Name: anatomically inspired three-dimensional micro-tissue engineered neural networks for nervous system reconstruction, modulation, and modeling
Uploaded: 2017-05-31T14:00:00
Description: Watch this Scientific Journal Video about Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling at JoVE.com

Financial support was provided by the National Institutes of Health U01-NS094340 (Cullen), T32-NS043126 (Harris), and F31-NS090746 (Katiyar)), the Michael J. Fox Foundation (Therapeutic Pipeline Program #9998 (Cullen)), the Penn Medicine Neuroscience Center Pilot Award (Cullen), the National Science Foundation (Graduate Research Fellowships DGE-1321851 (Struzyna and Adewole)), the Department of Veterans Affairs (RR&#38;D Merit Review #B1097-I (Cullen)), the American Association of Neurological Surgeons and Congress of Neurological Surgeons (2015-2016 Codman Fellowship in Neurotrauma and Critical Care (Petrov)), and the U.S. Army Medical Research and Materiel Command (#W81XWH-13-207004 (Cullen) and W81XWH-15-1-0466 (Cullen)).

All procedures involving animals were approved by the Institutional Animal Care and Use Committees at the University of Pennsylvania and the Michael J. Crescenz Veterans Affairs Medical Center and adhered to the guidelines set forth in the NIH Public Health Service Policy on Humane Care and Use of Laboratory Animals (2015).
1. Development of the Agarose Hydrogel (Acellular Component of micro-TENNs)
<ol>
	<li>Agarose solution preparation
	<ol>
		<li>Within a biosafety cabinet...

CNS injury and disease typically result in the loss or dysfunction of the long-distance axonal pathways that comprise the brain connectome, with or without concomitant neuronal degeneration. This is compounded by the limited capacity of the CNS to promote neurogenesis and regeneration. Despite the pursuit of repair strategies such as growth factor, cell, and biomaterial delivery as individual or combinatorial approaches, these techniques fail to simultaneously account for both the degeneration of neuronal cells and the l...

A common characteristic of disorders and diseases of the central nervous system (CNS), such as traumatic brain injury (TBI), spinal cord injury (SCI), stroke, Alzheimer&#39;s disease, and Parkinson&#39;s disease, is the disconnection of axonal pathways and neuronal cell loss1,2,3,4,5,6. For instance, when an ischemic stroke goes untreated, it is estimated that axons are lost at a rate of 7 miles of axons per minute5. In the case of TBI, which approximately 1.7 million people experience each year in the U.S. alone, axonal degeneration may continue to occur years after trauma, as the initial injury precipitates a long-term neurodegenerative state4. Aggravating these deleterious effects, the CNS has a severely limited capacity for regeneration1,7,8,9. Following injury, an inhibitory environment develops that is characterized by a lack of directed guidance to distant targets, the presence of myelin-associated inhibitors that hinder neurite outgrowth, and the formation of a glial scar by reactive astrocytes8,10,11,12. The glial scar serves as a biochemical and physical barrier to regeneration, with molecules such as chondroitin sulfate proteoglycans obstructing axon outgrowth8,11. Furthermore, even though neural stem cells have been found in the adult CNS, the production of new neurons is limited, as consistent evidence of neurogenesis has only been found in the olfactory bulb, the hippocampal subgranular zone, the periventricular area, and the central canal of the spinal cord13,14. These obstacles prevent the functional recovery of lost neurons and white matter architecture following injury or disease, resulting in the often life-changing and prolonged effects of these conditions.
Despite the lack of regenerative capacity in the adult CNS, it has been demonstrated that axonal regeneration is possible if adequate environmental cues are presented to host neurons15,16,17,18. Researchers have attempted to deliver and manipulate growth factors (e.g., nerve growth factor, epidermal growth factor, glial-dependent growth factor, and neurotrophic factor-3) and other guidance molecules to stimulate plasticity and axon regeneration14,18,19. Even though these studies have confirmed that adult axons are capable of responding to growth factors, these strategies are limited by the low permeability of the blood-brain barrier and the specific spatial and temporal gradients required to promote regeneration14,18,19. Other approaches have relied on the hyperactivation of regeneration-related transcription factors in CNS neurons. For example, overexpression of the Stat3 transcription factor stimulated axonal regeneration in the optic nerve20. Nevertheless, both biomolecule delivery and overexpression of transcription factors fail to replace lost neuronal populations. Cell-based strategies have mainly centered on transplanting neural stem cells (NSCs) into the CNS, taking advantage of their capacity to replace CNS neurons, release trophic factors, and support the attempts at neurogenesis that occur after injury17. In spite of this, there are still pressing challenges hindering this approach, including the hampered ability of transplanted neural cells to survive, integrate with the host, and remain spatially restricted to the injured area6,14,17,21. In addition, cell delivery alone is incapable of reinstating the cytoarchitecture of damaged or lost axonal pathways. An alternative approach that addresses the problems facing cell and drug/chemical delivery strategies is combining these approaches with the use of biomaterials14,22,23. Biomaterials such as hydrogels are capable of emulating the biochemical and physical properties of the extracellular matrix (ECM), aiding in cell delivery and retention within the injured area, and delivering growth factors and other bioactive molecules with controlled release22. The attractive characteristics of these biomaterial-based strategies have resulted in evidence of in vivo axonal regeneration after the transplantation of scaffolds to the lesioned area24,25,26,27,28,29,30. However, acellular biomaterial strategies do not replace lost neuronal populations; when used as delivery vehicles for neuronal, glial, or neuronal precursor cells, biomaterials are incapable of reconstituting long-distance axonal networks. The challenge of developing an approach that tackles both the axonal pathway degeneration and neuronal loss associated with CNS injury and disease still remains31.
Our research group previously reported the development of implantable micro-tissue engineered neural networks (micro-TENNs), which are a type of &#160;&#34;living scaffold&#34; consisting of neuronal cell bodies restricted to one or both ends of an agarose hydrogel-ECM micro-column, with aligned axonal tracts extending throughout the interior of this three-dimensional (3D) encasement1,10,31,32. One of the main differences between this technique and previous approaches is that the cytoarchitecture of micro-TENNs is created completely in vitro and is transplanted afterwards33,34,35,36,37,38,39,40,41. In vitro fabrication offers extensive spatial and temporal control of cellular phenotype and orientation, mechanical/physical properties, biochemical cues, and exogenous factors, which benefits the integration of these scaffolds with the host after implantation41,42. Micro-TENNs are anatomically inspired because they emulate brain neuroanatomy, displaying axonal tracts similar to those that bridge distinct functional regions of the brain (Figure 1A)1. Therefore, this strategy may be able to physically replace lost white matter tracts and neurons following implantation into a lesioned region. This technique is also inspired by developmental mechanisms in which &#34;natural living scaffolds&#34; formed by radial glial cells and pioneering axons act as pathfinding guides for cell migration from the subventricular zone and axonal outgrowth, respectively43. These mechanisms are recapitulated in the aligned axonal tracts of micro-TENNs, which can present living pathways for neural cell migration and axonal regeneration by axon-mediated axonal outgrowth (Figure 1C)43. Furthermore, this strategy takes advantage of synaptic integration between the micro-TENN neurons and native circuitry, forming new relays that may contribute to functional recovery (Figure 1B)43. The capacity for synapse formation may also grant this approach the ability to modulate the CNS and respond to host tissue according to network feedback. For example, optogenetically active neurons in the living scaffolds may&#160;be stimulated to modulate host neurons through synaptic interactions (Figure 1D).
In addition, the biomaterial-based tubular construction of micro-TENNs offers an adequate environment for cell adhesion, growth, neurite extension, and signaling, while the miniature dimensions of the constructs potentially permit minimally invasive implantation and provide a partially sequestered microenvironment for gradual integration into the brain. Indeed, recent publications have demonstrated the potential of micro-TENNs to mimic neural pathways following implantation into the rat brain. Following stereotaxic microinjection, we previously reported evidence of micro-TENN neuronal survival, maintenance of axonal tract architecture, and neurite extension into the host cortex out to at least 1 month in vivo10,31. Moreover, labeling with synapsin provided histological evidence of synaptic integration with native tissue10,31. Overall, micro-TENNs may be&#160;uniquely suited to reconstruct and modulate damaged CNS by replacing lost neurons, synaptically integrating with host circuitry, restoring lost axonal cytoarchitecture, and, in certain cases, providing regenerating axons with the appropriate pathfinding cues.
<img alt="Figure 1" src="/files/ftp_upload/55609/55609fig1.jpg" /> 
Figure 1: Principles and inspiration behind the development of micro-tissue engineered neural networks (micro-TENNs). (A) Micro-TENNs mimic the cytoarchitecture of the brain connectome (purple), in which functionally distinct regions are connected by long, aligned axonal tracts in a unidirectional (red, green) or bidirectional (blue) manner. As an example, micro-TENNs could reconstitute lost connections in corticothalamic and nigrostriatal pathways&#160;or in the perforant pathway from the entorhinal cortex to the hippocampus&#160;(adapted from Struzyna et al., 2015)1. (B) Diagram of a unidirectional and bidirectional micro-TENN (red and blue, respectively) synaptically integrating with the host circuitry (purple) to serve as a functional relay between both ends of a lesion. (C) Schematic of the axonal tracts of a unidirectional micro-TENN (green) serving as a guide for axon-facilitated regeneration of host axons (purple) towards a target with which the micro-TENN interacts. (D) Conceptual diagram of the use of optogenetically active micro-TENNS as neuromodulators, taking advantage of synaptic integration with excitatory or inhibitory neurons (bottom). <a href="http://cloudflare.jove.com/files/ftp_upload/55609/55609fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The current manuscript details the methodology utilized to fabricate micro-TENNs using embryonically derived cerebral cortical neurons. Notably, micro-TENNs could be fabricated with other types of neural cells. For example, the initial reports of successful micro-TENN development featured dorsal root ganglion (DRG) neurons32. The hydrogel micro-columns can be generated (Figure 2A) by adding liquid agarose to a custom-made, laser-cut cylindrical channel array or to capillary tubes, both containing aligned acupuncture needles. The needle forms the lumen and determines the inner diameter (ID) of the micro-column, while the capillary tube ID and the diameter of the cylinders in the laser-cut device dictate the outer diameter (OD) of the constructs. The OD and ID can be chosen according to the desired application by selecting different diameters for the device/capillary tubes and the acupuncture needles, respectively. The length of the micro-columns can also be varied; to date, we have reported the construction of micro-TENNs up to 20 mm in length10 and are actively pursuing even longer lengths. After the agarose gels and the acupuncture needles are removed, an ECM solution generally consisting of type I collagen and laminin is added to the lumen of the constructs (Figure 2C). The ECM core provides a scaffold to support neuronal cell adhesion and axonal outgrowth. Initially, primary rat cortical neurons were plated in the micro-columns using dissociated cell suspensions10,31,32. However, this approach did not produce the target cytoarchitecture in all cases, which was defined as the neuronal cell bodies restricted to the ends of the micro-columns, with the central lumen comprised of pure aligned axonal tracts. Since then, the use of a forced neuronal aggregation method (based on protocols adapted from Ungrin et al.) has enabled a more reliable and consistent fabrication of micro-TENNs with the ideal structure (Figure 2B)44. In addition to describing the current methodology, this article will show representative phase-contrast and confocal images of micro-TENNs that demonstrate the formation of axonal tracts over time, as well as the finalized target cytoarchitecture. This manuscript will also expand on noteworthy aspects of the protocol and remaining challenges and future directions of the micro-TENN technology.
<img alt="Figure 2" src="/files/ftp_upload/55609/55609fig2.jpg" /> 
Figure 2: Schematic diagram of the three-stage micro-TENN fabrication process. (A) Development of the agarose hydrogel: (i) Initially, a small acupuncture needle (e.g., 180-350 &#181;m in diameter) is inserted into the cylindrical channels of a custom-made, laser-cut mold or a capillary tube (e.g., 380-700 &#181;m in diameter). In the next step, liquid agarose in DPBS is introduced into the cylindrical channels or capillary tubes. (ii) After the agarose gels, the needle is removed and the mold is disassembled to yield the hollow agarose micro-columns. (iii) These constructs are then sterilized and stored in DPBS. (B) Primary neuron culture and the aggregate method: (i) Neuronal aggregation is performed in pyramidal micro-well arrays, cast from 3D-printed molds, that fit in the wells of a 12-well culture plate. (ii) Micro-TENNs include primary rat neurons dissociated from fetal brains of embryonic-day-18 rats. Following tissue dissociation with trypsin-EDTA and DNase I, a cell solution with a density of 1.0-2.0 x 106 cell/mL is prepared. (iii) 12 &#181;L of this solution are transferred to each well in the pyramidal micro-well array. The plate containing these micro-wells is centrifuged to produce cell aggregates. (iv) These are then incubated overnight prior to plating in the micro-columns. (C) ECM core fabrication and cell seeding: (i) Prior to cell seeding, an ECM solution containing 1 mg/mL type I collagen and 1 mg/mL laminin is transferred to the interior of the micro-TENNs and allowed to polymerize. (ii) Depending upon whether unidirectional or bidirectional micro-TENNs are being fabricated, an aggregate is placed at either one or both extremes of the micro-column, respectively. (iii) Following a period of incubation to promote adhesion, micro-TENNs are cultured in Petri dishes flooded with supplemented embryonic neuronal basal medium. (iv) After 3-5 days in culture, the final micro-TENN structure should demonstrate cell aggregates at the extremes of the micro-column, with axonal tracts spanning its length. <a href="http://cloudflare.jove.com/files/ftp_upload/55609/55609fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="1571">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Laser cutter</td>
			<td style="width:195px;">Universal Laser Systems</td>
			<td style="width:119px;">PLS4.75</td>
			<td style="width:944px;">Used to fabricate the laser-cut micro-channel mold.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Laser-cut micro-column fabrication device</td>
			<td style="width:195px;">Custom-made</td>
			<td style="width:119px;">--------------</td>
			<td>Contact our research group if interested. Dimensions and blueprints provided in the manuscript.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Screws</td>
			<td style="width:195px;">--------------</td>
			<td style="width:119px;">--------------</td>
			<td>#4-40 with a thread diameter of 3.05 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Nuts</td>
			<td style="width:195px;">--------------</td>
			<td style="width:119px;">--------------</td>
			<td>#4-40 with a thread diameter of 3.05 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Acupuncture needle (180 &#181;m diameter)</td>
			<td style="width:195px;">Lhasa Medical</td>
			<td>sj.16X40</td>
			<td>The diameter may be varied according to the desired size for the micro-column lumen.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Petri dish</td>
			<td style="width:195px;">Fisher</td>
			<td>08772B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td style="width:195px;">Invitrogen</td>
			<td>14200075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Polystyrene disposable serological pipet</td>
			<td style="width:195px;">Fisher</td>
			<td>13-678-11D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Agarose</td>
			<td>Sigma</td>
			<td>A9539-50G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Capillary tube (398 &#181;m diameter)</td>
			<td style="width:195px;">Fisher</td>
			<td>21170D</td>
			<td>The diameter may be varied according to the desired size for the micro-column shell.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Hot plate</td>
			<td style="width:195px;">Fisher</td>
			<td>SP88857200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Magnetic bar</td>
			<td style="width:195px;">Fisher</td>
			<td>1451352</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Micropipette</td>
			<td style="width:195px;">Sigma</td>
			<td>Z683884-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">25 mm gauge needle</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:119px;">14-826-49</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Microscalpel</td>
			<td style="width:195px;">Roboz Surgical</td>
			<td>RS-6270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Scissors</td>
			<td style="width:195px;">Fine Science Tools</td>
			<td>14081-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Forceps</td>
			<td>World Precision Instruments</td>
			<td>501985</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Hot bead sterilizer</td>
			<td style="width:195px;">Sigma</td>
			<td>Z378550-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Stereoscope</td>
			<td style="width:195px;">Nikon</td>
			<td>SMZ800N</td>
			<td>Used for all dissection steps and for micro-TENN fabrication.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Rat tail type I collagen</td>
			<td style="width:195px;">Corning</td>
			<td>354236</td>
			<td>Maintain at 4 &#186;C and remove only when needed.&#160; Use ice to preserve its temperature when in use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Microcentrifuge tube</td>
			<td style="width:195px;">Fisher</td>
			<td>02-681-256</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Mouse laminin</td>
			<td style="width:195px;">Corning</td>
			<td>354232</td>
			<td>Maintain at 4 &#186;C and remove only when needed.&#160; Use ice to preserve its temperature when in use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Neurobasal medium</td>
			<td style="width:195px;">Invitrogen</td>
			<td>21103049</td>
			<td>Basal medium for the culture of pre-natal and embryonic neuronal cells. Store at 4&#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sodium hydroxide (NaOH)</td>
			<td style="width:195px;">Fisher</td>
			<td>SS2661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Hydrochloric acid (HCl)</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:119px;">SA48-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Litmus paper</td>
			<td style="width:195px;">Fisher</td>
			<td>09-876-18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Hank&#39;s balanced salt solution (HBSS)</td>
			<td style="width:195px;">Invitrogen</td>
			<td>14170112</td>
			<td>Store at 4 &#186;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">0.25% Trypsin-EDTA</td>
			<td style="width:195px;">Invitrogen</td>
			<td>25200056</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Bovine pancreatic deoxyribonuclease (DNase) I</td>
			<td style="width:195px;">Sigma</td>
			<td>10104159001</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">B-27 Supplement</td>
			<td style="width:195px;">Invitrogen</td>
			<td>12587010</td>
			<td>Supplement added to Neurobasal medium for the culture of hippocampal and cortical neurons. Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">L-glutamine&#160;</td>
			<td style="width:195px;">Invitrogen</td>
			<td>35050061</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sprague Dawley embryonic day 18 rats</td>
			<td style="width:195px;">Charles River</td>
			<td style="width:119px;">Strain 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Pasteur pipette</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:119px;">22-042816</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">15 mL centrifuge tube</td>
			<td style="width:195px;">EMESCO</td>
			<td>1194-352099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Vortex</td>
			<td style="width:195px;">Fisher</td>
			<td>02-215-414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Centrifuge</td>
			<td style="width:195px;">Fisher</td>
			<td>05-413-115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Hemocytometer</td>
			<td style="width:195px;">Fisher</td>
			<td>02-671-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Objet30 3D-Printer</td>
			<td style="width:195px;">Stratasys&#160;</td>
			<td>--------------</td>
			<td>Used to fabricate the pyramidal micro-well molds.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">3D-printed pyramidal well mold</td>
			<td style="width:195px;">Custom-made</td>
			<td style="width:119px;">--------------</td>
			<td>Contact our research group if interested. Dimensions and blueprints provided in the manuscript.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Polydimethylsiloxane (PDMS) and curing agent</td>
			<td style="width:195px;">Fisher</td>
			<td>NC9285739</td>
			<td>Comes as kit with elastomer and curing agent. Use inside a chemical fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Funnel</td>
			<td style="width:195px;">Fisher</td>
			<td>10-348C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">1 ml pipette bulb</td>
			<td style="width:195px;">Sigma</td>
			<td>Z509035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Micro-spatula</td>
			<td style="width:195px;">Fisher</td>
			<td>S50821</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">12-well culture plate</td>
			<td style="width:195px;">EMESCO</td>
			<td>1194-353043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Oven</td>
			<td style="width:195px;">Fisher</td>
			<td>11-475-154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Incubator</td>
			<td style="width:195px;">Fisher</td>
			<td>13 998 076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AAV1.Syn.GCaMP6f.WPRE.SV40</td>
			<td style="width:195px;">UPenn Vector Core</td>
			<td style="width:119px;">36373</td>
			<td>Store at -80&#186;C. Commercially available adeno-associated virus (AAV) with the GCaMP6f calcium indicator.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Formaldehyde 40%</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:119px;">F77P-4</td>
			<td>Formaldehyde is a toxic compound known to be carcinogenic, and must be disposed of in a separate container.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Triton X-100</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">T8787</td>
			<td>Non-ionic surfactant used to permeabilize cell membranes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Horse serum</td>
			<td style="width:195px;">Gibco</td>
			<td style="width:119px;">16050-122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Mouse anti-Tuj-1/beta-III tubulin primary antibody</td>
			<td style="width:195px;">Sigma</td>
			<td>T8578-200UL</td>
			<td>Store at -20&#186;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Rabbit anti-synapsin 1 primary antibody</td>
			<td>Synaptic Systems</td>
			<td>106-001</td>
			<td>Store at -20&#186;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Donkey anti-mouse 568 secondary antibody</td>
			<td style="width:195px;">Invitrogen</td>
			<td style="width:119px;">A10037</td>
			<td>Store at 4&#186;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Donkey anti-rabbit 488 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td>Store at 4&#186;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342, Trihydrochloride</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>Store at 4&#186;C.&#160; Hoechst is a known mutagen that should be treated as a carcinogen.&#160; Therefore, it must be disposed of in a separate container.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A1RSI Laser Scanning Confocal Microscope&#160;</td>
			<td style="width:195px;">Nikon</td>
			<td style="width:119px;">--------------</td>
			<td>Used for taking the confocal reconstructions of immunolabeled constructs.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eclipse Ti-S Microscope&#160;</td>
			<td>Nikon</td>
			<td style="width:119px;">--------------</td>
			<td>Used for taking the phase-contrast images.&#160; With digital image acquisition using a QiClick camera interfaced with Nikon Elements Basic Research software (4.10.01).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">High-speed Fluorescence Microscope</td>
			<td style="width:195px;">Nikon</td>
			<td style="width:119px;">--------------</td>
			<td>Nikon Eclipse Ti microscope paired with an ANDOR Neo/Zyla camera for calcium imaging.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NIS Elements AR 4.50.00 Software</td>
			<td style="width:195px;">Nikon Instruments</td>
			<td style="width:119px;">--------------</td>
			<td>Used to identify calcium transients from the recordings taken with the high-speed fluorescence microscope.&#160;</td>
		</tr>
	</tbody>
</table>

anatomically inspired three-dimensional micro-tissue engineered neural networks for nervous system reconstruction, modulation, and modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

Micro-TENNs were monitored using phase-contrast microscopy to assess their cytoarchitecture and axonal outgrowth (Figure 4). Within unidirectional, 2 mm long micro-TENNs, neuronal aggregates were restricted to one end of the micro-column and projected a bundle of axons through the inner core. Axons spanned the entire length of the column by 5 days in vitro (DIV) (Figure 4A). There was a greater initial axonal growth rate in bidirectional 2 mm-lon...

Watch this Scientific Journal Video about Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling at JoVE.com

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

This protocol describes the fabrication of micro-tissue engineered neural networks or micro-TENNs, which are miniature three-dimensional constructs comprised of long aligned axonal tracts spanning discrete neuronal populations within the lumen of a tubular hydrogel. Micro-TENNs replicate the general systems level anatomy of the brain connectome, specifically, functionally similar groups of neurons connected by long axonal tracts. Because they are pre-grown to emulate the cytoarchitecture of brain pathways, micro-TENNs may be applied for the targeted reconstruction of neural circuitry lost due to trauma or neurodegenerative disease and as biofidelic models for neurobiological studies.One of the most challenging aspects of this protocol is working with the micron scale form factor, which is ultimately necessary to allow for a minimally invasive delivery into the brain. Begin by manually breaking glass capillary tubes into 2 to 2.5-centimeter fragments, and insert one acupuncture needle into each fragment. Transfer one milliliter of three percent agarose in DPBS to the surface of an empty petri dish.Holding the capillary tube in the introduced needle, place one end of the tube in contact with the liquid agarose pool to fill it by capillary action. When the liquid ceases to rise, remove the capillary tube from the pool, and place it horizontally on the surface of a petri dish. Next, place the thumb and index finger on either side of the tube and grasp tightly.Use the other hand to quickly pull the needle out while the thumb and index finger prevent the micro-column itself from sliding out of the tube. Then insert a 30-gauge needle into the capillary tube to slowly push the micro-column out into a dish containing DPBS. Carefully transfer one micro-column from DPBS to an empty dish using fine forceps.Add 10 microliters of DPBS to the top of the micro-column with a micropipetted to prevent drying. While working under a stereomicroscope, trip the micro-column with a micro scalpel to shorten it to the desired length. Then use fine forceps to transfer the trimmed micro-column to a different petri dish containing DBPS.Sterilize the micro-columns in the DPBS-containing petri dishes under ultraviolet light for 30 minutes. Use a drill bit to puncture 16 holes as a 4-centimeter 4 by 4 array with a 1-centimeter separation into the lid of a 10-centimeter petri dish. Inside a chemical fume hood, use the bottom piece of a petri dish to weigh 27 grams of PDMS and three grams of curing agent.Stir with a micro spatula to distribute the agent evenly. Then cover the PDMS curing agent with the punctured lid. Connect one end of a hose to the vacuum port of the fume hood, and insert the other end into the stem of a suitably sized funnel.Place a 1-milliliter pipette bulb on a 1-milliliter tip into the hose, and pull the hose upwards until the bulb seals the hose into the stem of the funnel. Next, place the funnel on the punctured dish lid and secure the hose with an available sturdy support. Open the vacuum valve for five minutes to suction and bring the air bubbles to the surface.After five minutes, close the valve, remove the funnel, and hit the petri dish against the surface of the fume hood a few times to burst any remaining air bubbles. Place a pyramidal well 3D printed mold into each of the wells in a 12-well culture plate with the pyramids pointing up. Then pour the PDMS curing agent on top of the molds until each well of the plate is filled.Transfer the covered plate to an oven for one hour at 60 degrees Celsius to dry. After sterilizing the PDMS arrays by autoclaving, and working in a biosafety cabinet, insert one micro-well array into each well of a sterile 12-well plate. To form the neuronal aggregates, use a micropipetted to add 12 microliters of cell suspension into each micro-well of the PDMS array.Centrifuge the plate at 200 times g for five minutes to force the aggregation of cells at the bottom of the micro-wells. Then carefully add approximately two milliliters of culture medium to the top of each PDMS array to cover all the seeded microwells. Incubate the plate for 12 to 24 hours at 37 degrees Celsius in 5%carbon dioxide.To begin CEM core fabrication, mix Type 1 collagen, laminin, and culture medium in a micro centrifuge tube and adjust the pH to 7.2 to 7.4. Then place the tube containing CEM on ice. Use sterile forceps to transfer the micro-columns to empty 35 or 60-millimeter petri dishes.Then, while working under a stereomicroscope, use a 10-microliter tip attached to a 1000-microliter tip to extract residual DPBS and air bubbles from the lumen. Quickly draw up four to five microliters of CEM in a micropipetted, then place the tip of the micropipetted at one end of a micro-column, and discharge enough CEM to fill the lumen. To prevent dehydration, add two microliters of CEM around each micro-column.Incubate the hydrogel CEM micro-columns in petri dishes at 37 degrees Celsius in 5%carbon dioxide for 15 minutes. Proceed to cell seeding immediately after incubation. Transfer approximately 10 to 20 microliters of culture medium to two free areas in the petri dishes holding the micro-columns.Use a micropipetted to collect the neuronal aggregates individually, and transfer them to the petri dish containing the constructs. Move the aggregates with forceps to one of the small pools of culture medium to preserve cell health. While observing under a stereomicroscope, use forceps to insert an aggregate at one end of the micro-columns for unidirectional micro-TENNs, or at each end for bi-directional architecture.Then move the seeded micro-column to the other small pool of culture medium to avoid dehydration and to preserve aggregate health. When all micro-columns have been loaded, incubate at 37 degrees Celsius in 5%carbon dioxide for 45 minutes to allow the aggregates to adhere to the CEM. After incubation, verify that the aggregates remain at the ends of the micro-columns using the stereomicroscope.Lastly, carefully flood the petri dishes containing the micro-TENNs with culture medium using a pipette. Place the dishes in an incubator at 37 degrees Celsius in 5%carbon dioxide for long-term culture. These phase contrast images show unidirectional 2-milliliter long micro-TENNs up to eight days in-vitro.In these images, notice the neuronal aggregates at the extremes of the micro-column while the aligned axonal tracts project across the lumen. As seen here, axonal tracts extend almost the entire length of the micro-column by five days in-vitro. For 5-millimeter bi-directional micro-TENNs, axonal tracts populate the length of the micro-column at 5 days in-vitro, and this architecture is maintained at least until 10 days in-vitro.The following two images can help troubleshoot problems with the procedure. This image shows a completely dehydrated dried hydrogel micro-column as a result of the complete removal and/or evaporation of DPBS, CEM, or culture medium during fabrication. This image shows a micro-column with the lumen lining the outer wall due to the lack of concentric alignment of the acupuncture needle with the capillary tube.Finally, this confocal image shows a bi-directional micro-TENN at 28 days in-vitro, stained for cell nuclei with Hoechst in blue, and with axons labeled with tudge one in red, and pre-synaptic boutons labeled with synapsin 1 in green. Cell migration from the aggregates along the axonal tracts is seen in the presence of pre-synaptic terminals as suggested. Micro-TENNs are self-contained, anatomically inspired constructs that emulate key aspects of connectome cytoarchitecture;therefore, micro-TENNs may provide a means to replace lost neural pathways upon implantation into the brain.Crucial components of this method include the tubular biomaterial encasement scheme, which allows for longitudinal axonal growth, and the aggregate method, which ensures obtaining the proper cytoarchitecture of cell bodies restricted to the ends of axonal tracts along the interior. Following mastery of these methods, the principles of this technique may be applied to build micro-TENNs with other cell phenotypes and biomaterial components, according to the specific application.

anatomically-inspired-three-dimensional-micro-tissue-engineered

Research

JoVE Journal

Neuroscience

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Three-Dimensional Motor Nerve Organoid Generation

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...

Reliable Acquisition of Electroencephalography Data during Simultaneous Electroencephalography and Functional MRI

Simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI), EEG-fMRI, combines the complementary properties of scalp EEG (good temporal resolution) and fMRI (good spatial resolution) to measure neuronal activity during an electrographic event, through hemodynamic responses known as blood-oxygen-level-dependent (BOLD) changes. It is a non-invasive research tool that is utilized in neuroscience research and is highly beneficial to the clinical community, especially for the management of neurological diseases, provided that proper equipment and protocols are administered during data acquisition. Although recording EEG-fMRI is apparently straightforward, the correct preparation, especially in placing and securing the electrodes, is not only important for safety but is also critical in ensuring the reliability and analyzability of the EEG data obtained. This is also the most experience-demanding part of the preparation. To address these issues, a straightforward protocol that ensures data quality was developed. This article provides a step-by-step guide for acquiring reliable EEG data during EEG-fMRI using this protocol that utilizes readily available medical products. The presented protocol can be adapted to different applications of EEG-fMRI in research and clinical settings, and may be beneficial to both inexperienced and expert operators.

This article provides a straightforward protocol for acquiring good quality electroencephalography (EEG) data during simultaneous EEG and functional magnetic resonance imaging by utilizing readily available medical products.

Simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI), EEG-fMRI, combines the complementary properties of scalp EEG ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...