The study was supported by the Swedish Research Council, the Swedish Rheumatism Asociation, Karolinska Institute Foundations, Stockholm County Council, The Wallenberg Foundation, and the GV 80 Years' Foundation for research. The authors would also like to thank Hannah Aucott for proofreading the manuscript.

Alle Tierversuche wurden nach den Richtlinien für die Pflege und Verwendung von Tieren von der lokalen Ethikkommission am Karolinska Institutet, Stockholm genehmigt durchgeführt. Die lokale Ethikkommission folgt den Richtlinien der Europäischen Union auf die Tierpflege. HINWEIS: Die wesentlichen Änderungen aus dem ursprünglichen Protokoll sind die Erholungszeit nach der Operation (6 h gegenüber 1 h) , und die Höhe der LPS injizierten (2 mg / kg im Vergleich zu 15 mg / kg). Andernfalls sich nicht geändert worden sind, die verschiedenen Einstellungen der Operation zusammen. 1. Vorbereiten Werkstoff für Stimulation <ol><li> Schalten Sie den Computer und dem Datenerfassungssystem zu der Stimulationselektrode (1A) verbunden ist . </li><li> Geben Sie das Quittieren Programm. </li><li> Vorbereiten einer Stammlösung von LPS in einer Konzentration von 5 mg / ml in 1X PBS, Aliquot es und lagern bei -20 ° C. Am Tag des Experiments, tauen eine aliquote und bereitet eine adeQuate Probe von LPS (0,5 mg / ml), um etwa 100 &amp; mgr; l in das Tier zu injizieren, nach dem Gewicht. </li></ol> 2. Vorbereitung des Tier für Anästhesie <ol><li> Verwenden Sie C57Bl / 6-Mäuse. Pflegen sie unter klimatisierten Bedingungen mit einer 12-Stunden - Hell / Dunkel - Zyklus, füttern sie Standard - Nagetierfutter, und geben ihnen Wasser ad libitum. </li><li> Führen Sie Operation auf Mäuse, die etwa 25 g am Tag des Experiments wiegen. HINWEIS: Bei Entzündungsreaktionen hervorgerufen werden, ist es besonders wichtig regelmäßige Vorsorgeuntersuchungen und Beobachtungen der klinischen Reaktionen bei den Tieren durchzuführen. Eine vorzeitige Euthanasie durch CO 2 Inhalation erforderlich ist , wenn der Zustand eines Tieres nicht die ethischen Kriterien nicht erfüllt. </li><li> Stellen Sie das Anästhesiegerät. <ol><li> Achten Sie darauf, dass der Schlauch richtig angeschlossen ist und nicht in irgendeiner Weise beschädigt. Achten Sie darauf, dass die belüfteten Bereich richtig funktioniert. Schließen Sie den Schlüssel Filter auf die Flasche Isofluran und füllen das vaporizer mit einer ausreichenden Menge von Isofluran. </li><li> Öffnen Sie die Gaszufuhr (Luft und Sauerstoff) und stellen Sie sicher, dass die Flaschen genug Gas für das Experiment enthalten. Ein Drei-Wege-Verbinder kann dann die Isofluran Strömung zur Induktionskammer oder die Maske zu senden. </li></ol></li><li> Drehen Sie den Drei-Wege-Anschluss mit dem Induktionskammer. Wählen Sie eine Maus aus dem Käfig und setzen Sie das Tier in die Kammer. Stellen Sie den Durchflußmengenregler auf 1,0 l / min Sauerstoff und 1,0 l / min Luft. Stellen Sie die Isofluran-Konzentration auf 4 bis 5%. </li><li> Wenn der gewünschte Grad der Anästhesie erreicht ist, wenn der gewünschte Grad der Anästhesie erreicht ist, schert das Operationsgebiet und das Tier aus der Kammer zu der Maske bewegen. Drehen Sie den Drei-Wege-Anschluss an die Maske fließen. Stellen Sie den Durchflußmengenregler auf 0,25 l / min Sauerstoff und 0,25 L / min Luft. <ol><li> Stellen Sie die Isofluran-Konzentration auf 1,5 bis 2,5%. Überprüfen Sie die Höhe der Anästhesie durch Reflex-Steuerung und Atemfrequenz vor der chirurgischen procedur Ausgangse. </li></ol></li><li> Fix die Beine der Maus auf die Werkbank Klebeband. Achten Sie darauf, dass die Nase des Tieres noch sorgfältig in der Maske positioniert ist. </li></ol> 3. Operation und Stimulation des Nervus vagus <ol><li> Desinfizieren das Operationsgebiet mit 70% Ethanol. </li><li> Unter Verwendung eines Skalpells, einzuschneiden sorgfältig die Haut auf der Höhe des Halses (Inzision von etwa 1 bis 1,5 cm). Hinweis: Im Protokoll endet die Chirurgie Verfahren hier für Sham-Tiere. Tatsächlich hat es sich gezeigt, dass nur mit einem Metallwerkzeug den Nerv berührt es bereits zu einem gewissen Grad stimuliert. Daher ein genaueres Operation Kontrolltier zu erhalten, wenn eine kleine Menge an LPS mit, bei diesem Schritt die Operation stoppen. </li><li> Mit Hilfe eines Mikroskops (12,5-fach-Objektiv), Isolieren des linken Vagusnerv aus der Arteria carotis mittels Dissektionszangen. Zuerst suchen Sie die Kopfnickers von Haut und Fettschichten zu entfernen und zurückziehen sie dann inbestellen die Zange setzen hinter sowohl den Nerv und die Arterie. Anmerkung: Der folgende Schritt sehr schwierig ist, da die Nerven und Arterie eng aneinander geklebt sind. Daher ist es sehr einfach, das Schiff und tötet das Tier zu schneiden. Doch durch die Zange sehr sorgfältig zwischen Nerv und Arterie platziert, sie schließlich zu trennen, und es ist möglich, den Nerv zu isolieren. </li><li> Die Elektrode (1B) , unter dem Vagusnerv. Die Nadelelektrode ist ziemlich lang, so dass selbst wenn der Nerv leicht während der Stimulation bewegt, wird es immer mit der Elektrode in Kontakt sein. </li><li> Führen Sie eine intraperitoneale (ip) Injektion von LPS (2 mg / kg) mit Hilfe einer Spritze (für das Auslesen auf Cytokinspiegel, dh die nach unten regulierenden Wirkung zu messen). </li><li> Warten 5 min vor Beginn der Stimulation. </li><li> Stimulieren des Vagusnervs für 5 min bei 5 V und 1 Hz durch die Starttaste in dem Programm Acknowledge drücken. </li><li> Rebewegen, um die Elektrode und die Wunde vernäht des Tieres mit chirurgischen Nähfadens. </li><li> Sprühen Sie einen No Sting Barrier Film (NSBF) auf der Wunde, um die Heilung zu verbessern und vor Infektionen zu schützen. </li></ol> 4. Wiederherstellung des Tieres <ol><li> Nach der Operation bewegt in seinen Heimkäfig das Tier zurück zum Erwachen und Erholung. Unter Infrarot-Licht, um die Körpertemperatur zu halten, stellen Sie sicher, dass das Tier zu überwachen, bis volles Bewusstsein wiedererlangt hat worden. </li><li> Lassen Sie das Tier für 6 Stunden vor der Tötung zur Analyse in seinem Käfig erholen. Hinweis: Die Wirkung der Vagusnervstimulation ist sehr schnell und hat auch durch den Experimentator festgelegt wird entsprechend die Bedürfnisse der Studie gezeigt wurde , kann seine lang anhaltende (bis zu 48 h), so dass die Recovery - Zeit. </li></ol> 5. Opfer für die weitere Analyse <ol><li> Legen Sie das Tier in einem an eine CO 2 Verabreichungsvorrichtung verbunden Käfig. </li><li> Stellen Sie das Gerät auf eine 5-min Zyklus von CO 2 Inhalation. </li><li> Wenn die Euthanasie durchgeführt wird, die Organe von Interesse sammeln und sie direkt für die weitere Analyse auf Trockeneis einfrieren (zB die Messung von Cytokinspiegel in Milz Extrakten eine Maus TH1 / TH2 9-Plex - Test unter Verwendung) 3. </li></ol>

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Die Autoren haben nichts zu offenbaren.

Seit seiner Entdeckung in den frühen 2000er Jahren wurden die Mechanismen der GAP gründlich untersucht. Wir haben jetzt ein gutes Bild des Weges, und insbesondere das Zielorgan, die Milz, wo NE, Gedächtnis - T - Zellen, Ach, und Makrophagen Arbeit als ein sehr effizientes Team herunterregulieren Entzündungsmediator 2. Wir haben vor kurzem auch Daten über die Bedeutung eines funktionellen Prostaglandinsystems bei Mäusen veröffentlicht, insbesondere PGE 2, die für die ACh - Freis...

Angeborene Immunität sorgt für eine sofortige erste Verteidigungslinie gegen Infektionen und Krankheiten in einem breiten Spektrum von Organismen. Es initiiert nicht nur die primäre Immunantwort die Gefahr zu beseitigen, aber es spielt auch eine zentrale Rolle bei der Aktivierung und die adaptive Immunität zu erziehen, die in einer pathogen-spezifischen Weise sekundäre Immunantworten führen. Die Entzündung wird durch eine Vielzahl von Zytokinen und Chemokinen orchestriert, was wiederum die Fähigkeit haben, andere Immunzellen an den Ort der Infektion zu gewinnen und die kardinalen Zeichen einer Entzündung, wie Rötung, Schwellung, Schmerzen, Verlust der Funktion und Fieber zu induzieren . Die Dauer und die Intensität der Entzündung, hängen von mehreren Faktoren ab, aber die Entzündung zu lösen und die Wiederherstellung der Homöostase ist ein wichtiger Schritt, um die Entstehung von chronischen entzündlichen Erkrankungen zu vermeiden. Jüngste Fortschritte auf dem Gebiet der Neurowissenschaften und Immunologie haben spezifische neuronale Mechanismen mit immensem therapeutischem Potential entwirren Einfl zu steuernammation sowohl im zentralen Nervensystem und in der Peripherie. Einer dieser Mechanismen ist der cholinerge antiinflammatorische Stoffwechselweg (CAP), das auch als Entzündungs reflex bekannt, die durch das autonome Nervensystem 4, 5 angesteuert wird. Es wird derzeit angenommen, dass Entzündungsmediator sensorische Nerven aktivieren und den Zustand der Entzündung auf das zentrale Nervensystem über Signale senden. Ein Reflexantwort wird dann durch die efferente Vagusnerv aktiviert. Eine umfangreiche Studie über die anatomischen Details der Kappe hat ein Parasympathikus-sympathisches Modell zweiere Nerven, den Vagusnerv und splenic Nerven bzw. 6 zusammengesetzt offenbart. In der Kappe endet die efferente cholinerge aktivierten Vagusnerv im celiac-mesenterialen Ganglion, was zur Aktivierung des adrenergen Nerven splenic durch einen Mechanismus noch erforscht werden. Die Milz- Nerven-, so aktiviert wird, um innere bekanntvate in unmittelbarer Nähe zu Immunzellen in der weißen Pulpa, Randzone, und roten Pulpa der Milz, der Haupt- und obligatorischen Organ der Kappe 7, 8. Norepinephrin (NE) aus dem Nervenendigungen splenic bindet an den β 2 -adrenergen Rezeptoren auf Milz - T - Lymphozyten exprimiert entspricht. Dies induziert Cholin - Acetyltransferase (ChAT) -vermittelten Acetylcholin (ACh) -Freisetzung, die wiederum α7 nikotinischen Acetylcholinrezeptoren (α7nACh) auf Makrophagen aktiviert, wodurch die Begrenzung der Cytokinproduktion und Entzündungen 2. Folglich ist es nun klar, dass das Nervensystem in der Lage ist eine Entzündung in den peripheren Geweben zu regulieren und lokale Immunhomöostase wiederherzustellen. Wie der Name des Weges schon sagt, ist das ACh-System von zentraler Bedeutung für das Funktionieren dieses neuro-immun regulierenden Weges. Interessanterweise beteiligten Mechanismen bei der Aktivierung vondie CAP scheint unterschiedlich in der Peripherie und im Zentralnervensystem zu sein. Während die Bedeutung von nikotinischen Rezeptoren (α7nAChR) in der Milz hat früher 9 gezeigt worden sind Muscarin - Rezeptoren (mAChR) zwingend für die zentrale Aktivierung des Weges 10, 11. In jüngerer Zeit, Muscarin periphere Verabreichung eines zentral wirkenden M1 - Agonisten signifikant unterdrückt Serum und Milz Tumornekrosefaktor α (TNF &amp; agr;) während lethal murine Endotoxämie, eine Aktion , die intakte Vagusnerv und splenic Nervensignale 12 erforderlich. Wir haben auch vor kurzem , dass Mäuse ohne Prostaglandin E 2 (PGE 2) waren nicht in der Lage zu reagieren Vagusnervstimulation und nicht herunterregulieren die LPS-induzierte Freisetzung von Zytokinen im Serum und Milz 3 gezeigt. Daher könnte die GAP auch von anderen Systemen als dem Haupt ACh pathw geregelt werdenay. Der Vagusnerv hat als solche wegen seiner Wanderung natürlich im Körper, Innervation Hauptorgane einschließlich der Leber, Lunge, Milz, Nieren und Darm 13 benannt. diese große Innervation und die sehr starke immunsuppressive Wirkung des Nervus vagus In Anbetracht könnte das therapeutische Potenzial der GAP eine Vielzahl von entzündlichen Erkrankungen abdecken. Der Nervus vagus kann elektrisch (oder mechanisch) aktiviert wird, die Kontrolle über die Spannung und die Frequenz, und im Gegensatz zur konventionellen Behandlung, ohne Arzneimittel auf den Körper gegeben. Studien laufen derzeit bei Rheumapatienten, zum Beispiel, die klinische Bedeutung der VNS zu testen , in 14 chronische Entzündung zu behandeln. Insgesamt ist die Neuroimmun-Kommunikation und Regulation der Entzündung derzeit untersucht, die eine mögliche alternative Behandlung zur konventionellen Therapie bieten. Daher Analyse des Nervus vagus stimulation-Effekt in den verschiedenen Organen innerviert, sondern auch die Charakterisierung der potentiellen therapeutischen Wirkung in Tiermodellen der chronischen Entzündung, würde auf jeden Fall Einblicke geben und für neue potenzielle therapeutische Ziele hoffen. Die ursprüngliche Methode , entwickelt von Tracey und Kollegen 4 konnte wegen Reizüberflutung der entzündlichen Reaktion auf unseren Bereich der Forschung umgesetzt werden (durch eine letale Dosis von LPS) und einem zu kurzem Zeitbereich zwischen CAP - Aktivierung und dem Auslesen. In der vorliegenden Arbeit präsentieren wir die auf den ursprünglichen Protokoll vorgenommene Änderungen, vergleichen die beiden unterschiedlichen Methoden auf Cytokinspiegel, und markieren Sie eine neue und entgegengesetzte Beobachtung auf dem Zielorgan (Milz).

<table border="0" cellpadding="0" cellspacing="0" width="665">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Computer</td>
			<td style="width:91px;">Toshiba</td>
			<td style="width:136px;">-</td>
			<td style="width:191px;">Any computer is actually compatible</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">MP-150 data acquisition system</td>
			<td style="width:91px;">Biopac Systems</td>
			<td rowspan="2" style="width:136px;">MP150WSW</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Acknowledge software</td>
			<td style="width:91px;">Biopac Systems</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Mice C57Bl/6</td>
			<td style="width:91px;">Charles River</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Anesthetic machine</td>
			<td style="width:91px;">Simtec Engineering</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Medical oxygen bottle</td>
			<td style="width:91px;">AGA</td>
			<td style="width:136px;">107563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Medical air bottle&#160;</td>
			<td style="width:91px;">AGA</td>
			<td style="width:136px;">108639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Vetflurane (1000mg/g)</td>
			<td style="width:91px;">Virbac</td>
			<td style="width:136px;">137317</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">LPS</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">L2630</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Saline</td>
			<td style="width:91px;">Merck Millipore</td>
			<td style="width:136px;">1024060080</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">PBS 10X</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">P5493</td>
			<td>Diluted 10 times for used concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Syringe (1 ml)</td>
			<td style="width:91px;">BD Plastipak</td>
			<td style="width:136px;">303172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Needles 23G</td>
			<td style="width:91px;">KD-FINE</td>
			<td style="width:136px;">900284</td>
			<td>0.6 x 30 mm (blue)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Microdissecting forceps (curved)</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">F4142</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Dissecting scissors</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">Z265969</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Surgical suture 4-0</td>
			<td style="width:91px;">Ethicon</td>
			<td style="width:136px;">G667G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Euthanasia unit</td>
			<td style="width:91px;">Euthanex Smartbox</td>
			<td style="width:136px;">EA-32000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Cavilon No Sting Barrier Film</td>
			<td style="width:91px;">3M Health Care</td>
			<td style="width:136px;">3346N</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">TH1/TH2 9-Plex assay, ultrasensitive kit</td>
			<td style="width:91px;">MesoScale Discovery</td>
			<td style="width:136px;">K15013C-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

increased recovery time and decreased lps administration to study the vagus nerve stimulation mechanisms in limited inflammatory responses

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). 
The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

Level von TNFa und Interleukin-1β (IL-1β) nach dem Zeitraffer nach der Operation Erhöhung und Verringerung der Dosis von LPS Wie vorher gezeigt, kann das ursprüngliche Protokoll verwenden, verringert VNS die Niveaus von TNF &amp; agr; (169,3 ± 24,9 pg / mg in SHAM gegenüber 39,7 ± 10,8 pg / mg in VNS, p &lt;0,001) und IL-1β (360,0 ± 40,21 pg / mg in SHAM gegenüber 191,7 ± 27,2 pg / mg in VNS, p &lt;0,01) in der Milz na...

Watch this Scientific Journal Video about Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses at JoVE.com

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

Erhöhte Erholzeit und abnehmend LPS-Verabreichung die Vagusnervstimulation Mechanisms in beschränkten Entzündungsreaktionen zu studieren

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

increased-recovery-time-decreased-lps-administration-to-study-vagus

Research

JoVE Journal

Immunology and Infection

9.8K Aufrufe.  Karolinska University Hospital. Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

Serie: Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Differenziert Functional Roles of Gene Expression von Immun-und Nicht-Immunzellen in Mouse Colitis von Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Forschung

Immunologie und Infektion

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Charakterisierung von Entzündungsreaktionen während der intranasalen Kolonisation mit Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

Transplantation von Haut-Schwanz zu allogene CD4-T-Zellantworten in Mäusen untersucht

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Hochdurchsatz-quantitative Echtzeit-RT-PCR-Assay zur Bestimmung Expressionsprofile der Typen I und III Interferon-Formationsglieder

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Die intranasale Verabreichung von rekombinantem Influenza Vaccines in chimären Maus-Modelle zu Mucosale Immunität Studieren

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Intratracheale Verabreichung von<em&gt; Haemophilus influenzae</em&gt; In Mausmodelle zur Untersuchung Atemwegsentzündung

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Retrovirale Transduktion von T-Helferzellen als genetischer Ansatz zur Untersuchung Mechanismen der Kontrolle ihrer Differenzierung und Funktion

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Ein IL-8 transientes transgenisiertes Mausmodell für die<em&gt; In vivo</em&gt; Langzeitüberwachung von entzündlichen Reaktionen

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

Screening-Assays zur Charakterisierung neuartiger endothelialer Regulators der entzündlichen Reaktion beteiligt

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.