This work was supported by the NIH grants (NS108763, NS100419, NS095064, and HD080429 to C.Y. K.; and NS106592 to Y.Y.S.).

This protocol is approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Virginia and follows the National Institutes of Health Guideline for Care and Use of Laboratory Animals. Figure 1A outlines the sequence of surgical procedures of this protocol.
1. Surgery setup
<ol>
	<li>Place a warming pad with temperature setting at 37 &#176;C on the small animal adaptor at least 15 minutes before the surgery. Prepare a nose-clip roll for adaptor which allows the animal head rotation. Prepare the anesthetics Ketamine (60 mg/kg)/ Xylazine (10 mg/kg).</li>
	<li>Sterilize the surgical tools including scissors, forceps, micro-needle holders, hemostats, cotton swabs and sutures with autoclave (121 &#176;C at 15 psi for 60 min). Prepare tissue glue and eye ointment. Prepare the 532 nm laser protection goggle for surgeons. 
	NOTE: This protocol describes a major survival surgery procedure and should be conducted using aseptic techniques.</li>
	<li>Set up the illumination system with a 532 nm laser source. Prepare a dental drill.</li>
	<li>Prepare the Rose Bengal solution in saline (10 mg/mL). Place an aliquot bovine thrombin (0.1 U/&#181;L) on ice-bucket.</li>
	<li>Inject Ketoprofen&#160;(4.0 mg/kg) subcutaneously to the mouse as analgesia at 30 min before surgery or use the analgesic regimen recommended by the local institutional guidelines.</li>
</ol>
2. Ligation of the ipsilateral common carotid artery
<ol>
	<li>Anesthetize 10-14 week-old male C57BL/6NCrl mice weighing 22 to 30 g by intramuscular injection of Ketamine (60 mg/kg) and Xylazine (10 mg/kg). 
	NOTE: The entire surgical procedure, encompassing the ligation of the ipsilateral common carotid artery through the monitoring of cerebral blood flow, is expected to take ~120 min. The anesthetic regimen will be typically effective for this entire duration, but the anesthetic depth should be reassessed at least every 15 min. While learning these procedures, it may be necessary to re-dose the anesthesia.</li>
	<li>Perform a toe-pinch to ensure the animal is fully anesthetized. Remove the hair on the left neck for CCA ligation and the head for skull thinning with the hair removal cream.</li>
	<li>Place the mouse on the small animal adaptor in the supine position. Sterilize the surgical area by wiping skin with three alternating swipes of povidone-iodine and 70% ethanol.</li>
	<li>Secure the mouse head using ear bars. Under a dissecting microscope, make a 0.5 cm left-cervical incision using a pair of micro-scissors and straight forceps at about 0.2 cm lateral to the midline.</li>
	<li>Use a pair of fine serrated forceps to pull apart the soft tissue and fascia to expose the left common carotid artery (LCCA). Carefully separate the left CCA from the vagal nerve using a pair of fine smooth forceps.</li>
	<li>Place a permanent double-knot suture around the LCCA using 5-0 silk suture cut into 20 mm segments, and then close the wound using sterile wound clips.</li>
</ol>
3. Skull thinning above the MCA branch and photoactivation
<ol>
	<li>Flip the mouse to prone position on the small animal adaptor. Rotate the nose-clip roll for 15&#176;. Sterilize the surgical area by wiping skin with three alternating swipes of betadine and 70% ethanol.</li>
	<li>Make a 0.8 cm incision in the scalp using a pair of micro-scissors and straight forceps along the left eye and ear to expose the temporalis muscle, which is located between the eye and the ear (Figure 1B).</li>
	<li>Under the dissecting microscope, make a 0.5 cm incision along the edge of temporalis muscle on left parietal bone by a pair of fine serrated forceps. Make a second 0.3 cm vertical incision on temporalis muscle by a micro scissors. Retract the temporal muscle to expose the edge of parietal bone and squamosal bone. Make sure to visualize the landmark of coronal suture between the frontal and the parietal bones (Figure 1B,C).</li>
	<li>Moisture the skull by applying sterile saline to reveal the left MCA. Mark the proximal MCA branch on the squamosal bone with a marker pen. Gently draw a circle for about 1 mm in diameter surrounding the marked area with the pneumatic dental drill (burr speed setting at 50% of speed controller), and then thin the skull about 0.2 mm in depth without touching the underneath dura. Stop the drilling until a very thin layer of bone is left.</li>
	<li>Mix the thrombin (T, 0.1 U/&#956;L, 80 U/kg) and Rose bengal (RB, 10 mg/mL, 50 mg/kg) solution based on the mouse&#8217;s body weight. For example, for a mouse of 25 g body weight, mix 20 &#956;L of thrombin (0.1 U/&#956;L) and 125 &#956;L of RB (10 mg/mL).</li>
	<li>Slowly inject T+RB solution (145 &#181;L per 25 g body weight) into the retro-orbital sinus with an insulin syringe (#31G needle). 
	NOTE: In pilot experiments, the mortality rate of increasing doses of thrombin mixed with the standard dose of RB dye (50 mg/kg) was examined for photoactivation. The mortality was 0% for 80 U/kg thrombin (n=13), 43% for 120 U/kg thrombin (n=7), and 100% for both 160 U/kg (n=5) and 200 U/kg thrombin (n=5). A dose of 80 U/kg thrombin was therefore chosen for this model. Laser speckle contract imaging was also used to exclude the possibility of rampant blood clotting near the orbital cavity after retro-orbital sinus injection of T+RB (Supplementary Figure 1), as well as, widespread fibrin deposition in the contralateral hemisphere that was not subjected to laser illumination (Supplementary Figure 2).</li>
	<li>Apply eye ointment on both eyes to prevent dryness.</li>
	<li>Apply the illuminator with a 532 nm laser light (with 0.5 mW energy) on the drilled site with 2-inch distance for 20 min. Visualize the illumination on the proximal branch of MCA through a laser protection goggle (Figure 1C,D). 
	NOTE: The MCA with 532 nm illumination shows red fluorescence under the goggle. The distal MCA will disappear after 10 min illumination. Exclude the animal if the distal MCA flow is still present after 20 min illumination.</li>
	<li>Stop the laser illumination after 20 min. Close the wound with sterile wound clips.</li>
</ol>
4. Intravital imaging (optional)
NOTE: To characterize the thrombus formation in-vivo, use intraviral imaging by a spin-disk confocal with photoactivation system23.
<ol>
	<li>Make a cranial window ~3 mm in diameter on the parietal bone of skull.</li>
	<li>Place a coverglass on the cranial window and locate the distal MCA (~50 &#956;m in diameter) under a 20x water-immersion objective.</li>
	<li>Label the circulating platelet by tail vein injection of DyLight488-conjugated anti-GPIb&#946; antibody (0.1 mg/kg) at 5 min before imaging.</li>
	<li>Inject the mixture solution of thrombin (80 U/kg) and Rose bengal (50 mg/kg) by retro-orbital at 5 min before imaging.</li>
	<li>Photoactivate the MCA using a 561 nm laser system with laser beam 10 &#956;m in diameter and record the image until the thrombus formation.</li>
</ol>
5. tPA administration
<ol>
	<li>Place the anesthetized animal on a 37 &#176;C warm pad. At the selected post-photoactivation time-point, wet a gauze with ~45 &#176;C warm water and wrap it on the tail for 1 min.</li>
	<li>Inject recombinant human tPA (10 mg/kg) through the tail vein with a 50% bolus and 50% over 30 min by infusion pump. 
	NOTE: Although the clinical dose of recombinant human tPA for acute ischemic stroke treatment is 0.9 mg/kg, a higher dose (10 mg/kg) is commonly used in rodents to compensate for reduced cross-species tPA reactivity. We also followed the standard protocol of tPA-administration in preclinical stroke models, using 50% as a bolus and 50% infused through the tail vein over 30 min.24</li>
</ol>
6. Monitor of cerebral blood flow (CBF)
NOTE: To confirm CBF recovery after tPA treatment, use a two-dimensional laser speckle contrast imaging system15 and record immediately after photothrombosis (step 3.9) or at 24 h after tPA treatment.
<ol>
	<li>Place anesthetized animal in the prone position and make a midline incision on the scalp with the skull exposed.</li>
	<li>Moisturize the skull with sterile saline and gently apply the ultrasound gel on the skull. Avoid any hair and bubble in the gel, which will interfere the CBF signal.</li>
	<li>Monitor CBF in both cerebral hemispheres under laser speckle contrast imager for 10 min.</li>
	<li>After recording the CBF image, close the scalp with tissue glue and return the animal to the cage.</li>
	<li>Analyze CBF in the selected regions and calculate the CBF recovery percentage compared to contralateral region.</li>
	<li>Then, place the animal back to a warm cage for recovery. Monitor the mice for 5-10 min until they recover from anesthesia. Place wetted food in the cage and return it to the animal care facility. 
	NOTE: Provide post-op analgesia as recommended by the local institutional guidelines.</li>
</ol>
7. Infarct volume measurement by triphenyl tetrazolium chloride (TTC) staining
<ol>
	<li>Twenty-four hours after photothrombosis, deeply anesthetize the animal per the local institutional guidelines for nonsurvival surgery. 
	NOTE: We administer tribromoethanol (avertin) 250 mg/kg via intraperitoneal (IP) injection.</li>
	<li>Perform transcardial perfusion with PBS, collect fresh brain and embed in 3% agar gel.</li>
	<li>Section the brain slice with 1 mm thickness by vibratome and incubate in 2% TTC solution for 10 min.</li>
	<li>Quantify the total infarct volume from 6 brain slices as the absolute volume by ImageJ software. 
	NOTE: Brain edema was not used as an outcome measurement for two reasons. First, the TTC stain measures tissue viability (via the mitochondrial reduction activity) which is a more severe consequence than edema. Second, as infarction proceeds, both vasogenic and cytotoxic edema occur and cannot be easily distinguished by the standard brain edema measurement methods. However, we have used anti-immunoglobin (IgG) labeling to assess the integrity of blood-brain-barrier (BBB), and found comparable IgG-extravasation at 6 h after photoactivation in both RB and T+RB stroke models (Supplementary Figure 3).</li>
</ol>
8. Thrombus formation measurement
NOTE: To measure the thrombus formation, collect the brain at 1 h and 2 h after photothrombosis for thrombus measurement in MCA by immunochemistry (IHC) and for fibrin measurement in brain hemisphere by immunoblot, respectively.
<ol>
	<li>Perform the IHC for the characterization of clot composition. Fix the brain with 4% paraformaldehyde overnight and then dehydrate the brain with 30% sucrose for the OCT embedding.</li>
	<li>Section the brain with sagittal orientation in 20 &#956;m thickness, and perform the IHC with specific antibodies against fibrinogen, platelet (glycoprotein IIb), red blood cell (TER119) and blood vessel (isolectin GS-IB4).</li>
	<li>Perform the measurement of fibrin in brain hemisphere by immunoblot with an antibody against fibrinogen.</li>
</ol>

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The authors have nothing to disclose.

The traditional RB photothrombotic stroke, introduced in 1985, is an attractive model of focal cerebral ischemia for simple surgical procedures, low mortality, and high reproducibility of brain infarction.5 In this model, the photodynamic dye RB rapidly activates platelets upon light excitation, leading to dense aggregates that occlude the blood vessel5,8,23. However, the small amount of fibrin in RB-indu...

Endovascular thrombectomy and tPA-mediated thrombolysis are the only two U.S. Food and Drug Administration (FDA)-approved therapies of acute ischemic stroke, which afflicts ~700,000 patients annually in the United States1. Because the application of thrombectomy is limited to large vessel occlusion (LVO), while tPA-thrombolysis may alleviate small vessel occlusions, both are valuable therapies of acute ischemic stroke2. Moreover, the combination of both therapies (e.g., initiation of tPA-thrombolysis within 4.5 hours of stroke onset, followed by thrombectomy) improves reperfusion and the functional outcomes3. Thus, optimizing thrombolysis remains an important goal for stroke research, even in the era of thrombectomy.
Thromboembolic models are an essential tool for preclinical stroke research aiming to improve thrombolytic therapies. This is because mechanical vascular occlusion models (e.g., intraluminal suture MCA occlusion) do not produce blood clots, and its fast recovery of cerebral blood flow after the removal of mechanical occlusion is overly idealized4,5. To date, major thromboembolic models include photothrombosis6,7,8, topical ferric chloride (FeCl3) application9, microinjection of thrombin into the MCA branch10,11, injection of ex vivo (micro)emboli into the MCA or common carotid artery (CCA)12,13,14, and transient hypoxia-ischemia (tHI)15,16,17,18. These stroke models differ in the histological composition of ensuing clots and the sensitivity to tPA-mediated lytic therapies (Table 1). They also vary in the surgical requirement of craniotomy (needed for in situ thrombin injection and topical application of FeCl3), the consistency of infarct size and location (e.g., CCA-infusion of microemboli yield very variable outcomes), and global effects on the cardiovascular system (e.g., tHI increases the heart rate and cardiac output to compensate for hypoxia-induced peripheral vasodilation).
The RB dye-based photothrombotic stroke (PTS) model has many attractive features, including simple craniotomy-free surgical procedures, low mortality (typically &#60; 5%), and a predictable size and location of infarct (in the MCA-supplying territory), but it has two major limitations.8 The first caveat is weak-to-nil response to tPA-mediated thrombolytic treatment, which is also a drawback of the FeCl3 model7,19,20. The second caveat of PTS and FeCl3 stroke models is that the ensuing thrombi consist of densely-packed platelet aggregates with a small amount of fibrin, which not only lead to its resilience to tPA-lytic therapy, but also deviates from the pattern of intermixed platelet:fibrin thrombi in acute ischemic stroke patients21,22. In contrast, the in situ thrombin-microinjection model mainly comprises polymerized fibrin and a uncertain content of platelets10.
Given the above reasoning, we hypothesized that admixture of RB and a sub-thrombotic dose of thrombin for MCA-targeted photoactivation through thinned skull may increase the fibrin component in the resultant thrombi and boost the sensitivity to tPA-mediated lytic treatment. We have confirmed this hypothesis,23 and herein we describe detail procedures of the modified (T+RB) photothrombotic stroke model.

<table>
	<tbody>
		<tr>
			<td>2,3,5-triphenyltetrazolium chloride (TTC)</td>
			<td>Sigma</td>
			<td>T8877</td>
			<td>infarct</td>
		</tr>
		<tr>
			<td>4-0 Nylon monofilament suture</td>
			<td>LOOK</td>
			<td>766B</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>5-0 silk suture</td>
			<td>Harvard Apparatus</td>
			<td>624143</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>543nm laser beam</td>
			<td>Melles Griot</td>
			<td>25-LGP-193-249</td>
			<td>photothrombosis</td>
		</tr>
		<tr>
			<td>adult male mice</td>
			<td>Charles River</td>
			<td>C57BL/6</td>
			<td>10~14 weeks old (22~30 g)</td>
		</tr>
		<tr>
			<td>Anesthesia bar for mouse adaptor</td>
			<td>machine shop, UVA</td>
			<td></td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Avertin (2, 2, 2-Tribromoethanol)</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td>euthanasia</td>
		</tr>
		<tr>
			<td>Dental drill</td>
			<td>Dentamerica</td>
			<td>Rotex 782</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Digital microscope</td>
			<td>Dino-Lite</td>
			<td>AM2111</td>
			<td>brain imaging</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus</td>
			<td>SZ40</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Fine curved forceps (serrated)</td>
			<td>FST</td>
			<td>11370-31</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>Fine curved forceps (smooth)</td>
			<td>FST</td>
			<td>11373-12</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>goat anti-rabbit Alexa Fluro 488</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Halsted-Mosquito hemostats</td>
			<td>FST</td>
			<td>13008-12</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>Heat pump with warming pad</td>
			<td>Gaymar</td>
			<td>TP700</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>infusion pump</td>
			<td>KD Scientific</td>
			<td>200</td>
			<td>thrombolytic treatment</td>
		</tr>
		<tr>
			<td>Insulin syringe with 31G needle</td>
			<td>BD</td>
			<td>328291</td>
			<td>photothrombosis</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>CCM, UVA</td>
			<td></td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Laser protective google 532nm</td>
			<td>Thorlabs</td>
			<td>LG3</td>
			<td>photothrombosis</td>
		</tr>
		<tr>
			<td>Meloxicam SR</td>
			<td>CCM, UVA</td>
			<td></td>
			<td>NSAID analgesia</td>
		</tr>
		<tr>
			<td>micro needle holders</td>
			<td>FST</td>
			<td>12060-01</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>micro scissors</td>
			<td>FST</td>
			<td>15000-03</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>MoorFLPI-2 blood flow imager</td>
			<td>Moor</td>
			<td>780-nm laser source</td>
			<td>Laser Speckle Contrast Imaging</td>
		</tr>
		<tr>
			<td>Mouse adaptor</td>
			<td>RWD</td>
			<td>68014</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Puralube Vet ointment</td>
			<td>Fisher</td>
			<td>NC0138063</td>
			<td>eye dryness prevention</td>
		</tr>
		<tr>
			<td>Retractor tips</td>
			<td>Kent Scientific</td>
			<td>Surgi-5014-2</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Rose Bengal</td>
			<td>Sigma</td>
			<td>198250</td>
			<td>photothrombosis</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T7513</td>
			<td>photothrombosis</td>
		</tr>
		<tr>
			<td>Tissue glue</td>
			<td>Abbott Laboratories</td>
			<td>NC9855218</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>tPA</td>
			<td>Genetech</td>
			<td>Cathflo activase 2mg</td>
			<td>thrombolytic treatment</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Stoelting</td>
			<td>51425</td>
			<td>TTC infacrt</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>CCM, UVA</td>
			<td></td>
			<td>anesthesia</td>
		</tr>
	</tbody>
</table>

a fibrin-enriched and tpa-sensitive photothrombotic stroke model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

First, we compared the fibrin content in RB versus T+RB photothrombosis-induced blood clots. Mice were sacrificed by transcardial perfusion of fixatives at 2 h after photoactivation, and brains were removed for immunofluorescence staining of the MCA branch in longitudinal and transverse planes. In RB photothrombosis, the MCA branch was densely packed with CD41+ platelets and little fibrin (Figure 2A,C). In contrast, the MCA branch in T+RB photothrombosis was occlu...

Watch this Scientific Journal Video about A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model at JoVE.com

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

a-fibrin-enriched-and-tpa-sensitive-photothrombotic-stroke-model

Research

JoVE Journal

Neuroscience

2.5K Views.  National Yang-Ming University School of Medicine. Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

Video: A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume three-dimensionally. While MRI is an extremely sensitive tool for detecting tissue abnormalities, association of signal changes with an underlying pathological process is usually not straightforward. In the central nervous system, for example, inflammation, demyelination, axonal damage, gliosis, and neuronal death may all induce similar findings on MRI. As such, interpretation of MRI scans depends on the context, and radiological-histopathological correlation is therefore of the utmost importance. Unfortunately, traditional pathological sectioning of brain tissue is often imprecise and inconsistent, thus complicating the comparison between histology sections and MRI. This article presents novel methodology for accurately sectioning primate brain tissues and thus allowing precise matching between histology and MRI. The detailed protocol described in this article will assist investigators in applying this method, which relies on the creation of 3D printed brain slicers. Slightly modified, it can be easily implemented for brains of other species, including humans.

The overall goal of this protocol is to accurately align magnetic resonance imaging (MRI) image volumes with histology sections via the creation of customized 3D-printed brain holders and slicer boxes.

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated &#937;-shape profile until it merges completely at the plasma membrane.
To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C &#948; (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal ...

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...