N Brossier is supported by the Francis S. Collins Scholars Program in Neurofibromatosis Clinical and Translational Research funded by the Neurofibromatosis Therapeutic Acceleration Program (NTAP, Grant # 210112). This publication was supported in part by funding from the NTAP at the Johns Hopkins University School of Medicine. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of The Johns Hopkins University School of Medicine. Additional support by the St. Louis Children&#39;s Hospital (FDN-2022-1082 to NMB) and the Washington University in St. Louis Diabetes Research Core (NIH P30 DK020579). Microscopy was performed through the use of the Washington University Center for Cellular Imaging (WUCCI), supported by the Washington University School of Medicine, The Children&#39;s Discovery Institute of Washington University, and St. Louis Children&#39;s Hospital (CDI-CORE-2015-505 and CDI-CORE-2019-813) and the Foundation for Barnes-Jewish Hospital (3770 and 4642). Nestin-CFPnuc35 mice were generously provided by Grigori Enikolopov (Renaissance School of Medicine, Stony Brook University, NY), and Nf1 mice heterozygous for either an R681X or C383X germline mutation32,38,39 were generously provided by David Gutmann (Washington University School of Medicine, St. Louis, MO). Figure 1 was created with BioRender.com.

All animal procedures in this study followed NIH guidelines and were approved by the Institutional Animal Care and Use Committee of Washington University in St. Louis. Animals were housed with standard 12 h light:dark cycling and free access to food and water.
1. Maternal diet
<ol>
	<li>Place female mice on control chow (CD) or obesogenic diet (Ob) at 4 weeks of age.</li>
	<li>At 8-12 weeks of age, set up timed mating by monitoring the mucus plug of the dam in the early morning to determine the correct collection age. To verify diet-induced obesity, weigh females at mating. 
	NOTE: A range of 8-12 weeks was used for the initial mating setup to ensure that the obesogenic diet-fed females were overweight compared to their counterparts, which did not always occur by 8 weeks of age. The day of the plug is considered embryonic day 1 (E1), and all the animals used in this study were collected at E19. The late fetal time point was chosen due to neurodevelopmental changes previously observed at this time in obesogenic diet-exposed animals (data not shown).</li>
</ol>
2. Fetus&#160;removal from the dam
<ol>
	<li>First, ensure that the dam appears gravid (abdominal shape and fullness, abdominal palpation, or fetal movement). If in doubt, weigh the female again to measure weight gain. 
	NOTE: Late-term pregnant mice should display at least 3-4 g of weight gain. To increase the likelihood of conception in the desired timeframe, dirty (urine- and pheromone-containing) sawdust from a male&#39;s cage can be added to the female&#39;s cage daily for 3 days prior to mating to induce estrus34.</li>
	<li>Euthanize the dam by placing it into a chamber with a paper towel containing 3 mL of isoflurane, being careful to avoid direct contact between dam and the anesthetic-soaked paper towel. After the animal is sedated, place it on a paper towel, hold it by the tail, place dissection scissors at the neck, and then force the scissors upward towards the head to perform cervical dislocation. Alternatively, perform decapitation after isoflurane sedation. 
	NOTE: Serum can be collected from the dam at this step, using a similar methodology as described below but with larger capillary tubes. If this is desired, decapitation rather than cervical dislocation should be performed. Care should be taken to ensure that the phenotype of interest is not altered by the administration of isoflurane immediately before euthanasia.</li>
	<li>Place the dam ventral side up in a 100 mm dish. Pinch the skin covering the abdomen and make a midline incision with dissection scissors. Make additional incisions perpendicular to the midline incision to create skin flaps to expose the uterus containing the embryos.</li>
	<li>Pull the uterus out of the abdominal cavity. Using scissors, disconnect the uterus from the dam. Place the intact embryonic sac (&#34;pearls on a string&#34;) containing the embryos into a dish with cold sterile 1x phosphate-buffered saline (PBS). Place the dish of fetuses on ice. 
	NOTE: As each litter typically contains 6-10 pups that will be harvested for both brains and serum, fetuses are kept on ice to avoid tissue degradation. Placing pups on ice additionally ensures adequate euthanasia of pups, as maternal euthanasia alone may not always be sufficient. If only serum collection is desired, room temperature collection may be preferred.</li>
</ol>
3. Fetal serum collection
<ol>
	<li>Remove the fetus from the embryonic sac by carefully dissecting with #5 forceps. Transfer the embryo to a dish using curved forceps with fresh cold sterile 1x PBS.</li>
	<li>Gently blot the animal on a paper towel to remove residual amniotic fluid and PBS from the specimen.</li>
	<li>Hold the animal by the abdomen. Partially decapitate the animal with micro scissors by making an incision at the ventral neck. Make sure the blood vessels are cut, but the skin keeping the head is still attached. 
	NOTE: The head must remain attached so that blood can be collected from the body and head simultaneously.</li>
	<li>Hold the body at a 45&#176; angle with the incision facing down. Hold the 50 &#181;L minivette (capillary tube) parallel to the incision site of the dam where the blood forms a droplet. Carefully allow the 50 &#181;L capillary tube to fill with blood. 
	NOTE: Capillary tube x2 or 100 &#181;L capillary tube can be used to collect up to ~75 &#181;L of blood.</li>
	<li>Place the head in a fresh 60 mm dish containing cold, sterile 1x PBS for further tissue collection (section 4).</li>
	<li>After the blood is within the capillary tube, press the plunger quickly into a 1.5 mL tube. Flick the tube or quickly spin it to make sure the blood is in the bottom of the tube.</li>
	<li>Repeat for the remainder of the litter. 
	NOTE: Keeping the pups on ice and moving quickly between is essential to reduce the risk of postmortem coagulation that will limit collection volume.</li>
	<li>Coagulate the blood at room temperature for 30 min.</li>
	<li>Centrifuge the blood samples at 4 &#176;C, 16,000 &#215; g&#160;for 20 min.</li>
	<li>Transfer the serum to fresh 500 &#181;L tubes with a 200 &#181;L pipette, without disturbing the blood cell pellet at the bottom of the tube. 
	NOTE: Serum should be straw-colored with no red debris. Each fetus should yield 15-30 &#181;L of serum.</li>
	<li>Store the collected serum at -80 &#176;C.</li>
</ol>
4. Fetal&#160;brain collection
<ol>
	<li>Looking through the dissecting microscope, hold the snout using curved forceps and use #5 forceps to carefully peel off the skin from the skull cap, keeping it attached at the snout for ease of holding. Angle #5 forceps to 45&#176; relative to the cranium, carefully puncture the clear membrane that is the developing skull, shift the forceps parallel to the cranium, insert it underneath the skull cap, and then pinch and peel to one side. After one side is removed, grasp the remaining side with #5 forceps and peel to the other side.</li>
	<li>Gently scoop the brain out of the skull using a micro spoon and transfer the brain to a fresh 60 mm dish containing cold 1x PBS. 
	NOTE: At this point, whole brains can be fixed and embedded for immunostaining. See previously published methodology32.</li>
	<li>If tissue must be snap-frozen for other molecular applications, sub-dissect the area of interest. Isolation of the periventricular tissue around the lateral, third, and fourth ventricles is described below.
	<ol>
		<li>Using the curved forceps, hold the brain at the junction of the hindbrain and forebrain with the non-dominant hand. With the dominant hand, hold the scalpel with the blade parallel to the surface of the brain. Press down firmly at the mid-cortical region (Figure 1).</li>
		<li>Still holding the hindbrain, make a second incision in front of the hindbrain to separate it from the forebrain (Figure 1). In the anterior-most section, look for two small semicircular spaces located within the horizontal plane, which are the lateral ventricles. Use the curved forceps placed in the center of each ventricle to hold the brain in place, carefully dissect the lining using #5 forceps, and remove to a fresh clean 1.5 mL microcentrifuge tube.</li>
		<li>In the middle section, look for a small linear space on the ventral surface, which is the third ventricle. Using #5 forceps, carefully pinch off the lining on either side of the ventricular surface, then remove to a fresh clean 1.5 mL microcentrifuge tube.</li>
		<li>In the posterior-most section, look for a small linear space central within the hindbrain, which is the fourth ventricle. Dissect the lining on both sides using #5 forceps, then remove to a fresh clean 1.5 mL microcentrifuge tube. 
		NOTE: If slicing the hindbrain is not a clean cut, the fourth ventricle tissue may be obscured and/or destroyed.</li>
		<li>For steps 4.3.2-4.3.4, immediately after the tissue has been placed into microcentrifuge tubes, place the tubes quickly into a cryo-safe container containing liquid nitrogen. 
		NOTE: Tubes can be kept in liquid nitrogen until the remaining samples have been dissected. CAUTION: Liquid nitrogen can cause severe frostbite. Do not stick hands directly in liquid nitrogen and avoid splashes.</li>
		<li>Place frozen tube(s) on dry ice and transfer frozen tissue into a -80 &#176;C freezer until use.</li>
	</ol>
	</li>
</ol>

Simultaneously Harvesting the Brain and Serum from the Murine Fetus

The authors do not have any conflicts of interest.

Traditional methods for collecting blood from mice include retrobulbar, tail vein, saphenous vein, facial vein, and jugular vein bleeding40,41,42. Unfortunately, these methods are not ideal for embryonic blood collection due to the size of the animal and small, delicate vasculature. Collection of blood via gravity-aided drainage after decapitation led to both low collection volumes and significant hemolysis in our samples. Previ...

Neurofibromatosis Type 1 (NF1) is considered a RASopathy, a group of disorders characterized by germline genetic mutations resulting in activation of the RAS/MAPK (RAt Sarcoma virus/Mitogen-activated Protein Kinase) signaling pathway. Patients with the NF1 RASopathy are at risk for developing many different manifestations, including both benign and malignant tumors of the central (optic pathway glioma1,2, high-grade glioma3,4) and peripheral (plexiform neurofibroma5,6, malignant peripheral nerve sheath tumor7,8) nervous system as well as bony dysplasias9 and skin pigmentary abnormalities10 (axillary freckling, caf&#233;-au-lait macules). The effect of this disorder on cognition and neurodevelopment is increasingly being recognized, with NF1 patients displaying an increased incidence of learning deficits, hyperactivity, and autism spectrum disorder11,12,13. However, there is significant heterogeneity in the development of these phenotypes between patients13,14,15,16,17, and it is unclear why some patients display significant cognitive impairments while others are unaffected. Maternal diet-induced obesity has been shown to similarly affect learning and behavior in the general population18,19,20,21,22,23,24,25,26,27,28, suggesting that differential maternal dietary exposures in NF1 could be one source of this clinical heterogeneity. In particular, children of obese mothers display an increased risk of developing hyperactivity18,19,20,23,25,26, autism19,24,27, executive function deficits21,23, and have lower full-scale IQ scores22,28. However, patients with NF1 have altered metabolic phenotypes compared to the general population, including decreased incidence of obesity and diabetes29,30,31, making it unclear whether they would respond similarly to dietary stimuli.
To address these questions, we wished to determine whether obesogenic-diet-induced changes to the macronutrient profile in fetal offspring with Nf1 contributed to neurodevelopmental changes. We have previously collected high-quality whole and regionally micro-dissected tissue appropriate for neurodevelopmental applications from the fetal brain32. However, fetal blood collection is challenging due to the small body size and low blood volume33. The collection of blood via gravity-aided drainage after decapitation led to low collection volumes and significant hemolysis in our samples, which can affect downstream application interpretation. Collection via aspiration from the fetal heart or thoracic vessels, as has been previously reported33, was technically challenging and also resulted in frequent hemolysis. We thus developed a method for fetal serum collection, which utilizes specialized capillary tubes to allow for higher volume collection without significant shear stress.
Here, we present this method to simultaneously collect embryonic brains and fetal serum from Nf1-heterozygous pups exposed to a high-fat, high-sugar diet versus a control diet in utero (Figure&#160;1 and Supplemental Table S1). Brains were cryo-embedded for subsequent analysis by immunofluorescence or regionally micro-dissected and flash-frozen for subsequent use in molecular biology applications. High-quality serum was obtained, suitable for downstream applications such as macronutrient profiling. Utilizing this method, we identified that maternal high-fat, high-sucrose dietary exposure leads to the elevation of serum cholesterol levels in both WT and Nf1-heterozygous pups.

<table><tbody><tr><td>#5/45 Forceps</td><td>Dumont&amp;nbsp;</td><td>11251-35</td><td>tip shape: angled 45&amp;deg;</td></tr><tr><td>4200 Tapestation</td><td>Agilent</td><td>G2991BA</td><td>Verify RNA integrity and quality, measurement of RIN values</td></tr><tr><td>Benchtop Liquid Nitrogen Container</td><td>Thermo Fisher</td><td>2122</td><td>Or other cryo-safe container</td></tr><tr><td>Control Chow</td><td>PicoLab</td><td>5053</td><td>Research diets D12328 (low-fat, low-sugar) may also be used.</td></tr><tr><td>Curved Forceps</td><td>Cole Parmer</td><td>UX-10818-25</td><td>Tip shape: curved 90&amp;deg;</td></tr><tr><td>Dissecting blade handle</td><td>Cole-Parmer Essentials</td><td>10822-20</td><td>SS Siegel-Type, #10 to #15 blades</td></tr><tr><td>EMS SuperCut Dissection Scissors</td><td>Electron microscope sciences</td><td>72996-01</td><td>5&amp;frac12;&quot; (139.7 mm), Straight</td></tr><tr><td>GFAP Antibody</td><td>Abcam</td><td>ab7260</td><td>Dilute 1:350. Block with 10% serum containing 0.3 M Glycine.</td></tr><tr><td>Glassvan Carbon Steel Surgical Blades, Size 11</td><td>MYCO medical</td><td>2001T-11</td><td>#11 blades allow straight, flat cut</td></tr><tr><td>Micro lab spoon</td><td>Az Scilab</td><td>A2Z-VL001</td><td>stainless steel, autoclavable</td></tr><tr><td>Micro scissors</td><td>Rubis</td><td>78180-1C3</td><td>model 1C300</td></tr><tr><td>Minivette&amp;nbsp;POCT neutral</td><td>Sarstedt</td><td>17.2111.050</td><td>nominal volume: 50 &amp;micro;L, without preparation</td></tr><tr><td>Nanorop</td><td>Thermo Fisher</td><td>13-400-519</td><td>Measure RNA concentration, 260/280 ratios</td></tr><tr><td>Obesogenic diet</td><td>Researchdiets.com</td><td>D12331</td><td>High-fat, high-sucrose</td></tr><tr><td>Total Cholesterol Reagent</td><td>Thermo Fisher</td><td>TR13421</td><td>Colorimetric detection</td></tr><tr><td>&amp;beta;-actin antibody</td><td>Cell Signaling</td><td>8457</td><td>Dilute 1:1,000.</td></tr></tbody></table>

concurrent collection of fetal murine brain and serum to assess effects of maternal diet on nutrition and neurodevelopment in neurofibromatosis type 1

Maternal diet-induced obesity has been demonstrated to alter neurodevelopment in offspring, which may lead to reduced cognitive capacity, hyperactivity, and impairments in social behavior. Patients with the clinically heterogeneous genetic disorder Neurofibromatosis Type 1 (NF1) may present with similar deficits, but it is currently unclear whether environmental factors such as maternal diet influence the development of these phenotypes, and if so, the mechanism by which such an effect would occur. To enable evaluation of how maternal obesogenic diet exposure affects systemic factors relevant to neurodevelopment in NF1, we have developed a method to simultaneously collect non-hemolyzed serum and whole or regionally micro-dissected brains from fetal offspring of murine dams fed a control diet versus a high-fat, high-sucrose diet. Brains were processed for cryosectioning or flash frozen to use for subsequent RNA or protein isolation; the quality of the collected tissue was verified by immunostaining. The quality of the serum was verified by analyzing macronutrient profiles. Using this technique, we have identified that maternal obesogenic diet increases fetal serum cholesterol similarly between WT and Nf1-heterozygous pups.

To illustrate the quality of brain tissue obtained via this technique, we show sample fetal brains from Nestin-CFPnuc mice35, immunostained for GFAP per a previously reported technique32. Nestin+ cells are seen lining the lateral ventricle (Figure 2A), with GFAP+ filaments extending from the surface. We did not observe differences between Nestin or GFAP expression in the lateral ventricle of CD versus Ob-exposed mice in ei...

Read this Scientific Article Protocol about Concurrent Collection of Fetal Murine Brain and Serum to Assess Effects of Maternal Diet on Nutrition and Neurodevelopment in Neurofibromatosis Type 1 at Jo

Author Spotlight: Collecting the Brain and Serum from the Same Mice Fetus to Study Brain Tumor Development

In this protocol, we describe a method for simultaneous collection of fetal brain tissue as well as high-quality, non-hemolyzed serum from the same mouse embryo. We have utilized this technique to interrogate how maternal dietary exposure affects macronutrient profiles and fetal neurodevelopment in mice heterozygous for Nf1 (Neurofibromatosis Type 1).

Concurrent Collection of Fetal Murine Brain and Serum to Assess Effects of Maternal Diet on Nutrition and Neurodevelopment in Neurofibromatosis Type 1

Maternal diet-induced obesity has been demonstrated to alter neurodevelopment in offspring, which may lead to reduced cognitive capacity, hyperactivity, ...

concurrent-collection-fetal-murine-brain-serum-to-assess-effects

Research

JoVE Journal

Genetics

509 Views.  Washington University in St. Louis. Onze groep is geïnteresseerd in zowel de neurologische ontwikkeling als de omgevingsfactoren die van invloed zijn op het al dan niet vormen van hersentumoren bij een kind. We onderzoeken hoe we zowel hersenen als serum van dezelfde embryonale muizen kunnen verkrijgen, zodat we het totale aantal dieren dat nodig is voor een bepaald onderzoek kunnen verminderen. De hersenen stellen ons in staat om de neurologische ontwikkeling te bestuderen, en vervolgens stel...

Video: Auteur Spotlight: de hersenen en het serum van dezelfde muizenfoetus verzamelen om de ontwikkeling van hersentumoren te bestuderen

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory ...

Cancer Research

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

Medicine

Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This is mostly due to the difficulty of microdissection of fetal brains and their sectioning for determination of apoptotic cells caused by prenatal exposure to alcohol. The experiments described here provide visualized techniques from mice breeding to the identification of cell death in fetal brain tissue. This study used C57BL/6 mice as the animal model for studying fetal alcohol exposure and the role of trophic peptide against alcohol-induced apoptosis. The breeding consists of a 2-hr matting window to determine the exact stage of embryonic age. An established fetal alcohol exposure model has been used in this study to determine the effects of prenatal alcohol exposure in fetal brains. This involves free access to alcohol or pair-fed liquid diets as the sole source of nutrients for the pregnant mice.
The techniques involving dissection of fetuses and microdissection of fetal brains are described carefully, since the latter can be challenging. Microdissection requires a stereomicroscope and ultra-fine forceps. Step-by-step procedures for dissecting the fetal brains are provided visually. The fetal brains are dissected from the base of the primordium olfactory bulb to the base of the metencephalon.
For investigating apoptosis, fetal brains are first embedded in gelatin using a peel-away mold to facilitate their sectioning with a vibratome apparatus. Fetal brains embedded and fixed in paraformaldehyde are easily sectioned, and the free floating sections can be mounted in superfrost plus slides for determination of apoptosis or cell death.
TUNEL (TdT-mediated dUTP Nick End Labeling; TdT: terminal deoxynucleotidyl transferase) assay has been used to identify cell death or apoptotic cells. It is noteworthy that apoptosis and cell-mediated cytotoxicity are characterized by DNA fragmentation. Thus, the visualized TUNEL-positive cells are indicative of cell death or apoptotic cells.
The experimental designs here provide information about the use of an established liquid diet for studying the effects of alcohol and the role of neurotrophic peptides during pregnancy in fetal brains. This involves breeding and feeding pregnant mice, microdissecting fetal brains, and determining apoptosis. Together, these visual and textual techniques might be a source for investigating prenatal exposure of harmful agents in fetal brains.

The experimental designs proposed here focus on studying the effects of alcohol exposure in apoptosis and the application of neurotrophic peptide during pregnancy in fetal brain. A detailed description from the breeding to the collection of fetal brains is described. Techniques for determination of apoptosis are also described in detail.

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This ...

Neuroscience

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Developmental Biology

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Immunology and Infection

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

Whole Central and Peripheral Nervous System Mice Dissection

Animal models represent the workhorse of the neuroscience field. Despite this, today, there is still no step-by-step protocol to dissect a complete rodent nervous system, nor is there a complete schematic representing it that is freely available. Only methods to harvest the brain, the spinal cord, a specific dorsal root ganglion, and the sciatic nerve (separately) are available. Here, we provide detailed pictures and a schematic of the central and peripheral murine nervous system. More importantly, we outline a robust procedure to perform its dissection. The 30 min pre-dissection step allows isolating the intact nervous system within the vertebra with muscles free of viscera and skin. A 2-4 h dissection follows it under a micro-dissection microscope to expose the spinal cord and the thoracic nerves, and finally peel the whole central and peripheral nervous system off the carcass. This protocol represents a significant step forward in studying the anatomy and pathophysiology of the nervous system globally. For example, the dissected dorsal root ganglions from a neurofibromatosis type I mice model can be further processed for histology to unravel changes in tumor progression.

Here, we present a protocol to complete the dissection of central and peripheral nervous systems of mice. The dissected tissues are further analyzed in downstream applications, such as taking gross pictures or histology.

Animal models represent the workhorse of the neuroscience field. Despite this, today, there is still no step-by-step protocol to dissect a complete rodent ...

Mouse Neonatal Brain Micro-CT Scanning and Image Processing

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains

Neuroimages are a valuable tool for studying brain morphology in experiments using animal models. Magnetic resonance imaging (MRI) has become the standard method for soft tissues, although its low spatial resolution poses some limits for small animals. Here, we describe a protocol for obtaining high-resolution three-dimensional (3D) information on mouse neonate brains and skulls using micro-computed tomography (micro-CT). The protocol includes those steps needed to dissect the samples, stain and scan the brain, and obtain morphometric measurements of the whole organ and regions of interest (ROIs). Image analysis includes the segmentation of structures and the digitization of point coordinates. In sum, this work shows that the combination of micro-CT and Lugol's solution as a contrast agent is a suitable alternative for imaging the perinatal brains of small animals. This imaging workflow has applications in developmental biology, biomedicine, and other sciences interested in assessing the effect of diverse genetic and environmental factors on brain development.

Author Spotlight: High-Resolution Imaging of Mouse Neonate Brains &#8211; A Micro-CT Protocol with Lugol&#39;s Solution Contrast Agent

This study describes the steps for obtaining high-resolution images of neonatal mouse brains by combining micro-computed tomography (micro-CT) and a contrast agent in ex vivo samples. We describe basic morphometric analyses to quantify brain size and shape in these images.

Neuroimages are a valuable tool for studying brain morphology in experiments using animal models. Magnetic resonance imaging (MRI) has become the standard ...

Concurrent Collection of Fetal Murine Brain and Serum to Assess Effects of Maternal Diet on Nutrition and Neurodevelopment in Neurofibromatosis Type 1

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