–764 Gut-Brain Axis: Probiotics 52
- 53. Neurological and physiological connections that enable the bidirectional communication between the gut and the brain Dietary fiber, prebiotics, and probiotics impact human health and the microbiota Much interest in understanding the complex relationships among diet, the microbiome, and health—identifying and utilizing key foods, nutrients, and microbes to modulate the microbiota for health benefit Summary 53
- 54. Questions?
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- Need picture of GI Tract ~ 1:1 ratio bacteria:human cells ~ 50% of colonic content, feces &gt; 1000 species Varies along GI tract, from lumen to mucosa
- The microbiome has a profound influence on human physiology and nutrition. GI microbes contribute to energy harvest from food Protective Functions Colonization resistance Nutrient competition Secretion of antimicrobials Immune development Structural Functions Mucus layer Tight junctions Intestinal villi and crypts Secretory IgA production
- Pathways involved in bidirectional communication between the gut microbiota and the brain. Multiple potential direct and indirect pathways exist through which the gut microbiota can modulate the gut–brain axis. They include endocrine (cortisol), immune (cytokines) and neural (vagus and enteric nervous system) pathways. The brain recruits these same mechanisms to influence the composition of the gut microbiota, for example, under conditions of stress. The hypothalamus–pituitary– adrenal axis regulates cortisol secretion, and cortisol can affect immune cells (including cytokine secretion) both locally in the gut and systemically. Cortisol can also alter gut permeability and barrier function, and change gut microbiota composition. Conversely, the gut microbiota and probiotic agents can alter the levels of circulating cytokines, and this can have a marked effect on brain function. Both the vagus nerve and modulation of systemic tryptophan levels are strongly implicated in relaying the influence of the gut microbiota to the brain. In addition, short-chain fatty acids (SCFAs) are neuroactive bacterial metabolites of dietary fibres that can also modulate brain and behaviour. Other potential mechanisms by which microbiota affect the brain are described in BOX 1. ACTH, adrenocorticotropic hormone; CRF, corticotropin-releasing factor.
- So far we have highlighted the complexity in micorbiota A bidirectional communication network between the brain and the gastrointestinal tract that includes the central nervous system (brain and spinal cord), the sympathetic (fight or flight response) and parasympathetic (rest and digest) arms of the autonomic nervous system, the neuroendocrine and neuroimmune systems, the enteric nervous system, and the collection of microorganisms and their genomes in the gut habitat.
- Neurotransmitters, also known as chemical messengers, are endogenous chemicals that enable neurotransmission. They transmit signals across a chemical synapse, such as a neuromuscular junction, from one neuron (nerve cell) to another &quot;target&quot; neuron, muscle cell, or gland cell. Neurotransmitters are released from synaptic vesicles in synapses into the synaptic cleft, where they are received by receptors on the target cells. Many neurotransmitters are synthesized from simple and plentiful precursors such as amino acids, which are readily available from the diet and only require a small number of biosynthetic steps for conversion. Neurotransmitters play a major role in shaping everyday life and functions. Their exact numbers are unknown, but more than 100 chemical messengers have been uniquely identified. Figure 1. Pathways involved in bidirectional communication between the gut microbiota and the brain. There are many potential direct and indirect pathways through which the gut microbiota can modulate the gut–brain axis. They include endocrine (cortisol), immune (cytokines), and neural (vagus and enteric nervous system) pathways. The gut microbiota and probiotic agents can alter the levels of circulating cytokines, and this can have a marked effect on brain function. Both the vagus nerve and modulation of systemic tryptophan levels are strongly implicated in relaying the influence of the gut microbiota to the brain. Stress at the level of the CNS can also impact on gut function and lead to perturbations of the microbiota. In addition, short-chain fatty acids (SCFAs) are neuroactive bacterial metabolites of dietary fibers that can also modulate brain and behavior. Harnessing such pathways may provide a novel approach to treat various brain disorders. Neurotransmitters: serotonin, dopamine, norepinephrine, GABA. Adapted from Cryan and Dinan (2015).
- Serotonin is perhaps the best known neurotransmitter. Alterations in serotonin transmission may underlie the pathological symptoms of both GI and some psychiatric disorders, and may explain their high comorbidity. Actually, selective serotonin reuptake inhibitors and tricyclic antidepressants modulating serotonergic neurotransmission, have been shown to be effective in the treatment of both affective and GI disorders such as irritable bowel syndrome (IBS)
- The bidirectional gut–brain axis. Four communication pathways (sensory neurons, cytokines, gut hormones and microbial factors) signal from the gut to the brain where they can modify cerebral function and behaviour. Two pathways (autonomic and neuroendocrine outputs) signal from the brain to the gut. EC, M and L denote different populations of endocrine cells in the gastrointestinal mucosa. The NPY-Y receptor system in the gut–brain axis. The graph shows the major sources of NPY, PYY and PP along the gut–brain axis and the Y receptor subtypes which mediate the effects of these peptides at the different levels of the gut–brain axis. The arrow symbols denote stimulation, the tack symbols denote inhibition.
- The bidirectional gut–brain axis. Four communication pathways (sensory neurons, cytokines, gut hormones and microbial factors) signal from the gut to the brain where they can modify cerebral function and behaviour. Two pathways (autonomic and neuroendocrine outputs) signal from the brain to the gut. EC, M and L denote different populations of endocrine cells in the gastrointestinal mucosa. The NPY-Y receptor system in the gut–brain axis. The graph shows the major sources of NPY, PYY and PP along the gut–brain axis and the Y receptor subtypes which mediate the effects of these peptides at the different levels of the gut–brain axis. The arrow symbols denote stimulation, the tack symbols denote inhibition.
- The bidirectional gut–brain axis. Four communication pathways (sensory neurons, cytokines, gut hormones and microbial factors) signal from the gut to the brain where they can modify cerebral function and behaviour. Two pathways (autonomic and neuroendocrine outputs) signal from the brain to the gut. EC, M and L denote different populations of endocrine cells in the gastrointestinal mucosa. The NPY-Y receptor system in the gut–brain axis. The graph shows the major sources of NPY, PYY and PP along the gut–brain axis and the Y receptor subtypes which mediate the effects of these peptides at the different levels of the gut–brain axis. The arrow symbols denote stimulation, the tack symbols denote inhibition.
- Need picture of brain. Of course NPY is one of many gut peptides that are influenced by microbiota and may influence the brain. A large family hormones released by the gut are known to directly impact the brain and influence subconscious as well as conscious actions in the brain. The gut can also communicate with the brain via hormonal signaling pathways that involve the release of gut peptides from enteroendocrine cells, which can act directly on the brain. Gut peptides, such as ghrelin, gastrin, orexin, pancreatic polypeptide, cholecystokinin, and leptin, modulate feeding behavior, energy homeostasis, circadian rhythm, sexual behavior, arousal, and anxiety. This may have been an evolutionary adaptation to increase activity/movement in service of food seeking behavior. Ghrelin which is released from the upper GI tract under conditions of hunger, reduces both anxiety-like and depression-related behavior (Lutter et al., 2008 and Schellekens et al., 2012). Leptin receptors can be found in limbic structures, and chronic leptin treatment reverses stress-induced behavioral deficits (Lu, Kim, Frazer, & Zhang, 2006), suggesting a potential role for this hormone in emotional processes (Finger, Dinan, & Cryan, 2010). The role of the gut hormonal response in the microbiota-gut-brain cross talk is clearly an area of research that demands more attention and may offer novel therapeutic targets for the brain-gut axis disorders.
- For the final 2 slides for this presentation I would like to talk spend a few minutes talking about the importance of bacterial metabolites, particularly SCFAs and their role in the microbiota-gut-brain-axis.
- SCFAs represent a potent dietary or broadly speaking a behavioral input that may have wide-ranging consequences from acute regulation of feeding behavior as is the case with appetite control and gut motility but chronic energy expenditure as well as anti-inflammatory agents that may prevent carcinogenesis. This is an area of active research.
- Correlation of diet and gut microbial taxa identified in the cross-sectional COMBO analysis. Columns correspond to bacterial taxa quantified using 16S rDNA tags; rows correspond to nutrients measured by dietary questionnaire. Red and blue denote positive and negative association, respectively. The intensity of the colors represents the degree of association between the taxa abundances and nutrients as measured by the Spearman’s correlations. Bacterial phyla are summarized by the color code on the bottom; lower-level taxonomic assignments specified are in fig. S1. The dots indicate the associations that are significant at an FDR of 25%. The FFQ data were used for this comparison (both FFQ and Recall dietary data are shown together in fig. S1). Columns and rows are clustered by Euclidean distance, with rows separated by the predominant
- a, b, Faecal concentrations of SCFAs from carbohydrate (a) and amino acid (b) fermentation (*P &lt; 0.05, two-sided Mann–Whitney U test; n = 9–11 faecal samples per diet arm; Supplementary Table 11). Median bulk bile acid concentrations are shown for all individuals on the plant-based (a) and animal-based (b) diets (error bars denote median absolute deviations). For detailed experimental protocols, see Methods. Bile acid levels did not significantly change on the plant-based diet relative to baseline levels (P &gt; 0.1, Mann–Whitney U test). However, bile acid levels trended upwards on the animal-based diet, rising from 1.48 μmol per 100 mg dry stool during the baseline period to 2.37 μmol per 100 mg dry stool (P &lt; 0.10, Mann–Whitney U test).
- Any open source pictures of toolboxes available?
- Any open source pictures available?
- Any open source pictures available?
- Any open source pictures available?
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