We acknowledge the Laboratory Animal Center staff at National Cheng Kung University (NCKU) for caring for the animals. This work was supported by the scholarship from Prof. Kun-Yen Huang Education Fund of CHENG-HSING Medical Foundation to C.-W.L.; the funds from the Ministry of Science and Technology (MOST) in Taiwan: (Undergraduate Research MOST 109-2813-C-006-095-B) to T.-H.Y.; (MOST 107-2320-B-006-072-MY3; 109-2314-B-006-046; 110-2314-B-006-114; 110-2320-B-006-018-MY3) to W.-L.W.; and the Higher Education Sprout Project, Ministry of Education to the Headquarters of University Advancement at NCKU to W.-L.W.

The authors have no conflicts of interest related to this work.

Gut-derived metabolites have been associated with brain-mediated diseases without much precise mechanism, partially due to their multiple binding sites in the body6,12,24. Previous reports indicated that SCFAs could serve as ligands for G protein-coupled receptors, epigenetic regulators, and sources for energy production at multiple sites in the body6,12. To bypass the c...

The human gastrointestinal tract harbors diverse microorganisms impacting the host1,2,3. These gut bacteria can secrete gut-derived metabolites during their utilization of dietary components consumed by the host4,5. Interestingly, the gut metabolites not metabolized in the periphery can be transported to other organs via circulation6. Of note, these secreted metabolites can serve as mediators for the gut-brain axis, defined as the bidirectional communication between the central nervous system and the gut7. Previous studies have shown that gut-derived metabolites can modulate complex behavior and emotion in animals8,9,10,11.
Short-chain fatty acids (SCFAs) are the main metabolites produced by gut microbiota during the fermentation of dietary fiber and indigestible carbohydrates6. Acetate, propionate, and butyrate are the most abundant SCFAs in the gut12. SCFAs serve as the energy source for cells in the gastrointestinal tract. Unmetabolized SCFAs in the gut can be transported to the brain through the portal vein, thus modulating brain and behavior6,12. Previous studies have suggested that SCFAs might play a critical role in neuropsychiatric disorders6,12. For example, intraperitoneal injection of butyrate in BTBR T+ Itpr3tf/J (BTBR) mice, an animal model of autism spectrum disorder (ASD), rescued their social deficits13. Antibiotic-treated rats receiving microbiota from depressive subjects showed an increase in anxiety-like behaviors and fecal SCFAs14. Clinically, alterations in fecal SCFAs levels were observed in people with ASD compared to typically developing controls15,16. People with depression have lower fecal SCFAs levels than healthy subjects17,18. These studies suggested that SCFAs can alter behavior in animals and humans through various routes.
Microbial metabolites exert diverse effects on multiple sites in the body, impacting host physiology and behaviors4,19, including the gastrointestinal tract, vagus nerve, and sympathetic nerve. It is difficult to pinpoint the precise role of gut-derived metabolites in the brain when administering the metabolites via peripheral routes. This paper presents a video-based protocol to investigate the effects of gut-derived metabolites in the brain of a freely moving mouse (Figure 1). We showed that SCFAs could be acutely given through the guide cannula during behavioral tests. The type, volume, and infusion rate of metabolites can be modified depending on the purpose. The site of cannulization can be adjusted to explore the impact of gut metabolites in a specific brain region. We aim to provide scientists with a method to explore the potential impact of gut-derived microbial metabolites on the brain and behavior.

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Advil Liqui-Gels Solubilized Ibuprofen A2:D41</td><td>Pfizer</td><td>n/a</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-rabbit</td><td>ThermoFisher Scientific</td><td>A-21206</td><td></td></tr><tr><td>Anti-Fluorescent Gold (rabbit polyclonal)</td><td>Millipore</td><td>AB153-I</td><td></td></tr><tr><td>Bottle Top Vacuum Filter, 500 mL, 0.22 &amp;mu;m, PES, Sterile</td><td>NEST</td><td>121921LA01</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C1016</td><td>ACSF: 0.14 g/L</td></tr><tr><td>Chlorhexidine scrub 2%</td><td>Phoenix</td><td>NDC 57319-611-09</td><td></td></tr><tr><td>Chlorhexidine solution</td><td>Phoenix</td><td>NDC 57319-599-09</td><td></td></tr><tr><td>Commercial dummy</td><td>RWD Life Science</td><td>62004</td><td>Single_OD 0.20 mm/ M3.5/G = 0.5 mm</td></tr><tr><td>Commercial guide cannul</td><td>RWD Life Science</td><td>62104</td><td>Single_OD 0.41 mm-27G/ M3.5/C = 2.5 mm&amp;nbsp;</td></tr><tr><td>Commercial injector</td><td>RWD Life Science</td><td>62204</td><td>Single_OD 0.21 mm-33G/ Mates with M3.5/C = 3.5 mm/G = 0.5 mm</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td>ACSF: 0.61 g/L</td></tr><tr><td>Dental acrylic</td><td>HYGENIC</td><td>n/a</td><td></td></tr><tr><td>Fixing screws</td><td>RWD Life Science</td><td>62521</td><td></td></tr><tr><td>Fluoroshield mounting medium with DAPI</td><td>Abcam</td><td>AB104139</td><td></td></tr><tr><td>Horse serum</td><td>ThermoFisher Scientific</td><td>16050130</td><td></td></tr><tr><td>Insulin syringes</td><td>BBraun</td><td>XG-LBB-9151133S-1BX</td><td>1 mL</td></tr><tr><td>Isoflurane&amp;nbsp;</td><td>Panion &amp;amp; BF biotech</td><td>DG-4900-250D</td><td></td></tr><tr><td>KCl&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P3911</td><td>ACSF: 0.19 g/L</td></tr><tr><td>Ketoprofen&amp;nbsp;</td><td>Swiss Pharmaceutical</td><td>n/a</td><td></td></tr><tr><td>Lidocaine&amp;nbsp;</td><td>AstraZeneca</td><td>n/a</td><td></td></tr><tr><td>Low melting point agarose</td><td>Invitrogen</td><td>16520</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M8266</td><td>ACSF: 0.19 g/L</td></tr><tr><td>Microscope cover slips</td><td>MARIENFELD</td><td>101242</td><td></td></tr><tr><td>Microscope slides</td><td>ThermoFisher Scientific</td><td>4951PLUS-001E</td><td></td></tr><tr><td>Mineral oil light, white NF</td><td>Macron Fine Chemicals</td><td>MA-6358-04</td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S9888</td><td>ACSF: 7.46 g/L</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO4&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S8282</td><td>ACSF: 0.18 g/L</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S5761</td><td>ACSF: 1.76 g/L</td></tr><tr><td>n-butyl cyanoacrylate adhesive (tissue adhesive glue)</td><td>3M</td><td>1469SB</td><td>3M Vetbond</td></tr><tr><td>Neural tracer&amp;nbsp;</td><td>Santa Cruz</td><td>SC-358883</td><td>FluoroGold</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Polyethylene tube</td><td>RWD Life Science</td><td>62329</td><td>OD 1.50, I.D 0.50 mm and OD 1.09, I.D 0.38 mm</td></tr><tr><td>Puralube Vet (eye) Ointment</td><td>Dechra&amp;nbsp;</td><td>12920060</td><td></td></tr><tr><td>Sodium acetate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S2889</td><td>SCFAs: 13.5 mM</td></tr><tr><td>Sodium azide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sodium butyrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>B5887</td><td>SCFAs: 8 mM</td></tr><tr><td>Sodium propionate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P1880</td><td>SCFAs: 5.18 mM</td></tr><tr><td>Stainless guide cannula</td><td>Chun Ta stainless steel enterprise CO., LTD.</td><td>n/a</td><td>OD 0.63 mm; Local vendor</td></tr><tr><td>Stainless injector</td><td>Chun Ta stainless steel enterprise CO., LTD.</td><td>n/a</td><td>OD 0.3 mm; dummy is made from injector; local vendor</td></tr><tr><td>Superglue</td><td>Krazy Glue</td><td>KG94548R</td><td></td></tr><tr><td>Triton X-100</td><td>Merck</td><td>1.08603.1000</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cannula holder</td><td>RWD Life Science</td><td>B485-68217</td><td></td></tr><tr><td>Ceiling camera</td><td>FOSCAM</td><td>R2</td><td></td></tr><tr><td>Digital stereotaxic instruments</td><td>Stoelting</td><td>51730D</td><td></td></tr><tr><td>Dissecting microscope</td><td>INNOVIEW</td><td>SEM-HT/TW</td><td></td></tr><tr><td>Glass Bead Sterilizer</td><td>RWD Life Science</td><td>RS1501</td><td></td></tr><tr><td>Heating pad</td><td>Stoelting</td><td>53800M</td><td></td></tr><tr><td>Leica microscope&amp;nbsp;</td><td>Leica</td><td>DM2500</td><td></td></tr><tr><td>Micro Dissecting Forceps</td><td>ROBOZ</td><td>RS-5136</td><td>Serrated, Slight Curve; Extra Delicate; 0.5mm Tip Width; 4&quot; Length&amp;nbsp;</td></tr><tr><td>Micro Dissecting Scissors</td><td>ROBOZ</td><td>RS-5918</td><td>4.5&quot; Angled Sharp</td></tr><tr><td>Microinjection controller</td><td>World Precision Instruments (WPI)</td><td>MICRO2T</td><td>SMARTouch Controller</td></tr><tr><td>Microinjection syringe pump</td><td>World Precision Instruments (WPI)</td><td>UMP3T-1</td><td>UltraMicroPump3&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Microliter syringe</td><td>Hamilton</td><td>80014</td><td>10 &amp;micro;L</td></tr><tr><td>Optical Fiber Cold Light with double Fiber</td><td>Step</td><td>LGY-150</td><td>Local vendor</td></tr><tr><td>Pet trimmer</td><td>WAHL</td><td>09962-2018</td><td></td></tr><tr><td>Vaporiser for Isoflurane</td><td>Step</td><td>AS-01</td><td>Local vendor</td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000S</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Animal behavior video tracking software</td><td>Noldus</td><td>EthoVision</td><td>Version: 15.0.1416</td></tr><tr><td>Leica Application Suite X software</td><td>Leica</td><td>LASX</td><td>Version: 3.7.2.22383</td></tr></tbody></table>

intracerebroventricular delivery of gut-derived microbial metabolites in freely moving mice

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have revealed that gut microbiota-derived metabolites modulate brain-mediated physiological functions through intricate gut-brain pathways in the host. Short-chain fatty acids (SCFAs) are the major bacteria-derived metabolites produced during dietary fiber fermentation by the gut microbiome. Secreted SCFAs from the gut can act at multiple sites in the periphery, affecting the immune, endocrine, and neural responses due to the vast distribution of SCFAs receptors. Therefore, it is challenging to differentiate the central and peripheral effects of SCFAs through oral and intraperitoneal administration of SCFAs. This paper presents a video-based method to interrogate the functional role of SCFAs in the brain via a guide cannula in freely moving mice. The amount and type of SCFAs in the brain can be adjusted by controlling the infusion volume and rate. This method can provide scientists with a way to appreciate the role of gut-derived metabolites in the brain.

The mouse was infused with SCFAs 1 week after recovery from the guide cannula implantation to evaluate locomotor activity in a novel cage. The mouse was placed in a novel cage and infused with 2,100 nL of SCFAs or ACSF in the first 5 min (delivery rate of 7 nL/s) into the brain through the commercial guide cannula implanted in the lateral ventricle of the brain. The locomotor activity in a novel cage was recorded for an additional 30 min after infusion. No difference was observed in the locomotor activity in the novel ca...

Watch this Scientific Journal Video about Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice at JoVE.com

Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice

Gut-derived microbial metabolites have multifaceted effects leading to complex behavior in animals. We aim to provide a step-by-step method to delineate the effects of gut-derived microbial metabolites in the brain via intracerebroventricular delivery via a guide cannula.

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have ...

intracerebroventricular-delivery-gut-derived-microbial-metabolites

Research

JoVE Journal

Neuroscience

3.1K Views.  National Cheng Kung University. De metabolieten die vrijkomen door darmmicrobiota produceren complexe effecten op de gastheer. Dit protocol presenteert een methode om de rol van darmmetabolieten in de hersenen en het gedrag van de muis te ondervragen. Deze techniek biedt een nauwkeurige, zorgvuldige en gevalideerde methode om darmmetabolieten in een brein af te leveren bij vrij bewegende muizen.We bieden een uitgebreide en fysiologische beste methodologie voor inzicht in darmhersenasonderzoek. Plaats de muis op het s...

Video: Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

Medicine

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Immunology and Infection

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

Probiotic Studies in Neonatal Mice Using Gavage

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.

This study details the process of gavaging precise amounts of probiotics to neonatal mice. The experimental set-up was optimized to include but is not limited to probiotic dosing, methods of administration, and quantification of bacteria in the intestines.

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult ...

Murine Fecal Isolation and Microbiota Transplantation

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal microbiota transplantation (FMT) is a valuable approach to delineating a direct role of the total microbiota or isolated species in disease pathophysiology. It is a safe treatment option for patients with recurrent Clostridium difficile infection. Preclinical studies demonstrate that manipulating the gut microbiota is a useful tool to study the mechanistic link between dysbiosis and disease. Fecal microbiota transplantation may help elucidate novel gut microbiota-targeted therapeutics for the management and treatment of cardiometabolic disease. Despite a high success rate in rodents, there remains translational changes associated with the transplantation. The goal here is to provide guidance in studying the effects of gut microbiome in experimental cardiovascular disease. In this study, a detailed protocol for the collection, handling, processing, and transplantation of fecal microbiota in murine studies is described. The collection and processing steps are described for both human and rodent donors. Lastly, we describe using a combination of the Swiss-rolling and immunostaining techniques to assess gut-specific morphology and integrity changes in cardiovascular disease and related gut microbiota mechanisms.

The goal here is to outline a protocol to investigate the mechanisms of dysbiosis in cardiovascular disease. This paper discusses how to aseptically collect and transplant murine fecal samples, isolate intestines, and use the "Swiss-roll" method, followed by immunostaining techniques to interrogate changes in the gastrointestinal tract.

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal ...

Improved Blood Sampling Technique to Assess Gut Microbial Metabolites in Mice

Sequential Blood Collection from Inferior Vena Cava Followed by Portal Vein to Evaluate Gut Microbial Metabolites in Mice

Gut microbial products are known to act both locally within the intestine and get absorbed into circulation, where their effects can extend to numerous distant organ systems. Short-chain fatty acids (SCFA) are one class of metabolites produced by gut microbes during the fermentation of indigestible dietary fiber. They are now recognized&#160;as important contributors to how the gut microbiome influences extra-intestinal organ systems via the gut-lung, gut-brain, and other gut-organ axes throughout the host. SCFAs are absorbed from the colon, through intestinal tissue,&#160;into the portal vein (PV). They then pass through the liver, and are consumed in various organs such as the brain, muscle, adipose tissue, and lungs. SCFAs are most easily measured in the expelled fecal material however, more accurate measurements have been obtained from intra-colonic fecal contents. Here we propose that sampling PV and systemic circulating plasma of a single subject may be preferable for studying the&#160;absorption, transport, and systemic levels of SCFAs in mice. We present a new technique for efficient blood sampling from the PV and inferior vena cava (IVC) that allows for the collection of relatively large volumes of blood from the portal and systemic circulations. This is accomplished by ligating the PV, thereby allowing&#160;for the dilation or enlargement of the PV as it backfills from the mesenteric veins that drain into it. Using this method, we were able to improve the rate of successful collection as well as the total amount of blood collected (up to 0.3 mL from IVC and 0.5 mL from PV).

Author Spotlight: Gut Microbiome-Lung Tissue Communication Through Short-Chain Fatty Acid Analysis

The protocol demonstrates a method to collect blood from portal veins and inferior vena cava from mice sequentially to evaluate the production and absorption of gut microbial metabolites.

Gut microbial products are known to act both locally within the intestine and get absorbed into circulation, where their effects can extend to numerous ...

Studying Gut-Brain Interaction via Transcranial Calcium Imaging in Mice

Real-time Analysis of Gut-brain Neural Communication: Cortex wide Calcium Dynamics in Response to Intestinal Glucose Stimulation

Communication between the gastrointestinal tract and the brain after nutrient absorption plays an essential role in food preference, metabolism, and feeding behaviors. Particularly concerning specific nutrients, many studies have elucidated that the assimilation of glucose within gut epithelial cells instigates the activation of many signaling molecules. Hormones such as glucagon-like peptide-1 are renowned as quintessential signaling mediators. Since hormones predominantly influence the brain through circulatory pathways, they slowly modulate brain activity.
However, recent studies have shown two expeditious gut-brain pathways facilitated by the autonomic nervous system. One operates via the spinal afferent neural pathway, while the vagus nerve mediates the other. Consequently, brain responses following glucose assimilation in the gastrointestinal tract are complicated. Moreover, as intestinal stimulation finally induces diverse cortical activities, including sensory, nociceptive, reward, and motor responses, it is necessary to employ methodologies that facilitate the visualization of localized brain circuits and pan-cortical activities to comprehend gut-brain neural transmission fully. Some studies have indicated precipitous alterations in calcium ion (Ca2+) concentrations within the hypothalamus and ventral tegmental area independently through different pathways after intestinal stimulation. However, whether there are changes in cerebral cortex activity has not been known.
To observe cerebral cortex activity after intragastric glucose injection, we developed an imaging technique for real-time visualization of cortex wide Ca2+ dynamics through a fully intact skull, using transgenic mice expressing genetically encoded Ca2+ indicators. This study presents a comprehensive protocol for a technique designed to monitor intestinal stimulation-induced transcranial cortex wide Ca2+ imaging following intragastric glucose injection via an implanted catheter. The preliminary data suggest that administering glucose solution into the gut activates the frontal cortex, which remains unresponsive to water administration.

Gut-brain communication, facilitated by the vagus nerve, is crucial for communication between the gastrointestinal endocrine system and the brain. However, whether intragastric glucose injection can change cortical activity is still not understood. Here, we offer a comprehensive protocol to observe changes in cortical activity after glucose injection into the duodenum.

Communication between the gastrointestinal tract and the brain after nutrient absorption plays an essential role in food preference, metabolism, and ...

An Intracerebroventricular Injection to Analyze Murine Cytomegalovirus Distribution in a Neonatal Mouse Brain

Source: Kawasaki, H., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54164">Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain.</a> J. Vis. Exp. (2016).
This video demonstrates the intracerebroventricular injection process for studying murine cytomegalovirus (MCMV) distribution in the neonatal mouse brain. MCMV, engineered to express an enhanced green fluorescent protein (EGFP) for visibility, is injected into the lateral ventricle of a mouse pup&#39;s brain. The injected virus circulates through the cerebrospinal fluid, infecting cells in the periventricular region, including the marginal zone, choroid plexus, and subventricular zone.

1. ICV Injection of MCMV and Fluorescent Microbeads into Neonatal Mice

	Maintain normal pregnant ICR mice in a temperature-controlled facility under a 12 ...

Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice

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