This research was supported by the Robert A. and Rene E. Belfer Family Foundation, NIA R21 AG038856 (JLJ), BrightFocus Foundation Alzheimer&#8217;s Disease research grant A2010097 (JLJ), and NIA Biology of Aging Training grant T32 AG000183 (support for SDG).

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	<li>De Vry, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+of+the+central+nervous+system:+a+non-viral+approach+for+targeted+gene+delivery.">In vivo electroporation of the central nervous system: a non-viral approach for targeted gene delivery.</a> Progress in neurobiology. 92, 227-244 (2010).</li><li>Sun, B., Gan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+gene+expression+in+the+central+nervous+system+with+lentiviral+vectors.">Manipulation of gene expression in the central nervous system with lentiviral vectors.</a> Methods Mol Biol. 670, 155-168 (2011).</li><li>Puntel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+rat+brain+using+adenoviral+vectors.">Gene transfer into rat brain using adenoviral vectors.</a> Curr Protoc Neurosci. 4, (2010).</li><li>Passini, M. A., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+gene+delivery+and+structure-specific+patterns+of+expression+in+the+brain+after+intraventricular+injections+of+neonatal+mice+with+an+adeno-associated+virus+vector.">Widespread gene delivery and structure-specific patterns of expression in the brain after intraventricular injections of neonatal mice with an adeno-associated virus vector.</a> J Virol. 75, 12382-12392 (2001).</li><li>Passini, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraventricular+brain+injection+of+adeno-associated+virus+type+1+(AAV1)+in+neonatal+mice+results+in+complementary+patterns+of+neuronal+transduction+to+AAV2+and+total+long-term+correction+of+storage+lesions+in+the+brains+of+beta-glucuronidase-deficient+mice.">Intraventricular brain injection of adeno-associated virus type 1 (AAV1) in neonatal mice results in complementary patterns of neuronal transduction to AAV2 and total long-term correction of storage lesions in the brains of beta-glucuronidase-deficient mice.</a> J Virol. 77, 7034-7040 (2003).</li><li>Kim, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+transduction+of+the+neonatal+brain+delivers+controllable+genetic+mosaicism+for+visualising+and+manipulating+neuronal+circuits+in+vivo.">Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualising and manipulating neuronal circuits in vivo.</a> Eur J Neurosci. 37, 1203-1220 (2013).</li><li>Chakrabarty, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capsid+serotype+and+timing+of+injection+determines+AAV+transduction+in+the+neonatal+mice+brain.">Capsid serotype and timing of injection determines AAV transduction in the neonatal mice brain.</a> PloS one. 8, (2013).</li><li>Croyle, M. A., Cheng, X., Wilson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+formulations+that+enhance+physical+stability+of+viral+vectors+for+gene+therapy.">Development of formulations that enhance physical stability of viral vectors for gene therapy.</a> Gene therapy. 8, 1281-1290 (2001).</li><li>Bauer, H. C., Bauer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+induction+of+the+blood-brain+barrier:+still+an+enigma.">Neural induction of the blood-brain barrier: still an enigma.</a> Cellular and molecular neurobiology. 20, 13-28 (2000).</li><li>Cearley, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanded+repertoire+of+AAV+vector+serotypes+mediate+unique+patterns+of+transduction+in+mouse+brain.">Expanded repertoire of AAV vector serotypes mediate unique patterns of transduction in mouse brain.</a> Molecular therapy : the journal of the American Society of Gene Therapy. 16, 1710-1718 (2008).</li><li>Grimm, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+gene+therapy+vector+evolution+via+multispecies+interbreeding+and+retargeting+of+adeno-associated+viruses.">In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses.</a> J Virol. 82, 5887-5911 (2008).</li></ol>

חוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

שתארנו הליך תכליתי עבור המניפולציה ביטוי גנים עצבי באמצעות AAV ככלי למסירה נרחבת למוח העכבר בילוד. בהשוואה לשיטות אחרות של transgenesis העצבי כגון electroporation ברחם 1 או הזרקה תוך גולגולתי stereotaxic 2,3, הזרקה נגיפית בילוד היא קלה ופשוט יחסית. ההליך הבסיסי יכול להתבצע בתו...

שיטות מסורתיות לשינוי ביטוי גנים עצבי דורשות זמן רב ומניפולציות בתאי מין יקרות. אלטרנטיבי דה נובו גישות כגון electroporation ברחם או הזרקת lentiviral stereotaxic להניב תוצאות מהירות יותר ופחות יקרים, אבל יש לי החסרון של צורך בהתערבות כירורגית מורכבת 1-3. יתר על כן, יש ביטוי transgene מגוון מרחבי מוגבל עם שיטות אלה. בזאת, אנו מתארים שיטה מהירה, קלה, וחסכונית למניפולציה עצבית נרחבת באמצעות הזרקה תוך חדרים של נגיף Adeno הקשורים (AAV) לתוך מוח העכבר בילוד. השיטה תוארה לראשונה על ידי ג&#39;ון וולף ומרקו Passini ב2001, שבו הם הציעו גודל חלקיקים קטן של AAV אפשר לה לפזר בתוך נוזל השדרה מוחין כשהוא עובר מחדרים לרוחב דרך מחסום ependymal לא בשלה ולתוך parenchyma המוח 4, 5. הזרקה תוך חדרים של AAV בתוך first 24 שעות לאחר תשואות לידת התמרה ויראלית נרחבת של תת עצביים הכולל את כל אזור של המוח, מנורות חוש הריח לגזע המוח 6,7. transgenes נמסר-באופן ויראלי בא לידי ביטוי ופעיל בתוך ימים של הזרקה ולהימשך עד שנה לאחר התמרה. לפיכך, מניפולציה תכליתית זה מאפשרת לימודים החל מהתפתחות המוח לאחר לידה מוקדמת להזדקנות וניוון במבוגרים. בהתאמת הטכניקה לצרכי הניסוי הספציפי שלנו, יש לנו התמקדנו בעיקר בserotype AAV8 כי זה הוא יעיל ביותר בtransducing תאי עצב 6. אנו מראים כי כייל נגיף יכול להיות מדוללים כדי לשלוט בצפיפות של נוירונים transduced להשלכות תא פנימי לבדיקת ניסויים של מניפולציה גנטית. בנוסף, אנו מראים כי שני וירוסים יכולים להיות שותף הזריקו לייצר דפוסי ביטוי שמוטים כלפי קבוצות שונות או חופפות של תאי עצב, בהתאם לזנים אל נבחרו לאריזת ויראלי. העבודה שלנו מרחיבה את הרבגוניות של טכניקה זו לשימוש במגוון רחב של הגדרות מדעי מוח ניסיוני.

<table><tbody><tr><td>ICR outbred mice</td><td>Harlan</td><td>Hsd:ICR (CD-1)</td><td>This strain is also known as CD-1</td></tr><tr><td>FVB inbred mice</td><td>The Jackson Laboratory</td><td>1800</td><td>5-6 weeks of age</td></tr><tr><td>Nestlets</td><td>Lab Supply</td><td>NESTLETS</td><td></td></tr><tr><td>Shepherd shacks</td><td>Lab Supply</td><td>SS-mouse</td><td></td></tr><tr><td>High fat rodent chow</td><td>Purina Mills</td><td>PicoLab Mouse diet 20, #5058</td><td>This is our standard breeder chow</td></tr><tr><td>High fat rodent chow (alternative)</td><td>Harlan Laboratories</td><td>Teklad Global 19% protein rodent diet #2019S</td><td>If low phytoestrogen, autoclavable diet is needed</td></tr><tr><td>Injection syringe</td><td>Hamilton</td><td>7653-01</td><td>10 ml syringe</td></tr><tr><td>Injection needles</td><td>Hamilton</td><td>7803-04, RN 6PK PT4, 0.375&quot;</td><td>32 G, for standard P0 injections</td></tr><tr><td>Metal plate for cryoanesthesia</td><td>McMaster Carr</td><td>8975K439</td><td>Raw aluminum plate, 6&amp;rdquo; x 12&amp;rdquo;, 0.25&amp;rdquo; thick, will need to be cut into 3 equal pieces and edges sanded by local machine shop</td></tr><tr><td>Small animal stereotaxic device with digital readout</td><td>David Kopf Instruments</td><td>Model 940</td><td></td></tr><tr><td>Universal syringe holder with needle support foot</td><td>David Kopf Instruments</td><td>Model 1772-F1</td><td></td></tr><tr><td>Neonatal frame</td><td>Stoelting</td><td>51625</td><td>Officially called a mouse and neonatal rat adaptor</td></tr><tr><td>Biohazard disposal bags with sterile indicator</td><td>VWR</td><td>14220-030</td><td>Important! &amp;ndash; Check with local veterinary and environmental safety staff to learn your institute&amp;rsquo;s protocol for biohazard waste disposal</td></tr></tbody></table>

intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction

עם קצב התקדמות מדע בהאצה במהירות, שיטות חדשות יש צורך למדעי מוח ניסיוני לתמרן במהירות ובקלות ביטוי גנים במוח העכבר. כאן אנו מתארים טכניקה הוצגה לראשונה על ידי Passini ווולף למסירה תוך גולגולתי ישירה של transgenes באופן ויראלי בקידוד לתוך מוח העכבר בילוד. בצורתו הבסיסית ביותר שלה, ההליך דורש רק דלי קרח ומזרק microliter. עם זאת, הפרוטוקול יכול גם להיות מותאם לשימוש עם מסגרות stereotaxic כדי לשפר את העקביות לחוקרים חדשים לטכניקה. השיטה מסתמכת על יכולתו של נגיף Adeno הקשורים (AAV) לנוע בחופשיות מחדרי הלב המוחי לparenchyma המוח בזמן שבטנת ependymal היא עדיין לא בשלה במהלך 12-24 לשעה הראשונה שלאחר לידה. הזרקה תוך חדרים של AAV בתוצאות בגיל זה בתמרה נרחבת של תאי עצב במוח. ביטוי מתחיל בתוך ימים של הזרקה ונמשך lifetimדואר של בעלי החיים. כייל נגיף יכול להיות מותאם כדי לשלוט בצפיפות של נוירונים transduced, תוך שיתוף ביטוי של חלבון פלואורסצנטי מספק תווית חיונית של תאי transduced. עם הזמינות העולה של מתקני גרעין נגיפיים לספק חומרים כימיים שני, ארוזים מראש מחוץ למדף והכנה נגיפית מותאם אישית, גישה זו מציעה בזמן שיטה לטיפול בביטוי גנים במוח העכבר שהוא מהיר, קל, והרבה פחות יקר מ הנדסה בתאי מין מסורתית.

תשואות מוצלחות תוך חדרי הזרקה נגיפית ביטוי עצבי נרחב וחזק. כאן אנו הערכנו התמרה ויראלית באמצעות YFP או tdTomato גני ניאון תחת השליטה של ​​אמרגן אקטין בטא עוף (אמרגן CBA). מבנים אלה היו ארוזים לAAV8 ומוזרקים לתוך החדרים לרוחב של עכברים בילוד (P0) ICR. titers הנגיפי גבוה (10 10 חלקי...

Watch this Scientific Journal Video about Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction at JoVE.com

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

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.

הזרקת נגיפי Intracerebroventricular של ילודים עכבר המוח לתמרה עצבית מתמשכת ונרחבת

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

intracerebroventricular-viral-injection-neonatal-mouse-brain-for

Research

JoVE Journal

Neuroscience

58.6K Views.  Baylor College of Medicine. 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.

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

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

אולטרסאונד מונחה Microinjection לתוך forebrain העכבר בתוך הרחם ב E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

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.

מסירה של סוכני טיפולית באמצעות Intracerebroventricular (ICV) ו תוך ורידי (IV) הזרקה של עכברים

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

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

שימוש<em&gt; בvivo</em&gt; הדמיה למעקב אחר ההתקדמות של זיהום ציטומגלווירוס עכבר ניסיוני בילודים

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Immunology and Infection

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

התרחבות של תאי גזע עצביים עובריים ומבוגרים על ידי<em&gt; ברחם</em&gt; Electroporation או הזרקת Stereotaxic ויראלי

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

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.

הזרקת intracerebroventricular ו intravascular של חלקיקים נגיפיים ו פלורסנט microbeads לתוך המוח בילוד

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 ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

ניתוח stereotaxic מניפולציה גנטית בתאים Striatal של מוח העכבר Neonatal

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Targeting the Corticospinal Tract in Neonatal Rats with a Double-Viral Vector using Combined Brain and Spine Surgery

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric spinal cord injuries (SCIs) compared to adult SCIs. In addition, reliably introducing therapies into the target cells of the central nervous system (CNS) can be challenging, and inaccuracies can compromise the efficacy of the study or therapy. This protocol combines viral vector technology with a novel surgical technique&#160;to accurately introduce gene therapies into neonatal rats at postnatal day 5. Here, a virus engineered for retrograde transport (retroAAV2) of Cre is introduced at the axon terminals of corticospinal neurons in the spinal cord, where it is subsequently transported to the cell bodies. A double-floxed&#160;inverted orientation (DIO) designer receptor exclusively activated by designer drug(s) (DREADD) virus is then injected into the somatomotor cortex of the brain. This double-infection technique promotes the expression of the DREADDs only in the co-infected corticospinal tract (CST) neurons.&#160;Thus, the simultaneous co-injection of the somatomotor cortex and cervical CST terminals is a valid method for studying the chemogenetic modulation of recovery following cervical SCI models in neonatal rats.

This protocol demonstrates a novel method for applying gene therapies to subpopulations of cells in neonatal rats at postnatal ages 5-10 days by injecting an anterograde chemogenetic modifier into the somatomotor cortex and a retrogradely transportable Cre recombinase into the cervical spinal cord.

התמקדות במערכת הקורטיקוספינלית בחולדות ילודים עם וקטור דו-ויראלי באמצעות ניתוח משולב של המוח ועמוד השדרה

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

זריקות תוך ורידים בעכברים בילוד

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Biology

פרוטוקול מקיף למתן זריקות תוך-תאיות בעכברי יילודים

Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and treatments. By directly delivering medications and treatments into the cerebrospinal fluid of the central nervous system, this method achieves higher localized drug concentrations while reducing systemic side-effects compared to other routes such as intravenous, subcutaneous, or intramuscular injections. Its importance extends beyond clinical settings, as intrathecal injection plays a vital role in preclinical studies focused on treating neurogenetic disorders in rodents and other large animals, including non-human primates. However, despite its widespread application, intrathecal injection in young, particularly neonatal pups, poses significant technical challenges due to their small size and fragile nature. Successful and reliable administration of intrathecal injections in newborn mice requires meticulous attention to detail and careful consideration of various factors. Thus, there is a crucial need for a standardized protocol that not only provides instructions but also highlights key technical considerations and good laboratory practices to ensure procedural consistency, as well as the safety and welfare of the animals.
To address this unmet need, we present a detailed and comprehensive protocol for performing intrathecal injections specifically in newborn pups on postnatal day 1 (P1). By following the step-by-step instructions, researchers can confidently perform intrathecal injections in neonatal pups, enabling the accurate delivery of drugs, antisense oligos, and viruses for gene replacement or genome editing-based treatments. Furthermore, the importance of adhering to good laboratory practices is emphasized to maintain the well-being of animals and ensure reliable experimental outcomes. This protocol aims to address the technical challenges associated with intrathecal injections in neonatal mice, ultimately facilitating advances in the field of neurogenetic research that aims to develop potential therapeutic interventions.

Author Spotlight: Intrathecal Injection – שיטת העברה יעילה ואמינה לבדיקת יעילות עריכת גנים במוחות עכברים בילודים

The present protocol outlines step-by-step instructions for performing intrathecal injections in neonatal mice for gene editing and drug delivery.

הזרקה תוך-תטית של עכבר שזה עתה נולד לצורך עריכת גנום ואספקת תרופות

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and ...

הזרקת נגיפי Intracerebroventricular של ילודים עכבר המוח לתמרה עצבית מתמשכת ונרחבת

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אולטרסאונד מונחה Microinjection לתוך forebrain העכבר בתוך הרחם ב E9.5

מסירה של סוכני טיפולית באמצעות Intracerebroventricular (ICV) ו תוך ורידי (IV) הזרקה של עכברים

שימוש

התרחבות של תאי גזע עצביים עובריים ומבוגרים על ידי

הזרקת intracerebroventricular ו intravascular של חלקיקים נגיפיים ו פלורסנט microbeads לתוך המוח בילוד

ניתוח stereotaxic מניפולציה גנטית בתאים Striatal של מוח העכבר Neonatal

התמקדות במערכת הקורטיקוספינלית בחולדות ילודים עם וקטור דו-ויראלי באמצעות ניתוח משולב של המוח ועמוד השדרה

זריקות תוך ורידים בעכברים בילוד

הזרקה תוך-תטית של עכבר שזה עתה נולד לצורך עריכת גנום ואספקת תרופות

הזרקה תוך-תטית של עכבר שזה עתה נולד לצורך עריכת גנום ואספקת תרופות