The authors thank Jayden Smith for critically reviewing and proof editing the manuscript. This work has received support from the National Multiple Sclerosis Society (NMSS, partial grants RG-4001-A1), the Italian Multiple Sclerosis Association (AISM, grant 2010/R/31), the Italian Ministry of Health (GR08-7), Wings for Life, Banca Agricola Popolare di Ragusa (BAPR), the European Research Council (ERC) under the ERC- 2010-StG Grant agreement no&#160;260511-SEM_SEM and the European Community (EC) 7th Framework Program (FP7/2007-2013) under Grant Agreement n*deg; 280772- iONE.

<ol>
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The authors have nothing to disclose.

Somatic stem cell based therapies are emerging as one of the most promising strategies for treating chronic inflammatory CNS disorders such as MS211. While the mechanisms sustaining their therapeutic effects still need to be completely elucidated, the significant impact of NPC transplantation in different experimental models of neurodegenerative diseases has given rise to the somewhat provocative belief that stem cells may soon be applied into human studies. However, before envisaging any potential human appli...

Strong evidence has arisen from in vivo studies attesting to the therapeutic efficacy of the transplantation of somatic neural stem/precursor cells (NPCs) in animal models of CNS disorders1-8. Nevertheless, a number of issues relating to the delivery of stem cells into the host require careful consideration before these experimental results can be translated into clinical applications. A particularly substantial hurdle towards the development of (nonhematopoietic) restorative stem cell therapies for multifocal, chronic inflammatory brain diseases is the identification of the ideal route of NPC injection. A firm understanding of the pathophysiology of the targeted disease (focal or multifocal; primary inflammatory or primary degenerative), and a cautious analysis of feasibility and risk issues associated with the delivery techniques are in identifying the optimal protocol for stem cell delivery.
While the focal (e.g. into the nervous system parenchyma) stem cell transplantation is a logical approach to the treatment of CNS diseases characterized by spatially confined areas of damage (e.g. Parkinson's and Huntington's disease, brain and spinal cord traumatic injuries, and stroke), the very same approach may prove to be practically not feasible in conditions such as MS, where a multifocal, chronic, and spatially disseminated CNS damage accumulates over time. In this latter case, targeting focal cell injections to individual lesions is also hindered by the limited capacity of transplanted NPCs to migrate over long distances within the CNS parenchyma, thus prompting the identification of alternative, more suitable methods of CNS targeting with less invasive NPC transplants.
Great promise emerged from the observations that NPCs target an intracranial tumor (e.g. glioma) in mice when injected intravascularly outside the CNS9. Following this seminal in vivo evidence of the stem cell pathotrophism10, extensive data have been accumulated pertaining to the feasibility and therapeutic efficacy of the systemic transplantation of NPCs in laboratory animals with experimental autoimmune encephalomyelitis (EAE), as a model of inflammatory CNS damage, via either intravenous (i.v.) or intracerebroventricular (i.c.v.) NPC injection1,2,5,6,8.. We have first shown that this is dependent on the capability of transplanted NPCs to target and enter the inflamed CNS, and to subsequently engage multiple intercellular communications programs within specific microenvironments in vivo11. In order to specifically target the CNS, NPCs are delivered directly into the cerebrospinal fluid (CSF) circulation by i.c.v. injection, or into the bloodstream via i.v. injection. Once entering either the bloodstream or CSF, transplanted NPCs actively interact with the blood brain (BBB) or blood cerebrospinal fluid (BCSFB) barriers and enter the CNS parenchyma. This interaction between the NPC graft and the BBB (or BCSFB) is regulated by specific set of NPC surface cell adhesion molecules (CAMs) and facilitated by the expression of high levels of CAM counter-ligands on activated endothelial/ependymal cells12-14. Examples of these CAMs include the receptor for hyaluronate, CD44, and the intercellular adhesion molecule (ICAM)-1 ligand very late antigen (VLA)-45,15,16 (that, in leukocytes, are responsible of the interaction with activated ependymal and endothelial cells), and to a much lower extent Lymphocyte function-associated antigen (LFA)-1 and P-selectin glycoprotein ligand (PSGL)-1. NPCs also express a wide range of chemokine receptors, including CCR1, CCR2, CCR5, CXCR3, and CXCR4 (but do not express CCR3 and CCR7), which are functionally active, both in vitro and in vivo5,16. Thus, systemically injected NPCs use these CAMs, along with G-protein coupled receptor (GPCRs), to accumulate at the level of the inflamed CNS. Conversely, NPCs injected systemically into healthy mice do not enter the CNS via vascular or cerebrospinal fluid space routes2. CNS inflammation, or endothelial/ependymal cell activation following systemic cytokine or lypopolisaccharide (LPS) injection as a model of chemically induced encephalitis, is therefore necessary for the accumulation of systemically injected NPCs into the brain and spinal cord2. Thus, successful targeting of the CNS with systemic NPC therapies is dependent on the identification of a disease specific window of Opportunity (WoO) in which the brain and spinal cord environment are conducive to the accumulation and transendothelial migration of NPCs. Such conditions generally arise in the context of acute and subacute inflammation17. Once having entered the CNS, transplanted undifferentiated NPCs have been shown to ameliorate the clinico-pathological features of mice as well as larger, nonhuman primates with EAE. This has been described to be dependent from minimal cell replacement2 and remarkable secretion of immune regulatory and neuroprotective paracrine factors within perivascular CNS2,5,6,18 vs non-CNS inflamed areas19,20 (e.g. lymph nodes) in response to the inflammatory cell signaling elicited by infiltrating immune cells5.
Herein we describe the key methodological aspects of the systemic injection of somatic NPCs into a mouse model of chronic EAE. More specifically, we define the protocols that we have established to (i) derive, expand and prepare for transplantation somatic NPCs from the subventricular zone (SVZ) of adult C57BL/6 mice; (ii) induce chronic EAE in such mice and (iii) perform therapeutically efficacious systemic (i.v. or i.c.v) NPC transplantation into EAE mice.

<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

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

NPC derivation and characterization 
SVZ dissections are performed on pools (n= 5-7 mice/pool) of 6-8 week old C57Bl/6 mice by means of mechanical and enzymatic dissociation (Figure 1A). After a few days of culturing in CGM, free-floating neurospheres begin forming (Figures 1A and 1B). Primary spheres are collected and mechanically passaged every 4-5 DIV. Upon passaging, numbers of live and dead cells are ascertained and cumulative cell numbers plotted to generate a...

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

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

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

17.6K Views.  University of Cambridge, UK. The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

Video: 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.

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

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.

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.

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.

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.

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.

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.

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 ...

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.

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.

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

Quantifying NET Formation Using Dual-Dye Live Cell Analysis

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

Author Spotlight: Quantifying Neutrophil Extracellular Traps in Disease and Drug Screening Using Dual-Color Live-Cell Imaging

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.