Name: intratracheal instillation of stem cells in term neonatal rats
Uploaded: 2020-05-04T14:30:00.000+00:00
Duration: 4 min 27 s
Description: Watch this Scientific Journal Video about Intratracheal Instillation of Stem Cells in Term Neonatal Rats at JoVE.com

This study was partly supported by a grant from Meridigen Biotech Co., Ltd. Taipei, Taiwan (A-109-008).

This procedure was approved by the Animal Care and Use Committee at Taipei Medical University.
NOTE: Human MSCs stably transfected with green fluorescent protein (GFP) and firefly luciferase genes (Fluc) were obtained from a commercial company (Table of Materials).
1. Characterization of human MSCs with firefly luciferase and green fluorescent protein
<ol>
	<li>Maintain human MSCs transfected with GFP and Fluc in complete media (minimum essential medium eagle-alpha modification [&#945;MEM], supplemented with 10&#8722;15% fetal bovine serum [FBS], 2 mM L-glutamine, 1 ng/mL basic FGF, and PSF) at 37 &#176;C with saturated humidity and 5% CO2. Passage cells at ~70&#8722;90% confluence.</li>
	<li>Observe MSCs under a fluorescence phase contrast microscope (Figure 1A) and analyze the expression levels of Fluc and GFP14.</li>
	<li>Characterize the MSCs by analyzing the expression of CD markers including CD44, CD73, CD90, CD105 using flow cytometry (Figure 1B). Induce trilineage differentiation of stem cells to adipocytes, chondrocytes, and osteocytes, and confirm trilineage differentiation (Figure 1C) by von Kossa, oil red O, and Alcian blue staining following a commercial protocol15,16.</li>
</ol>
2. Anesthetization of rat pups
<ol>
	<li>Allow time-dated pregnant Sprague&#8211;Dawley rats to deliver vaginally at term.</li>
	<li>Remove the rat pups from the cage on postnatal day 5.</li>
	<li>Anesthetize the rat pups using gas anesthesia (i.e., 2% isoflurane) in an anesthesia chamber.</li>
	<li>Confirm adequate anesthesia by checking breathing and reflexes. 
	NOTE: Breathing should become shallow and reflexes should diminish. Rat pups remain unconscious for at least ~10 min with this isoflurane concentration.</li>
</ol>
3. Intratracheal instillation
<ol>
	<li>Once anesthetized, restrain the rat pups on an intubation stand angled at ~60&#176; and hold pups in place with laboratory labeling tape on all four limbs.</li>
	<li>Apply tape below the nose to fix the head and pinpoint the neck for puncture tracheotomy.</li>
	<li>Disinfect the incision area (i.e., neck) with a 75% alcohol prep pad.</li>
	<li>Make a 0.3 cm vertical midline neck incision above the trachea with microscissors to avoid damaging the carotid arteries.</li>
	<li>Dissect the fat and muscle layers away to locate the trachea with curved tip tapered tweezers without a hook.</li>
	<li>Hold the trachea with the curved tip tapered tweezers.</li>
	<li>Hold a 100 &#956;L syringe upright and slowly inject 30 &#956;L of normal saline (i.e., control) or 30 &#956;L of Fluc-GFP labeled MSCs (1 x 105 cells) into the trachea through a 30 G needle syringe during inspiratory phase.</li>
	<li>Close the incision with one 6-0 silk stitch, tie the knot as small as possible, and cut the ends as short as possible.</li>
	<li>Place the rats under infrared light or on a heating pad to keep warm and allow them to recover from anesthesia.</li>
	<li>Confirm the rats are warm, pink, and capable of spontaneous movement before returning the rats to the cage.</li>
</ol>
4. Monitoring the pulmonary distribution of MSCs
<ol>
	<li>To track the transplanted human MSCs, intraperitoneally inject the rats with luciferin potassium salt in phosphate buffered saline (PBS) at a dose of 125 mg/kg of body weight 15 min after MSC injection.</li>
	<li>Anesthetize the rats using 2% isoflurane through nose cones.</li>
	<li>Acquire sequential images at 5&#8211;15 s intervals 10 min after luciferin administration with medium binning, 1 f/stop, and a 26 cm field of view using a small-animal imaging system (Table of Materials).</li>
	<li>Quantify the luminescence activity from the lungs based on the automatic regions of interest using imaging software (Table of Materials)17.</li>
</ol>

<ol>
	<li>Ramanathan, R., Bhatia, J. J., Sekar, K., Ernst, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+in+preterm+infants+with+respiratory+distress+syndrome+treated+with+poractant+alfa,+calfactant+or+beractant:+a+retrospective+study.">Mortality in preterm infants with respiratory distress syndrome treated with poractant alfa, calfactant or beractant: a retrospective study.</a> Journal of Perinatology. 33, 119-125 (2013).</li><li>Gien, J., Kinsella, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+treatment+of+bronchopulmonary+dysplasia.">Pathogenesis and treatment of bronchopulmonary dysplasia.</a> Current Opinion in Pediatrics. 23, 305-313 (2011).</li><li>Pasha, A. B., Chen, X. Q., Zhou, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchopulmonary+dysplasia:+Pathogenesis+and+treatment.">Bronchopulmonary dysplasia: Pathogenesis and treatment.</a> Experimental and Therapeutic. 16, 4315-4321 (2018).</li><li>Committee on Fetus and Newborn. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+corticosteroids+to+treat+or+prevent+chronic+lung+disease+in+preterm+infants.">Postnatal corticosteroids to treat or prevent chronic lung disease in preterm infants.</a> Pediatrics. 109, 330-338 (2002).</li><li>Watterberg, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+Academy+of+Pediatrics;+Committee+on+Fetus+and+Newborn.+Policy+statement-postnatal+corticosteroids+to+prevent+or+treat+bronchopulmonary+dysplasia.">American Academy of Pediatrics; Committee on Fetus and Newborn. Policy statement-postnatal corticosteroids to prevent or treat bronchopulmonary dysplasia.</a> Pediatrics. 126, 800-808 (2010).</li><li>Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marrow+stromal+cells+as+stem+cells+for+nonhematopoietic+tissues.">Marrow stromal cells as stem cells for nonhematopoietic tissues.</a> Science. 276, 71-74 (1997).</li><li>Nemeth, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+attenuate+sepsis+via+prostaglandin+E(2)-+dependent+reprogramming+of+host+macrophages+to+increase+their+interleukin-10+production.">Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)- dependent reprogramming of host macrophages to increase their interleukin-10 production.</a> Nature Medicine. 15 (2), 42-49 (2009).</li><li>Chou, H. C., Li, Y. T., Chen, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cells+attenuate+experimental+bronchopulmonary+dysplasia+induced+by+perinatal+inflammation+and+hyperoxia.">Human mesenchymal stem cells attenuate experimental bronchopulmonary dysplasia induced by perinatal inflammation and hyperoxia.</a> American Journal of Translational Research. 8, 342-353 (2016).</li><li>Chen, C. M., Chou, H. C., Lin, W., Tseng, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactant+effects+on+the+viability+and+function+of+human+mesenchymal+stem+cells:+in+vitro+and+in+vivo+assessment.">Surfactant effects on the viability and function of human mesenchymal stem cells: in vitro and in vivo assessment.</a> Stem Cell Research &amp; Therapy. 8, 180 (2017).</li><li>Kim, Y. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratracheal+transplantation+of+mesenchymal+stem+cells+simultaneously+attenuates+both+lung+and+brain+injuries+in+hyperoxic+newborn+rats.">Intratracheal transplantation of mesenchymal stem cells simultaneously attenuates both lung and brain injuries in hyperoxic newborn rats.</a> Pediatric Research. 80, 415-424 (2016).</li><li>Fleurence, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+efficacy+of+intratracheal+adeno-associated+virus+administration+to+newborn+rats.">Comparative efficacy of intratracheal adeno-associated virus administration to newborn rats.</a> Human Gene Therapy. 16, 1298-1306 (2005).</li><li>Waszak, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+intratracheal+adenoviral+vector+administration+on+lung+development+in+newborn+rats.">Effect of intratracheal adenoviral vector administration on lung development in newborn rats.</a> Human Gene Therapy. 13, 1873-1885 (2002).</li><li>O'Reilly, M., Th&#233;baud, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+bronchopulmonary+dysplasia.+The+term+rat+models.">Animal models of bronchopulmonary dysplasia. The term rat models.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 307, 948 (2014).</li><li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP+labeling+and+hepatic+differentiation+potential+of+human+placenta-derived+mesenchymal+stem+cells.">GFP labeling and hepatic differentiation potential of human placenta-derived mesenchymal stem cells.</a> Cellular Physiology and Biochemistry. 35, 2299-2308 (2015).</li><li>Dominici, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8, 315-317 (2006).</li><li>Zhang, Z. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superior+osteogenic+capacity+for+bone+tissue+engineering+of+fetal+compared+with+perinatal+and+adult+mesenchymal+stem+cells.">Superior osteogenic capacity for bone tissue engineering of fetal compared with perinatal and adult mesenchymal stem cells.</a> Stem Cells. 27, 126-137 (2009).</li><li>Huang, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+surfactant+and+budesonide+on+pulmonary+distribution+of+fluorescent+dye+in+mice.">Effect of surfactant and budesonide on pulmonary distribution of fluorescent dye in mice.</a> Pediatric and Neonatology. 56, 19-24 (2015).</li><li>Keyaerts, M., Caveliers, V., Lahoutte, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging:+looking+beyond+the+light.">Bioluminescence imaging: looking beyond the light.</a> Trends in Molecular Medicine. 18, 164-172 (2012).</li><li>Yeh, T. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-tracheal+administration+of+budesonide/surfactant+to+prevent+bronchopulmonary+dysplasia.">Intra-tracheal administration of budesonide/surfactant to prevent bronchopulmonary dysplasia.</a> American Journal of Respiratory and Critical Care Medicine. 193, 86-95 (2016).</li><li>Nguyen, J. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratracheal+inoculation+of+Fischer+344+rats+with+Francisella+tularensis.">Intratracheal inoculation of Fischer 344 rats with Francisella tularensis.</a> Journal of Visualized Experiments. (127), e56123 (2017).</li><li>de Almeida, P. E., van Rappard, J. R., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+for+tracking+cell+fate+and+function.">In vivo bioluminescence for tracking cell fate and function.</a> American Journal of Physiology-Heart and Circulatory Physiology. 301, 663-671 (2011).</li><li>Kim, J. E., Kalimuthu, S., Ahn, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cell+tracking+with+bioluminescence+imaging.">In vivo cell tracking with bioluminescence imaging.</a> Nuclear Medicine and Molecular Imaging. 49, 3-10 (2011).</li></ol>

The authors have nothing to disclose.

Newborn infants with respiratory distress commonly require intratracheal surfactant and/or corticosteroid treatment19. Human phase I clinical trials have demonstrated the safety of intratracheal MSCs in preterm infants8. These studies suggest that intratracheal administration of drugs is an important option for newborn infants with respiratory distress. Animal model studies are most helpful if the model features are directly pertinent to humans. Term...

Supplemental oxygen is often required to treat newborn infants with respiratory distress1. However, hyperoxia therapy in infants has adverse long-term effects. Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD)2. BPD is a major complication of hyperoxia treatment that can occur in spite of early surfactant therapy, optimal ventilation procedures, and increased use of noninvasive positive pressure ventilation in premature infants. While many treatment strategies have been reported for BPD3, no known therapy can reduce this complication.
Corticosteroid use is one potential treatment to prevent BPD, because pulmonary inflammation plays a crucial role in its pathogenesis. However, systemic corticosteroid therapy is not usually recommended for preterm infants due to long-term adverse effects4,5.
Mesenchymal stromal cells (MSCs) have pluripotent characteristics and can differentiate into various cell types, including bone, cartilage, adipose tissue, muscle, and tendons6. MSCs have immunomodulatory, anti-inflammatory, and regenerative effects7, and animal studies show the therapeutic benefits of MSCs and their secreted components in hyperoxia-induced lung injury in rodents8,9. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and corticosteroid therapy might be a potential treatment strategy to treat newborns with respiratory disorders. Preclinical studies have used intratracheal administration of stem cells and adeno-associated virus in newborn rats10,11,12. However, a step-by-step presentation of the technique and in vivo tracking of the transplanted stem cells is not available. The newborn rat is appropriate for studying the effects of intratracheal administration on preterm infants with respiratory distress because the saccular stage of the rat lung at birth is equivalent to that of the human lung at 26&#8722;28 week of gestation13. An effective method for administration into the rat trachea is crucial for successful pulmonary distribution. The technique presented here allows for the study of intratracheal administration of cells and/or drugs for treatment of neonatal pulmonary diseases using rats as a model for humans.

<table><tbody><tr><td>6-0 silk</td><td>Ethicon</td><td>1916G</td><td></td></tr><tr><td>Alcohol Prep Pad</td><td>CSD</td><td>3032</td><td></td></tr><tr><td>BD Stemflow hMSC Analysis Kit</td><td>BD Biosciences</td><td>562245</td><td>CD markers</td></tr><tr><td>CMV-Luciferase-EF1&amp;alpha;-copGFP BLIV 2.0 Lentivector for In Vivo Imaging</td><td>SBI</td><td>BLIV511PA-1</td><td></td></tr><tr><td>CryoStor10</td><td>BioLife Solutions</td><td>640222</td><td></td></tr><tr><td>Human MSCs</td><td>Meridigen Biotech Co., Ltd. Taipei, Taiwan</td><td></td><td></td></tr><tr><td>Infrared light</td><td>JING SHANG</td><td>JS300T</td><td></td></tr><tr><td>Isoflurane</td><td>Halocarbon</td><td>26675-46-7</td><td></td></tr><tr><td>IVIS-200 small animal imaging system</td><td>Caliper LifeSciences, Hopkinton, MA</td><td></td><td></td></tr><tr><td>Luciferin potassium salt</td><td>Promega, Madison, WI</td><td></td><td></td></tr><tr><td>Micro-scissors, straight</td><td>Vannas</td><td>H4240</td><td></td></tr><tr><td>Normal saline</td><td>TAIWAN BIOTECH CO., LTD.</td><td>113531</td><td>Isotonic Sodium Chloride Solution</td></tr><tr><td>Small Hub RN Needle, 30 gauge</td><td>Hamilton Company, Reno, NV</td><td>7799-06</td><td></td></tr><tr><td>Syringe (100 &amp;micro;l)</td><td>Hamilton Company, Reno, NV</td><td>81065</td><td></td></tr><tr><td>Xenogen Living Image 2.5 software</td><td>Caliper LifeSciences, Hopkinton, MA</td><td>N/A</td><td></td></tr></tbody></table>

intratracheal instillation of stem cells in term neonatal rats

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26&#8722;28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

The pulmonary distribution of intratracheal instillation of stem cells in the term neonatal rats was determined by firefly luciferase (Fluc)-labeled stem cells. MSCs were labeled with Fluc and tagged with green fluorescent protein through lentiviral transduction. Figure 1A demonstrates a high level of GFP expression in human MSCs, and 93.7% of the population showed GFP positive expression detected by flow cytometry. MSCs were characterized by analyzing the expression of CD markers (i.e., CD ...

Watch this Scientific Journal Video about Intratracheal Instillation of Stem Cells in Term Neonatal Rats at JoVE.com

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...

intratracheal-instillation-of-stem-cells-in-term-neonatal-rats

Research

JoVE Journal

Medicine

5.6K Views.  Taipei Medical University. Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

Video: Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell therapy may alleviate many of the symptoms associated with lung disease and induce alveolar repair in adults. Concurrent with the ongoing search for stem cells applicable for human treatment, precise delivery and homing (to the site of disease) must be reassured for successful therapy. Here, I report that stem cells can safely be instilled via the trachea opening a non-stop route to the lung. This method involves a skin incision, caudal insertion of a cannula into and along the tracheal lumen, and injection of a stem cell vehicle mixture into airways of the lung. A broad range of media solutions and stabilizers can be instilled via tracheotomy, resulting in the ability to deliver a wider range of cell types. With alveolar epithelium confining these cells to the lumen, lung expansion and negative pressure during inhalation may also assist in stem cell integration. Tracheal delivery of stem cells, with a quick uptake and the ability to handle a large range of treatments, could accelerate the development of cell-based therapies, opening new avenues for treatment of lung disease.

Significant breakthroughs in stem cell identification are continuously being made. To translate these discoveries, however, novel methods for cellular delivery must be devised. Here I report that the airways provide a safe route for stem cell transplantation to the lungs.

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell ...

Biology

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo &#8220;pre-conditioning&#8221; stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations.

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

This article describes the methodology for administering short periods of intermittent hypoxia to postnatal day 1-8 mouse or rat pups. This approach effectively elicits a robust tissue level &#8220;priming effect&#8221; on cultured neural progenitor cells that are harvested within 30 min of hypoxia exposure.

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

Developmental Biology

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Direct Tracheal Instillation of Solutes into Mouse Lung

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice. Using this approach, one can target delivery of test solutes or solids (such as lung therapeutics, surfactants, viruses, and small oligonucleotides) into the distal lung. Tracheal instillations may be the preferred methodology, over inhalation protocols that may primarily target the upper respiratory tract and possibly expose the investigator to potentially hazardous substances. Additionally, in using the tracheal instillation protocol, animals can fully recover from the non-invasive procedure. This allows for making subsequent physiological measurements on test animals, or reinstallation using the same animal. The amount of instillate introduced into the lung must be carefully determined and osmotically balanced to ensure animal recovery. Typically, 30-75 &mu;L instillate volume can be introduced into mouse lung.

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice.

Intratracheal instillations deliver solutes directly into the lungs.  This procedure targets the delivery of the instillate into the distal regions of the ...

A Novel Minimally Invasive Slow Injection Method for Reducing Tissue Injury

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 &#176;C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

Author Spotlight: A Novel Cell Injection Method with Minimum Invasion  

This method eliminates any major invasion during cell injections caused by the cell suspension solution.

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...

Bioengineering

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Neuroscience

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered drug. This has direct translational impact because surfactant and drugs are administered locally into the lungs. Though the literature has many publications describing minimally invasive transoral intubation of adult mice and rats in therapeutic experiments, this approach in neonatal rat pups is lacking. The small size of orotracheal region/pharynx in the pups makes visualization of laryngeal lumen (vocal cords) difficult, contributing to the variable success rate of intratracheal drug delivery. We hereby demonstrate effective oral intubation of neonatal rat pup - a technique that is non-traumatic and minimally-invasive, so that it can be used for serial administration of drugs. We used an operating otoscope with an illumination system and a magnifying lens to visualize the tracheal opening of the rat neonates. The drug is then instilled using a 1 mL syringe connected to a pipette tip. The accuracy of the delivery method was demonstrated using Evans blue dye administration. This method is easy to get trained in and could serve as an effective way to instill drugs into trachea. This method could also be used for administration of inoculum or agents to simulate disease conditions in animals and, also, for cell-based treatment strategies for various lung diseases.

This technique of instilling drugs directly into the trachea of neonatal rodents is important in studying the impact of locally administered drugs or biologicals on neonatal lung diseases. Additionally, this method can also be used for inducing lung injury in animal models.

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered ...

Simplifying Transcutaneous Intratracheal Drug Delivery in the Newborn Preterm Rabbit

Intratracheal (IT) drug delivery allows the direct delivery of pharmaceutical substances to the lung, maximizing potential pulmonary benefit and minimizing systemic drug exposure. The transcutaneous technique is simple and allows for the IT delivery of substances to the lung of prematurely born rabbits shortly after birth. Newborn pups are anesthetized with inhaled Isoflurane before being placed in a supine position with the neck extended. The larynx is identified and stabilized before transcutaneous placement of a 26-gauge (G) catheter into the trachea. Following catheterization of the trachea, a 30 G blunt needle attached to a Hamilton syringe is introduced into the IT catheter and is used for delivering a precise volume into the trachea during spontaneous respiration. After the IT injection is completed, the needle and catheter are withdrawn, and the pup is allowed to recover from anesthesia. Transcutaneous IT injection delivers a large proportion of the injected substance to the lung, with the majority remaining in the lung 3 hours after the intervention. The injections are well tolerated from the day of birth and can be repeated for multiple consecutive days without influencing survival. This technique can be used to investigate the effect of pharmaceutical agents on lung development and in the prevention of neonatal lung injury in preterm rabbits.

Transcutaneous intratracheal injection allows for effective intrapulmonary drug delivery during spontaneous respiration. Single and multiple injections are well tolerated with no effect on survival. The technique is simple to perform and can examine the effect of substances on lung development and the prevention of lung injury in newborn rabbits.

Intratracheal (IT) drug delivery allows the direct delivery of pharmaceutical substances to the lung, maximizing potential pulmonary benefit and ...

In Vivo Heart Transplantation to Study Endothelial-to-Mesenchymal Transition

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.
To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.
A neonatal rat heart is transplanted into an adolescent rat&#39;s abdomen and connected to the recipient&#39;s infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.
This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

Author Spotlight: A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition  

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and ...

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Direct Tracheal Instillation of Solutes into Mouse Lung

A Novel Cell Injection Method with Minimum Invasion

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease

Simplifying Transcutaneous Intratracheal Drug Delivery in the Newborn Preterm Rabbit

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition