The authors acknowledge funding sources responsible for this work: 5K12HD055929 (HHR), 5R01NS080180-02 (DDF).

NOTE: All animal procedures in this protocol are conducted with the approval of University of Florida Institutional Animal Care and Use Committee (IACUC) and are in compliance with the &#39;Guide for the Care and Use of Laboratory Animals&#39;.
1. Basic Experimental Set up Before Intermittent Hypoxia Administration
<ol>
	<li>Expose pups12 to the different gas mixes using a whole-body mouse plethysmograph chambers in much the same manner as adult rodents for other experimental purposes5,13,14. The chambers have a 4 inch diameter (volume = 450 ml), which is of ample size to accommodate 3-4 mouse pups or 1-2 rat pups. Here, AIH protocols are delivered to neonates aged postnatal day 1-8 (P1-P8).</li>
	<li>Use compressed air gas tanks to deliver the 21% &#8220;baseline&#8221; gas, which maintains chamber oxygenation at 21%. To achieve &#8220;relative hypoxia&#8221;, use a 10% oxygen/90% nitrogen gas tank. 
	NOTE: Plastic tubing (3/16&#8221; diameter) connects the gas tanks to a flow meter. Thus, the input to the flow meter consists of one tube each for baseline and hypoxia airflow.</li>
	<li>Configure the gas tubing as follows.
	<ol>
		<li>Run plastic tubing (1/8&#8221; diameter) from the flow meter to a bias flow regulator, which enables flows of 1 L/min to each chamber (i.e., 4 L/min if 4 chambers are used).</li>
		<li>Run plastic tubing (1/8&#8221; diameter) from the bias flow regulator to the plethysmography chambers. Airflow at 1 L/min to each chamber is a predetermined flow rate at which gas exchange is deemed to be non-stress-inducing to the animals based on physiological and behavioral observations.</li>
	</ol>
	</li>
	<li>Seal all unused connections to and from the bias flow unit and all unused openings to the chambers with caps so that no gas flow escapes to other chambers not in use, or out of the plethysmography chambers during the protocol.</li>
	<li>Place the chamber(s) into an incubator prior to putting animals in the chamber to allow equilibration to 37 &#176;C, or body temperature.</li>
</ol>
2. Calibration of Cycle Times for Intermittent Hypoxia
<ol>
	<li>Inspect all tubing, between air tanks and flow meter, flow meter and bias flow unit, and bias flow unit and plethysmography chamber(s) for proper connections. The connections are set up as described in Step 1. Verify the connection by a step-wise check of all described lines and closures: e.g., checking unused lines out of the bias flow unit and any chamber openings not in use.</li>
	<li>Connect an ambient O2 sensor with a hand-held O2 meter and then attach the sensor to the plethysmography chamber lid.</li>
	<li>Open both gas tanks and attach one pair of surgical, long handled hemostats insulated with plastic tubing to clamp the off-cycle flow of gas. In this way, only one gas tank at a time is delivering the 1 L/min gas flow per chamber. Clamp the &#8220;baseline&#8221; gas mix while the &#8220;hypoxic&#8221; gas mix is delivered to the chamber, and vice versa.</li>
	<li>Record the time required for the chamber O2 to reach the target levels of 10% and return to 21%. Utilize the recorded times for subsequent protocol delivery.</li>
</ol>
3. Administration of Acute Intermittent Hypoxia
<ol>
	<li>Inspect all tubing and connections as described in Step 2.1.</li>
	<li>Place neonates in the plethysmography chamber(s) and house the plethysmography chamber lids into the chamber base. Confirm a proper seal to the O-ring seal is as well as closure of any unused chamber connections. These steps are critical to ensuring the appropriate delivery of gas mixes.</li>
	<li>Place the chambers holding the pups to be treated into the 37 &#176;C incubator and close the door. Allow chamber to equilibrate to 37 &#176;C prior to starting the protocol. Maintain control animals at 37 &#176;C in the incubator at room air oxygenation.</li>
	<li>Open both gas tanks and use one pair of surgical, long handled hemostats insulated with plastic tubing to clamp the off-cycle flow of gas. In this way, only one gas tank at a time is delivering the 1 L/min gas flow per chamber.</li>
	<li>Use a hand-held timer to precisely time cycles and to indicate the time to switch the hemostats between tubes, thus resulting in the alternation of gas flow to the chambers.</li>
	<li>Record the completion of each cycle using a treatment log such as the one provided in Figure 1 to mark off all steps in the 2-step, 20 cycle protocol.</li>
	<li>Monitor the incubator temperature at every cycle change to ensure animals are maintained at the designated range (33-35 &#176;C, which is the setting on the incubator that results in 37 &#176;C).</li>
	<li>Closely monitor pup activity throughout the protocol to ensure that animals tolerate cycles without visible distress such as vocalizations, altered level of limb activity or rolling.</li>
</ol>
4. Isolation and Culture of Stem/Progenitor Cells from the Subventricular Zone
NOTE: The change in neurosphere formation following AIH compared to controls is an example of an endpoint that demonstrates the efficacy of this protocol to elicit tissue- and cellular-level changes.
<ol>
	<li>To isolate neurospheres forming cells, remove mouse or rat pups from the chamber immediately after AIH is completed. Sacrifice the animals according to IACUC protocols within 30 min of protocol termination.</li>
	<li>Harvest the SVZ tissue, place into neurosphere culture and conduct standard passage and expansion of derivative neurospheres, as previously published in methods chapters15,16 and a separate video protocol8.</li>
</ol>

<ol>
	<li>Studer, L., 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=Enhanced+proliferation,+survival,+and+dopaminergic+differentiation+of+CNS+precursors+in+lowered+oxygen.">Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen.</a> J Neurosci. 20 (19), 7377-7383 (2000).</li><li>Chen, H. L., 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=Oxygen+tension+regulates+survival+and+fate+of+mouse+central+nervous+system+precursors+at+multiple+levels.">Oxygen tension regulates survival and fate of mouse central nervous system precursors at multiple levels.</a> Stem Cells. 25 (9), 2291-2301 (2007).</li><li>Ling, L., 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=Chronic+intermittent+hypoxia+elicits+serotonin-dependent+plasticity+in+the+central+neural+control+of+breathing.">Chronic intermittent hypoxia elicits serotonin-dependent plasticity in the central neural control of breathing.</a> J Neurosci. 21 (14), 5381-5388 (2001).</li><li>Mitchell, G. S., 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=Invited+review:+Intermittent+hypoxia+and+respiratory+plasticity.">Invited review: Intermittent hypoxia and respiratory plasticity.</a> J Appl Physiol. 90 (6), 2466-2475 (2001).</li><li>Fuller, D. D., Zabka, A. G., Baker, T. L., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phrenic+long-term+facilitation+requires+5-HT+receptor+activation+during+but+not+following+episodic+hypoxia.">Phrenic long-term facilitation requires 5-HT receptor activation during but not following episodic hypoxia.</a> J Appl Physiol. 90 (5), 2001-2006 (2001).</li><li>Fuller, D. D., Johnson, S. M., Olson, E. B., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+pathways+to+phrenic+motoneurons+are+enhanced+by+chronic+intermittent+hypoxia+after+cervical+spinal+cord+injury.">Synaptic pathways to phrenic motoneurons are enhanced by chronic intermittent hypoxia after cervical spinal cord injury.</a> J Neurosci. 23 (7), 2993-3000 (2003).</li><li>Baker-Herman, T. L., 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=BDNF+is+necessary+and+sufficient+for+spinal+respiratory+plasticity+following+intermittent+hypoxia.">BDNF is necessary and sufficient for spinal respiratory plasticity following intermittent hypoxia.</a> Nat Neurosci. 7 (1), 48-55 (2004).</li><li>Zhu, L. L., 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=Neurogenesis+in+the+adult+rat+brain+after+intermittent+hypoxia.">Neurogenesis in the adult rat brain after intermittent hypoxia.</a> Brain Res. 1055, 1-6 (2005).</li><li>Zhang, J. X., Chen, X. Q., Du, J. Z., Chen, Q. M., Zhu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+exposure+to+intermittent+hypoxia+enhances+mice+performance+in+water+maze+and+8-arm+radial+maze+tasks.">Neonatal exposure to intermittent hypoxia enhances mice performance in water maze and 8-arm radial maze tasks.</a> Journal of neurobiology. 65 (1), 72-84 (2005).</li><li>Zhu, L. L., Wu, L. Y., Yew, D. T., Fan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hypoxia+on+the+proliferation+and+differentiation+of+NSCs.">Effects of hypoxia on the proliferation and differentiation of NSCs.</a> Mol Neurobiol. 31 (1-3), 231-242 (2005).</li><li>Ross, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+intermittent+hypoxia+elicits+enhanced+expansion+and+neuronal+differentiation+in+cultured+neural+progenitors.">In vivo intermittent hypoxia elicits enhanced expansion and neuronal differentiation in cultured neural progenitors.</a> Exp Neurol. 235 (1), 238-245 (2012).</li><li>Fuller, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+recovery+of+hypoxic+phrenic+responses+in+adult+rats+exposed+to+hyperoxia+for+the+first+month+of+life.">Induced recovery of hypoxic phrenic responses in adult rats exposed to hyperoxia for the first month of life.</a> J Physiol. 536 (Pt 3), 917-926 (2001).</li><li>Fuller, D. D., Fregosi, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatiguing+contractions+of+tongue+protrudor+and+retractor+muscles:+influence+of+systemic+hypoxia.">Fatiguing contractions of tongue protrudor and retractor muscles: influence of systemic hypoxia.</a> J Appl Physiol. 88 (6), 2123-2130 (2000).</li><li>Baker, T. L., Fuller, D. D., Zabka, A. G., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+plasticity:+differential+actions+of+continuous+and+episodic+hypoxia+and+hypercapnia.">Respiratory plasticity: differential actions of continuous and episodic hypoxia and hypercapnia.</a> Respir Physiol. 129 (1-2), 25-35 (2001).</li><li>Marshall, G. 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=Production+of+neurospheres+from+CNS+tissue.">Production of neurospheres from CNS tissue.</a> Methods Mol Biol. 438, 135-150 (2008).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+expansion+of+the+adult+mouse+neural+stem+cells+using+the+neurosphere+assay.">Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay.</a> J Vis Exp. (45), (2010).</li></ol>

The authors have nothing to disclose.

This work reports the development of a protocol to expose neonatal rodents to AIH. The parameters described here effectively alter in situ neural stem cell biology, which is observable over several rounds of cell passage. Specifically, AIH increases the number of non-adherent neurospheres, the expansion of cells within each neurosphere (refected by sphere diameter), the expansion of adherent NPC populations, and the presence of neuroblasts in both non-adherent and adherent populations. It should be emphasized that the method described herein has been used in multiple trials, and the impact of AIH on in vitro cell biology is robust and reproducible. Over the last several years, more than 60 mouse and rat pups have been exposed to AIH, with increased growth and differentiation observed in each experiment. This is particularly interesting proof of concept, methodological data when considering the development of AIH protocols as a therapeutic modality, for example following neural injury and in conjunction with cell therapies. 
During the calibration and delivery of this protocol, precision in several steps is critical to achieve the consistent delivery and cycling of O2 conditions. Therefore, a rigorous pre-treatment protocol is performed to ensure ample gas in both air tanks, proper connections in the entire system&#8212;from air tank to plethysmography chamber, precise gas flow into each chamber via air flow regulator checks, and a proper seal on all chambers. Finally, maintaining treatment chambers at a constant temperature ensures that animals are kept as a comfortable body temperature. 
One limitation to this method is the lack of a vacuum-based removal of cycling gas, which would decrease the time for each cycle to achieve the limits of the exposure range, thus providing a more rapid method of cycling parameters. Another limitation is that the detailed mechanisms by which this protocol elicit the observed changes is not completely understood, something which this group continues to explore. Prior work demonstrated that HIF1-alpha target genes, EPO and VEGF, appear to be involved in both expansion and morphological changes in these cells11. Further, this report outlined that changes in expansion, in particular, become more pronounced with increased time in culture. This highlights the critical need to carefully document the post-treatment cell culture time course in order to standardize experimental results. 
Taken together, these technical results describe a novel application of acute intermittent hypoxia delivered via whole body plethysmography chambers. A significant future application is the continued development of AIH as a non-invasive and powerful tool to encourage the health of both injured CNS tissue and regenerative therapeutic tools such as stem cells destined for transplant.

The goal of this method is to reproducibly and effectively deliver intermittent bouts of systemic lowered ambient oxygen to neonatal rodents. The rationale for using intermittent hypoxia (IH) to manipulate stem cell biology originates from in vitro cell culture experiments in which O2 content of the growth media is altered. Specifically, when compared to &#8220;standard&#8221; conditions of 20% O2, extended culture of stem/progenitor cell populations cells in 3% O2 results in increased proliferation, decreased apoptosis and increased neuronal yield1,2.
This group has significant experience with the administration of systemic IH, and has conducted extensive studies on the role of IH in respiratory plasticity3-7. This work, and the recent finding that chronic IH increased neurogenesis in the rodent CNS8-10, forms the basis for the exploration of acute in vivo hypoxia as a preconditioning stimulus (i.e., prior to tissue harvest) on the subsequent culture of neural stem/progenitor cells (NPCs)11. Remarkably, when mouse pups were exposed to a brief (&#60;1 hr) period of acute intermittent hypoxia (AIH), cells that were harvested from the subventricular zone (SVZ) had significantly increased capacity for expansion as neurospheres or adherent monolayer cells. The AIH protocol was also associated with increased expression of a &#8220;neuronal fate&#8221; transcription factor (Pax6).
Accordingly, in vivo AIH protocols may provide a means to &#8220;prime&#8221; NPCs prior to culture. For example, applications for this approach could include expanding cell populations prior to transplantation into the injured central nervous system, or simply increasing the neuronal differentiation of cultured cells prior to in vitro experiments. Further, because this is a systemic delivery, any organ, tissue or cell is a candidate for similar study. Therefore, the protocol as written is potentially applicable to a wide range of studies on intermittent oxygen manipulation in small mammals.
There are certain advantages to this approach. In other published work, neonates were treated as a litter with the dam in hypobaric chambers, which allows for chronic dosing, less handling prior to treatment, and maintained maternal contact during treatment9. The current approach bypasses repeated treatments to a breeding female, or the use of a different dam for each experiment. This protocol also allows study of precise litter-matched and age-matched neonates. Representative data demonstrate another key strength of this protocol, namely the rapidity with which AIH, as delivered, elicits a powerful and consistent biological response in neural stem cell biology. This establishes a precedent for this protocol to elicit tissue- and cellular-level biological changes that alter cell biology.
This report will outline the detailed procedures used for exposing rodent pups to AIH as well as the population analysis of SVZ cells grown as neurospheres.

<table><tbody><tr><td>Mouse plethysmography chambers</td><td>Buxco</td><td>PLY4211</td><td></td></tr><tr><td>Flow meter&amp;nbsp;</td><td>Porter</td><td>F150</td><td></td></tr><tr><td>Bias flow unit</td><td>AFPS</td><td></td><td></td></tr><tr><td>Baseline Gas Mix</td><td>Airgas</td><td>AIZ300</td><td>Compressed Air</td></tr><tr><td>Hypoxic Gas Mix</td><td>Airgas</td><td>X03NI72C2000189</td><td>10% Oxygen, balance nitrogen</td></tr><tr><td>Oxygen Meter</td><td>Teledyne</td><td>AX-300</td><td></td></tr></tbody></table>

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.

The initial experiments, based on historical data, were conducted using 1 min cycle lengths. Based on the subsequent calibrations performed in Step 2 above, it was determined that the O2 level in the chamber was 13% at 1 min post-hypoxia flushing and, that it took a similar time to return to the 21% baseline. However, a 2 min&#160;cycle was sufficient to both achieve 10% oxygen and a return to 21% during the &#8220;baseline&#8221; cycle. Subsequently, 2 min cycle lengths have been used. The protocol duration consisted of 20 cycles alternating between baseline and hypoxia (80 min overall treatment time). A 20 cycle duration was chosen due to previous work showing that such cycle duration is sufficient to elicit changes in respiratory outcome measures11. Using the described set up (Figure 2), neonates subjected to AIH had no apparent adverse events and did not exhibit behaviors suggestive of pain and discomfort during the 80 min protocol. Specifically, no visible apnea was observed, excessive limb or head movements, or vocalizations. Following AIH, subventricular zone-derived neural stem/progenitor cell populations cultured as neurospheres for 14 days (and also as adherent monolayer populations, data not shown) exhibit a nearly two-fold increase in diameter, demonstrating increased expansion within each forming sphere (Figure 3). Cells that are next plated in permissive conditions (i.e., absence of mitogenic factors) yield significantly more beta-3-positive cells (an increase from &#60;10% to &#62;45%) which indicates more extensive neuronal differentiation as compared to cells harvested from normoxic treated (control) pups (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/52527/52527fig1.jpg" /> 
Figure 1.&#160;This acute IH record sheet is used to document animal and experimental details, provide a check list for before starting and following completion of the IH protocol, and serve as a tracking document for accurate tally of all completed cycles.
<img alt="Figure 2" src="/files/ftp_upload/52527/52527fig2.jpg" /> 
Figure 2.&#160;Intermittent hypoxia set up for neonatal rodents. (A) Incubator to maintain environment at 37 &#176;C. Plethysmography chamber is shown within the incubator, and again with the chamber door ajar (B). (C) Right, flow meter adjusted to 1 L/min for each chamber. Gas flow is directed via the bias flow unit splitter unit (center) to any connected chamber (left, within incubator). (D) Hemostat control of baseline (red tape) and hypoxia (yellow tape) input lines. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52527/52527fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52527/52527fig3.jpg" /> 
Figure 3.&#160;Control neurospheres (A) demonstrate a smaller diameter than AIH neurospheres (B). Images taken at 10X objective, scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52527/52527fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52527/52527fig4.jpg" /> 
Figure 4.&#160;Control neurospheres (A) demonstrate fewer beta-3-tubulin-positive neuroblasts than AIH neurospheres (B). Images taken at 20X objective, scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52527/52527fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures at JoVE.com

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.

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 ...

delivery-vivo-acute-intermittent-hypoxia-neonatal-rodents-to-prime

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.2K Views.  University of Florida. 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 “priming effect” on cultured neural progenitor cells that are harvested within 30 min of hypoxia exposure.

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

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

Genetics

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

Biology

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Neuroscience

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Medicine

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 ...

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.

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 ...

Intravenous Injections in Neonatal Mice

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

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

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

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 ...

Rat "Brain Milking" for Neural Stem and Oligodendrocyte Progenitor Cell Isolation

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new neurons and glia. One such niche is the subependymal zone (SEZ; also called the ventricular-subventricular zone), which is located across the lateral walls of the lateral ventricles, adjacent to the ependymal cell layer. Oligodendrocyte progenitor cells (OPCs) are abundantly distributed throughout the central nervous system, constituting a pool of proliferative progenitor cells that can generate oligodendrocytes.
Both NSCs and OPCs exhibit self-renewal potential and quiescence/activation cycles. Due to their location, the isolation and experimental investigation of these cells is performed postmortem. Here, we describe in detail &#34;brain milking&#34;, a method for the isolation of NSCs and OPCs, amongst other cells, from live animals. This is a two-step protocol designed for use in rodents and tested in rats. First, cells are &#34;released&#34; from the tissue via stereotaxic intracerebroventricular (i.c.v.) injection of a &#34;release cocktail&#34;. The main components are neuraminidase, which targets ependymal cells and induces ventricular wall denudation, an integrin-&#946;1-blocking antibody, and fibroblast growth factor-2. At a second &#34;collection&#34; step, liquid biopsies of cerebrospinal fluid are performed from the cisterna magna, in anesthetized rats without the need of an incision.
Results presented here show that isolated cells retain their endogenous profile and that NSCs of the SEZ preserve their quiescence. The denudation of the ependymal layer is restricted to the anatomical level of injection and the protocol (release and collection) is tolerated well&#160; by the animals. This novel approach paves the way for performing longitudinal studies of endogenous neurogenesis and gliogenesis in experimental animals.

Author Spotlight: Collecting Neural Stem and Progenitor Cells from Live Animals Using a Novel Brain Milking Protocol 

A method for the isolation of neural stem cells and oligodendrocyte progenitor cells from the brains of live rats is presented here in experimental detail. It allows multiple collections of these cells from the same animals without compromising their well-being.

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new ...

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

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Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Neonatal Subventricular Zone Electroporation

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

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

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Intravenous Injections in Neonatal Mice

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

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats