Die Autoren danken Jayden Smith für die kritische Prüfung und der Nachweis der Bearbeitung des Manuskripts. Diese Arbeit Unterstützung von der National Multiple Sclerosis Society (NMSS, Teilstipendien RG-4001-A1) erhalten hat, die Italienische Multiple Sklerose Vereinigung (AISM gewähren 2010/R/31), dem italienischen Gesundheitsministerium (GR08-7), Wings for Life, die Banca Agricola Popolare di Ragusa (BAPR), des European Research Council (ERC) im Rahmen des ERC-2010-StG Finanzhilfevereinbarung Nr. 260511-SEM_SEM und die Europäische Gemeinschaft (EG) 7. Rahmenprogramm (FP7/2007-2013) unter Grant Agreement n * deg; 280772 - Ione.

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Die Autoren haben nichts zu offenbaren.

Somatische Stammzelltherapien sind als einer der vielversprechendsten Strategien zur Behandlung von chronisch-entzündlichen ZNS-Erkrankungen wie MS2 11 entstehen. Während die Mechanismen Erhaltung ihrer therapeutischen Wirkungen vollständig aufgeklärt werden müssen, noch hat die wesentlichen Auswirkungen der NPC-Transplantation in verschiedenen experimentellen Modellen neurodegenerativer Erkrankungen Anlass zu der etwas provokanten Überzeugung, dass Zellen können bald in Studien am Menschen angewendet ...

Starke Beweise von in vivo-Studien zur Bestätigung der therapeutischen Wirksamkeit der Transplantation von neuralen somatischen Stammzellen / Vorläuferzellen (NPC) in Tiermodellen für Erkrankungen des ZNS 1-8 entstanden ist. Dennoch, eine Reihe von Fragen im Zusammenhang mit der Lieferung von Stammzellen in das Wirts erfordern eine sorgfältige Überlegung, bevor diese experimentellen Ergebnisse in klinische Anwendungen umgesetzt werden. Eine besonders erhebliche Hürde für die Entwicklung der (nicht-hämatopoetischen) Stammzellen restaurative Therapien für multifokale, chronisch-entzündliche Erkrankungen des Gehirns ist die Identifizierung der idealen Route der NPC-Injektion. Ein Unternehmen Verständnis der Pathophysiologie der Zielerkrankung (fokale oder multifokale; primäre entzündliche oder degenerative) und einer vorsichtigen Analyse der Machbarkeit und Risikofragen mit den Liefer Techniken verbunden sind, sind bei der Ermittlung der optimalen Protokoll für die Stammzell Lieferung. Während der Schwerpunkt ( <em&gt; ZB. in das Nervensystem Parenchym) Stammzell-Transplantation ist ein logischer Ansatz für die Behandlung von ZNS-Erkrankungen, die durch räumlich begrenzten Bereichen von Schäden (z. B. Parkinson-und Huntington-Krankheit, Gehirn und Rückenmark traumatische Verletzungen und Schlaganfall), kann die gleichen Ansatz zu beweisen unter Bedingungen, wie MS, wo eine multifokale, chronische und räumlich verbreitet ZNS-Schädigung akkumuliert im Laufe der Zeit praktisch nicht durchführbar. In diesem letzteren Fall wird Targeting Brennzellinjektionen an einzelne Läsionen auch durch die begrenzte Fähigkeit der transplantierten NPC gehinderte lange Strecken innerhalb des ZNS wandern über Parenchym, so veranlasst die Identifizierung von alternativen, geeigneten Methoden von ZNS-Targeting mit weniger invasiven NPC Transplantationen . Große Versprechen, entstand aus den Beobachtungen, die NPCs Ziel eine intrakranielle Tumor (z. B.. Gliom) bei Mäusen, wenn außerhalb der CNS9 intravasal injiziert. Nach dieser bahnbrechendenIn-vivo-Nachweis der Stammzellen pathotrophism 10, umfangreiche Daten akkumuliert worden sind in Bezug auf die Machbarkeit und die therapeutische Wirksamkeit des systemischen Transplantation von NSC in Labortieren mit experimentellen autoimmunen Enzephalomyelitis (EAE), ein Modell für entzündliche ZNS-Schädigung, entweder über intravenöse (iv) oder intracerebroventricular (ICV). NPC Injektion 1,2,5,6,8 .. Wir haben zuerst gezeigt, dass dies abhängig von der Fähigkeit der transplantierten NPCs zum Ziel und geben Sie die entzündeten ZNS, und anschließend mehrere inter engagieren Kommunikationsprogramme in spezifischen Mikroumgebung in vivo 11. Um speziell auf die ZNS, NPCs sind direkt in die Rückenmarksflüssigkeit (CSF) Zirkulation durch icv Injektion oder in die Blutbahn über iv Injektion verabreicht. Nach Eingabe entweder die Blutbahn oder CSF, transplantiert NPCs interagieren aktiv mitdie Blut-Hirn-(BBB) ​​oder Blut Liquor (BCSFB) Barrieren und geben Sie die ZNS-Parenchym. Diese Wechselwirkung zwischen dem Transplantat und der NPC BBB (oder BCSFB) von bestimmten Satz von NPC Oberfläche Zelladhäsionsmoleküle (CAMs) geregelt und durch die Expression von großen Mengen von CAM Gegenliganden auf aktivierten Endothelzellen / Ependymzellen 12-14 erleichtert. Beispiele für diese Nocken umfassen, die den Rezeptor für Hyaluronat, CD44, und die interzelluläre Adhäsionsmolekül (ICAM) -1 Ligand sehr späten Antigens (VLA) -4 5,15,16 (dh, in Leukozyten, sind verantwortlich für die Interaktion mit aktivierten Ependymzellen und Endothelzellen), und zu einem viel geringeren Ausmaß Lymphozytenfunktion-assoziiertes Antigen (LFA) -1 und P-Selektin-Glykoprotein-Liganden (PSGL) -1. NPCs eine breite Palette von Chemokin-Rezeptoren, einschließlich CCR1, CCR2, CCR5, CXCR3 und CXCR4 Ausdruck auch (aber nicht ausdrücken, CCR3 und CCR7), die funktionell aktiv sind, sowohl in vitro als auch in vivo 5,16. So SYSTEMICALLY injiziert NSC verwenden diese Nocken, zusammen mit G-Protein-gekoppelten Rezeptor (GPCR), die auf Ebene des entzündeten ZNS ansammeln. Umgekehrt injiziert NPCs systemisch in gesunden Mäusen nicht in das ZNS über vaskuläre oder Zerebrospinalflüssigkeit Platz zwei Routen. CNS-Entzündung, oder Endothelzellen / ependymalen Zellaktivierung nach einer systemischen Cytokin oder lypopolisaccharide (LPS) Injektion als Modell für chemisch induzierte Enzephalitis, ist daher für die Akkumulation von systemisch injiziert NSC in das Gehirn und das Rückenmark 2 erforderlich. So erfolgreich Targeting des ZNS mit systemischen Therapien NPC abhängig von der Identifikation eines krankheitsspezifischen Zeitfenster (woo) in das Gehirn und das Rückenmark Umwelt sind förderlich für die Akkumulation und die transendotheliale Migration von NSC. Solche Bedingungen entstehen in der Regel im Rahmen der akuten und subakuten Entzündungs ​​17. Sobald er die CNS eingegeben, transplantierten undifferenzierten NPCsEs ist gezeigt worden, um die klinisch-pathologischen Merkmale von Mäusen als auch größere, nicht-menschlichen Primaten, die mit EAE verbessern. Dies wurde beschrieben abhängig von minimalen Zellenwechsel 2 und bemerkenswerte Sekretion von immunregulatorische und neuroprotektive parakrine Faktoren innerhalb perivaskuläre CNS 2,5,6,18 vs nicht-entzündeten Bereichen ZNS 19,20 (zB Lymphknoten) in Reaktion auf das zu sein, Entzündungszellsignalisierung durch Infiltration Immunzellen 5 ausgelöst. Hier beschreiben wir die wichtigsten methodischen Aspekte der systemischen Injektion von somatischen NPCs in einem Mausmodell der chronischen EAE. Genauer gesagt, definieren wir die Protokolle, die wir haben, um (i) gegründet ableiten, erweitern und die Vorbereitungen für die Transplantation somatischer NPCs aus dem Subventrikularzone (SVZ) der erwachsenen C57BL / 6 Mäusen; (Ii) induziert chronische EAE in solchen Mäusen und (iii) durchzuführen therapeutisch wirksame systemische (iv oder ICV) NPC Transplantation into EAE Mäusen.

<table border="0" cellpadding="0" cellspacing="0" width="1474">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:753px;">Cell culture</td>
			<td style="width:300px;"></td>
			<td style="width:149px;"></td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EBSS</td>
			<td>Sigma</td>
			<td>E2888</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Cystein</td>
			<td>SIGMA-ALDRICH CO LTD</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papain</td>
			<td>WORTHINGTON</td>
			<td>30H11965</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Fisher scientific</td>
			<td>D/0700/50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse NeuroCult basal medium</td>
			<td>Stem Cell technologies</td>
			<td>05700</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NeuroCult proliferation supplements</td>
			<td>Stem Cell technologies</td>
			<td>05701</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Basic fibroblast growth factor</td>
			<td>Peprotech</td>
			<td>100-18B-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epidermal growth factor</td>
			<td>Peprotech</td>
			<td>AF-100-15-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td>1514012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel (coating solution)</td>
			<td>BD biosciences</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NeuroCult&#174; Differentiation Kit (Mouse)</td>
			<td>Stem cell technologies</td>
			<td>05704</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accumax</td>
			<td>eBioscience</td>
			<td>00-4666-56</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s PBS (DPBS) (10x) without Ca&#38; Mg</td>
			<td>PAA LABORATORIES LTD</td>
			<td>H15-011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Myco trace</td>
			<td>PAA LABORATORIES LTD</td>
			<td>Q052-020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide (DMSO)</td>
			<td>SIGMA</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">immunofluorescence</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal goat serum</td>
			<td>PAA LABORATORIES LTD</td>
			<td>B11-035</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether</td>
			<td>SIGMA-ALDRICH CO LTD</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti Nestin</td>
			<td>Abcam</td>
			<td>ab11306</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti GFAP</td>
			<td>DAKO</td>
			<td>203344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti Histone H3 (phospho S10)&#160;</td>
			<td>Abcam</td>
			<td>ab14955</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti MAP-2</td>
			<td>Abcam</td>
			<td>ab32454</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat anti MBP</td>
			<td>AbD SEROTEC</td>
			<td>MCA409S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-O4 Antibody, clone 81 | MAB345</td>
			<td>Millipore</td>
			<td>MAB345</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mounting solution</td>
			<td>DAKO</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EAE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Freund&#39;s Adjuvant Incomplete</td>
			<td>SIGMA-ALDRICH CO LTD</td>
			<td>F5506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mycobacterium tuberculosis&#160;</td>
			<td>DIFCO</td>
			<td>H37Ra</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MOG(35&#8211;55)&#160;</td>
			<td>Espikem</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pertussis toxin</td>
			<td>List Biological Laboratories</td>
			<td>181</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue processing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iris scissor straight</td>
			<td>Fine Sciences Tolls</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Blunt/bended forceps</td>
			<td>Fine Sciences Tolls</td>
			<td>11080-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Brain slicer</td>
			<td>Zivic instruments</td>
			<td>BSMAS005-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical blades</td>
			<td>Swann-Morton</td>
			<td>324</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P200, P1000 pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamine (Vetalar)</td>
			<td style="width:300px;">Boehringer Ingelheim</td>
			<td>01LC0030&#160;&#160;</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylazine (Rompun)</td>
			<td>Bayer</td>
			<td>32371</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereotaxic frame</td>
			<td>KOPF</td>
			<td>Model 900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hamilton syringe</td>
			<td>Hamilton</td>
			<td>7762-04</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde (PFA)</td>
			<td>SIGMA</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VECTASTAIN Elite ABC Kit</td>
			<td>vector laboratories</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
	</tbody>
</table>

systemic injection of neural stem/progenitor cells in mice with chronic eae

Neuronale Stammzellen / Vorläuferzellen (NPCs) sind eine vielversprechende Quelle für die Stammzelltransplantation Ansätze mit dem Ziel Gehirn Reparatur oder Wiederherstellung in der regenerativen Neurologie. Diese Richtlinie wurde von der umfangreichen Beweise dafür, dass Gehirn Reparatur nach fokaler oder systemische NPC Transplantation in mehreren präklinischen Modellen von neurologischen Erkrankungen erreicht entstanden. Diese experimentellen Daten wurden die Zellabgabe Route als eine der wichtigsten Hürden der restaurativen Stammzellen Therapien für Erkrankungen des Gehirns, die dringende Beurteilung erfordert identifiziert. DruckmessungVentrikuläre Stammzellentransplantation stellt einen logischen Ansatz zu diesen Pathologien gekennzeichnet durch isolierte und zugänglich Hirnläsionen wie Rückenmarksverletzungen und der Parkinson-Krankheit. Leider ist dieses Prinzip schlecht für Zustände, die durch eine multifokale, entzündliche und verbreitet (sowohl in Zeit und Raum) Art, einschließlich multipler Sklerose (MS). Als solche Gehirn-Targeting durch systemic NPC Lieferung hat sich zu einem niedrigen invasive und therapeutisch wirksame Protokoll zu den Zellen in das Gehirn und Rückenmark von Nagern und Primaten durch experimentelle chronisch entzündliche Schädigung des zentralen Nervensystems (ZNS) betroffen zu liefern. Dieses alternative Verfahren der Zellabgabe beruht auf der NPC pathotropism, insbesondere ihre angeborene Fähigkeit, (i) Erfassen der Umwelt über funktionelle Zelladhäsionsmoleküle und inflammatorische Zytokin und Chemokin-Rezeptoren; (Ii) überqueren Sie die undichte anatomische Barrieren nach intravenöser (iv.) Oder intracerebroventricular (ICV) Injektion; (Iii) sammeln sich an der Höhe des perivaskulären mehreren Stelle (n) von entzündlichen Hirn-und Rückenmarksverletzungen; und (iv) ausüben bemerkenswerte trophischen Gewebe-und Immun regulatorische Effekte auf verschiedenen Host-Zielzellen in vivo. Hier beschreiben wir die Methoden, die wir für die iv entwickelt. und &lt;em&gt; ICV Lieferung von syngenen NPCs in Mäusen mit experimentellen autoimmunen Enzephalomyelitis (EAE), als Modell der chronischen entzündlichen ZNS-Demyelinisierung und sehen die systemische Stammzell Lieferung als wertvolle Technik für die selektive Ausrichtung der entzündeten Gehirn in der regenerativen Neurologie.

NPC Ableitung und Charakterisierung SVZ Dissektionen an Pools von 6-8 Wochen alte C57BL / 6 Mäuse durch mechanische und enzymatische Spaltung (Fig. 1A) (n = 5-7 Mäuse / Pool). Nach ein paar Tagen der Kultivierung in CGM, beginnen frei schwebenden Neurosphären bilden (1A und 1B). Primäre Kugeln werden gesammelt und mechanisch agiert alle 4-5 DIV. Nach der Passage werden Zahlen von lebenden und toten Zellen ermittelt und kumulative Zellzahlen aufgetragen, um ei...

Watch this Scientific Journal Video about Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE at JoVE.com

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Die Transplantation von neuronalen Stammzellen / Vorläuferzellen (NPCs) hält große Versprechungen in der regenerativen Neurologie. Die systemische Verabreichung von NPCs ist in effektive, invasiv und therapeutisch sehr wirksam Protokoll wandte sich Stammzellen im Gehirn und Rückenmark von Nagern und Primaten durch experimentelle chronisch entzündliche Schädigung des zentralen Nervensystems betroffen zu liefern.

Die systemische Injektion von Neural Stem / Progenitor-Zellen in Mäusen mit chronischer EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

systemic-injection-neural-stemprogenitor-cells-mice-with-chronic

Research

JoVE Journal

Immunology and Infection

17.5K Aufrufe.  University of Cambridge, UK.  Die Transplantation von neuronalen Stammzellen / Vorläuferzellen (NPCs) hält große Versprechungen in der regenerativen Neurologie. Die systemische Verabreichung von NPCs ist in effektive, invasiv und therapeutisch sehr wirksam Protokoll wandte sich Stammzellen im Gehirn und Rückenmark von Nagern und Primaten durch experimentelle chronisch entzündliche Schädigung des zentralen Nervensystems betroffen zu liefern. 

Serie: Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

Identifizierung von DNA-Mutationen in hämatopoetischen Stammzellen Gereinigtes / Vorläuferzellen

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Forschung

Immunologie und Infektion

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Langzeitchronik<em&gt; Pseudomonas aeruginosa</em&gt; Airway-Infektion bei Mäusen

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

Myelin Oligodendrozyten Glykoprotein (MOG<sub&gt; 35-55</sub&gt;) Induzierten experimentellen autoimmunen Enzephalomyelitis (EAE) in C57BL / 6 Mäuse

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Einem Mausmodell für Pathogen-induzierten chronischen Entzündung an lokalen und systemischen Seiten

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Die phänotypische und funktionale Analyse von aktivierten regulatorischen T-Zellen isoliert von chronischer lymphatischer Choriomeningitisvirus-infizierte Mäuse

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Analyse von Lymphozyten-Extravasation einer Verwendung<em&gt; In Vitro</em&gt; Modell des Barrier Menschliches Blut-Hirn

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable dry eye animal model to test novel therapeutics to treat autoimmune dry eye conditions. This protocol describes a chronic autoimmune dry eye rat model. Lewis rats were immunized with an emulsion containing lacrimal gland extract, ovalbumin, and complete Freund&#39;s adjuvant. A second immunization with the same antigens in incomplete Freund&#39;s adjuvant was administered two weeks later. These immunizations were administered subcutaneously at the base of the tail. To boost the immune response at the ocular surface and lacrimal glands, lacrimal gland extract and ovalbumin were injected into the forniceal subconjunctiva and lacrimal glands 6 weeks after the first immunization. The rats developed dry eye features, including reduced tear production, decreased tear stability, and increased corneal damage. Immune profiling by flow cytometry showed a preponderance of CD3+ effector memory T cells in the eyeball.

This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later.

Ein chronisches Autoimmun-Trockenaugen-Rattenmodell mit Zunahme im Effekt-Speicher T-Zellen in Augapfel-Gewebe

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Adoptive Immuntherapie von iNKT-Zellen in Glucose-6-Phosphat-Isomerase (G6PI)-Induzierte RA-Mäuse

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to draining lymph nodes (LNs). Interruption of lymph flow is an important method when studying the function of lymphatic vessels. The afferent lymphatic vessels from the murine footpad to the popliteal lymph nodes (pLNs) are well-defined as the only routes for lymph drainage into the pLNs. Suturing these afferent lymphatic vessels can selectively prevent lymph flow to the pLNs. This method allows for interference in lymph flow with minimal damage to the lymphatic endothelial cells in the draining pLN, the afferent lymphatic vessels, as well as other lymphatic vessels around the area. This method has been used to study how lymph impacts high endothelial venules (HEV) and chemokine expression in the LN, and how lymph flows through the adipose tissue surrounding the LN in the absence of functional lymphatic vessels. With the growing recognition of the importance of lymphatic function, this method will have broader applications to further unravel the function of lymphatic vessels in regulating the LN microenvironment and immune responses.

A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented.

Blockierung des Lymphflusses durch Suieren von afferenten Lymphgefäßen bei Mäusen

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to ...

A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice

Intrathymic injection in mouse models is an important technique for studying thymic and immune function, including genetic and acquired T cell disorders. This requires methods for the direct deposition of reagents and/or cells into the thymus of living mice. Traditional methods of intrathymic injection include thoracic surgery or minimally invasive percutaneous blind injections, both of which have significant limitations. Ultra-high frequency ultrasound imaging devices have made image-guided percutaneous injections possible in mice, greatly improving the injection accuracy of the percutaneous injection approach and enabling the injection of smaller targets. However, image-guided injections rely on the utilization of an integrated rail system, making this a rigid and time-consuming procedure. A unique, safe, and efficient method for percutaneous intrathymic injections in mice is presented here, eliminating reliance on the rail system for injections. The technique relies on using a high-resolution micro-ultrasound unit to image the mouse thymus noninvasively. Using a free-hand technique, a radiologist can place a needle tip directly into the mouse thymus under sonographic guidance. Mice are cleaned and anesthetized before imaging. For an experienced radiologist adept at ultrasound-guided procedures, the learning period for the stated technique is quite short, typically within one session. The method has a low morbidity and mortality rate for the mice and is much faster than current mechanically assisted techniques for percutaneous injection. It allows the investigator to efficiently perform precise and reliable percutaneous injections of thymuses of any size (including very small organs such as the thymus of aged or immunodeficient mice) with minimal stress on the animal. This method enables the injection of individual lobes if desired and facilitates large-scale experiments due to the time-saving nature of the procedure.

The present protocol describes an interventional radiology procedure established for intrathymic injection in mice to avoid the risk of open surgery and improve the accuracy of blind percutaneous injections.

Eine minimal-invasive, genaue und effiziente Technik zur intrathymischen Injektion bei Mäusen

Intrathymic injection in mouse models is an important technique for studying thymic and immune function, including genetic and acquired T cell disorders. ...