Funded by NHLBI R01HL091905 (SE), the United Leukodystrophy Foundation CADASIL research grant (FD) and AHA 15POST247200 (PWP). The authors would like to thank Samantha P. Ahchay for providing the image on Figure 1, and Dr. Gerry Herrera, Ph.D., for providing critical comments on the manuscript.

1. Cannula and Chamber Preparation
<ol>
	<li>Insert clean borosilicate glass capillaries (outer diameter: 1.2 mm; internal diameter: 0.69 mm; 10 mm in length) into the grooves of a pipette puller with a platinum filament (diameter: 100 &#181;m).</li>
	<li>Using appropriate settings, pull the capillary to generate a cannula with a long and thin tip (Figure 2) using a micropipette puller. The settings used are: Heat - 700, Pull - 100, Velocity - 50, Time - 10.</li>
	<li>Insert cannula into the holder of pressure myograph chamber. Align the cannulas appropriately.</li>
	<li>Carefully break the tips of the cannulas by using a forceps under the dissecting microscope to the desired diameter. Here we show a cannula with a 10 &#181;m tip (Figure 2).</li>
	<li>Fill both cannulas with artificial cerebrospinal fluid (aCSF) containing 1.8 mM Ca2+; fill the chamber with Ca2+-free aCSF supplemented with 1% bovine serum albumin (BSA) + 10 &#181;M Diltiazem (all solutions should be prepared fresh before the start of the experiment). Depending on the size of the chamber, vary the volume of aCSF between 5 to 20 ml. Store the chamber at 4 &#7506;C until ready to start cannulation. 
	NOTE: Diltiazem is a reversible L-type voltage gated calcium inhibitor that will cause dilation of the arterioles, which facilitates cannulation</li>
</ol>
2. Isolation of Parenchymal Arterioles
<ol>
	<li>Euthanize the animal immediately prior to dissection using standard protocols in the laboratory that are approved by the institution&#39;s animal care committee. The use of barbiturates or carbon dioxide is preferred, as other commonly used anesthetics may have vasodilatory effects in the cerebral circulation 15. All animal procedures shown here have been approved by Institutional Animal Care and Use Committee from the University of Nevada School of Medicine, and are in agreement with the National Institutes of Health &#34;Guiding Principles in the Care and Use of Animals&#34;.</li>
	<li>Euthanize the animal using carbon dioxide. Before decapitation, ensure that the animal has stopped breathing and is unresponsive when pinching its paws.</li>
	<li>Decapitate the animal using a sharp scissor (mouse) or guillotine (rat). Remove the skin from the animal&#39;s head and cut along the midline cranial suture using a fine tip scissor. Using a blunt forceps (mouse) or a bone plier carefully remove the cranial bones and expose the cerebrum. Remove the brain slowly and carefully as to not damage surface pial arteries. Place the brain into a container filled with 20 ml of Ca2+-free aCSF supplemented with 1% BSA on ice.</li>
	<li>Fill a dissection dish containing a pad with ice-cold Ca2+-free aCSF supplemented with 1% BSA and transfer the brain with the ventral surface facing upwards to this dish (Figure 2A). Place the dish under a dissecting microscope (Figure 3A). Pin the brain to the pad using 27-gauge needles. Be careful to avoid placing pins near the middle cerebral artery (MCA).</li>
	<li>Localize the Circle of Willis and the MCA branching from it. Using sharp and aligned Vannas spring scissors, cut a small rectangle around the MCA. Ensure that rectangle is close to 5 mm across through the entire length of the MCA. The top part of the rectangle should be past the branching point from the Circle of Willis (proximal to the Circle of Willis, Figure 2B, arrow).</li>
	<li>Using the Vannas spring scissors, perform an undercut into the depth of the tissue for approximately 2 - 3 mm. 
	NOTE: This part is critical for good isolation of parenchymal arterioles, and it may take a few trials to get it right. If the cut is note made deep enough, the isolated parenchymal arterioles will be short; if the cut is too deep, the arterioles will break at their branching point when the dissected tissue is removed.</li>
	<li>Pin down the most distal end of the brain slice into the dissecting dish with the MCA facing upwards (distal from the Circle of Willis) using insect pins. Make a shallow incision to cut the pia near the pin, being careful not to cut too deep into the underlying cerebral cortex (otherwise the pins will not hold).</li>
	<li>Carefully grab each side of the pia with small forceps and start peeling the pia from cortex. If necessary, hold on to the MCA, ensuring that the surrounding pial membrane is not damaged.</li>
	<li>Keep peeling until the pia containing the MCA is free from the cortex. Note that the resistance against peeling will increase close to the Circle the Willis. Be extra careful at this point, because the longer parenchymal arterioles will be in this region.</li>
	<li>Once the pia is free, place it carefully on top of the pad on the dissecting dish with the surface opposite to parenchymal arterioles facing down (Figure 2B).</li>
	<li>Using Vannas spring scissors, cut out the dissected arterioles free from the MCA, making sure the ends of the vessel are cut bluntly.</li>
	<li>Collected arterioles can be used for pressure myography, molecular analysis, or isolation of native smooth muscle or endothelial cells.</li>
</ol>
3. Pressure Myography
<ol>
	<li>Prepare the suture ahead of time. Cut the dark green nylon thread into 5 mm pieces and place on the sticky side of a double-sided tape on a Petri dish. Under the dissecting microscope, separate each piece into its filaments using fine forceps, and loosely prepare a simple half-hitch knot by passing one end the filament into the other.</li>
	<li>Using forceps, pick a knot off the double-sided tape, making sure not to grab it with too much pressure. Pull the suture into bath solution, slide knot onto cannula and tighten it at a distance from the tip of the cannula. Repeat this process to have 2 sutures on each cannula.</li>
	<li>Coat a glass micropipette with a 10% BSA solution and then rinse off the excess with Ca2+-free aCSF supplemented with 1% BSA.</li>
	<li>Pull 50 &#181;l of solution into the glass micropipette, pull up a parenchymal arteriole and transfer to the pressure myography chamber. Push plunger in one fluid motion to dispense the parenchymal arteriole into the chamber to prevent it from sticking to the internal chamber of the glass micropipette.</li>
	<li>Using two super fine forceps, open the lumen of the PA, being careful to only touch the very end of the vessel.</li>
	<li>Using the forceps to hold the edges of the parenchymal arteriole, carefully pull the vessel onto the cannula. Pull far enough on the cannula so that the vessel is not at the very tapered end of the cannula (Figure 3A).</li>
	<li>Loosen the 2 sutures on the cannula and tighten them on the vessel (Figure 3B). Space the sutures apart a little on the vessel, and pull toward the operator while tighten it. Make sure that any branches are tied off before closing the opposite end of the parenchymal arteriole.</li>
	<li>Turn chamber around and close the opposite end of the parenchymal arteriole as a blind-sac. To achieve that, open the ties on the opposite cannula and pass it onto the arteriole. Slowly close the knot in such a way that the parenchymal arteriole will be tied to the side of the cannula. Avoid longitudinal stretch of the arteriole. (Figure 3C). 
	NOTE: If the goal of the study is to perfuse drugs through the lumen, cannulate the opposite end instead of tying the parenchymal arteriole to the side of the cannula. Make sure the cannulas do not have any salt crystals or internal build-ups that block intraluminal flow. Regulate flow rate appropriately in order to maintain shear stress within physiological levels. A study by Shih et al. shows flow rates inside penetrating arterioles in rats 8, and can serve as an initial guide for pilot studies.</li>
	<li>Carefully transfer the chamber to the stage of the microscope to be used for recording vessel diameter. Attach the chamber onto the microscope stage, connecting temperature probe and inlet and outlets for perfusion to the correct tubes. Pressurize the arteriole to 40 mmHg intraluminal pressure for mouse parenchymal arterioles or 50 mmHg for rat arterioles, using a pressure system available in the laboratory (water column, peristaltic pump coupled to a pressure transducer, etc.). Figure 4D shows an example of a peristaltic pump coupled to a pressure transducer to pressurize the cannulated arteriole.</li>
	<li>Turn on the system used to detect changes in diameter (Figure 3E). Adjust the settings on the microscope, such as illumination and contrast, in order to obtain the best detection possible. Ideally the walls of the arteriole should be dark and the lumen translucent. Once the detection is appropriate, start recording the experiment.</li>
	<li>Start the superfusion system. Wash the cannulated, pressurized parenchymal arteriole with warm (37 &#7506;C) aCSF containing 1.8 mM Ca2+ for 15 to 20 min at a flow rate between 3 - 5 ml/min. This aCSF should not contain Diltiazem or BSA.</li>
	<li>Replace the regular aCSF with 60 mM KCl aCSF to evaluate the viability of the preparation. At this point PAs should exhibit 10 - 20% constriction to 60 mM KCl. If the arteriole does not constrict to that extent, remove and cannulate another arteriole.</li>
	<li>Wash out the 60 mM KCl with regular aCSF. Wait until generation of spontaneous myogenic tone, which could take up to one hour. If arteriole does not have myogenic tone by then, replace it with another arteriole. 
	NOTE: Myogenic tone is observed as a gradual, and sometimes slow, reduction in the lumen diameter of the vessel without stimulation by contractile agonists or high KCl solution. Myogenic tone is calculated by the following formula: (1 - (active lumen diameter/passive lumen diameter)) x&#160;100 5. An appropriate amount of myogenic tone may vary according to treatments, strains, species, transgenics, etc. In general, physiological myogenic tone ranges from 15 - 30%5.</li>
</ol>
4. Example Pressure Myography Experiments: Agonist-induced Constriction and Myogenic Reactivity
<ol>
	<li>Prepare a series of dilutions of an agonist of choice in aCSF using appropriate concentrations. For the current example the thromboxane A2 receptor agonist U46619 was used. A stock solution of 1 ml of U46619 at a concentration of 1 mM was prepared. Transfer 100 &#181;l of the 1 mM solution to a new tube containing 900 &#181;l of aCSF to make a 10-fold dilution of the drug, resulting in a solution containing 100 &#181;M U46619. Repeat this process for 6 dilutions, ranging from 1 mM to 100 nM to prepare a concentration-response constrictor curve.</li>
	<li>Add the entire volume of the solution containing the agonist (1 ml for the first dose, followed by 900 &#181;l of all subsequent doses) to 100 ml of superfusing aCSF. This will lead to an extra 100-fold dilution of the agonist. Thus, the final concentrations of agonist in the bath range from 10 pM to 10 &#181;M. Allow arterioles to incubate for ~10 min in each concentration, until luminal diameter reaches a steady-state.</li>
	<li>Wash out the agonist by superfusing PAs with aCSF without any drugs until the PA diameter is back to the original value. Incubate the parenchymal arteriole in Ca2+-free aCSF (without BSA) supplemented with 10 &#181;M Diltiazem + 2 mM EGTA to induce maximum dilation and record passive diameter of the arteriole.</li>
	<li>Remove the parenchymal arteriole from the cannula by carefully pulling it out while holding into the end of the ties. Wash the vessel chamber with double deionized water to remove debris and excess of agonist. Fill the chamber with Ca2+-free aCSF + BSA and cannulate another arteriole. It is not recommended to perform more than one experiment per arteriole.</li>
	<li>To perform a myogenic reactivity experiment, allow the parenchymal arteriole to equilibrate at 40 mmHg intraluminal pressure until spontaneous myogenic tone is generated (described above). Reduce the intraluminal pressure to 5 mmHg and allow PA to equilibrate for ~5 - 10 min, until luminal diameter reaches steady state. Increase the intraluminal pressure stepwise using the interval of choice (for example from 5 to 140 mmHg in 20 mmHg increments).</li>
	<li>Do not expose the arteriole to intraluminal pressure below 5 mmHg, as that could cause collapse and damage the endothelium, thus altering the outcomes of the experiment.</li>
	<li>At the end of the pressure curve, superfuse the parenchymal arteriole with Ca2+-free aCSF (without BSA) supplemented with 10 &#181;M Diltiazem + 2 mM EGTA and repeat the pressure steps, starting at the lowest pressure. 
	NOTE: This will give the passive diameters of the PA, which are necessary to calculate % myogenic tone, defined by the formula: % tone = (1 - (active diameter/passive diameter)) x 100.</li>
</ol>

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The authors have nothing to disclose.

Cerebral parenchymal arterioles are high resistance arterioles with few anastomoses and branches that perfuse distinct neuronal populations. These specialized blood vessels are central players in cerebrovascular autoregulation and neurovascular coupling through astrocyte-mediated vasodilation 1. The importance of these specialized blood vessels in cerebral vascular disease has been known for approximately 50 years, when the pioneering work of Dr. Miller Fisher described structural alterations in parenchymal ar...

The cerebral circulation is uniquely organized to support the metabolic demands of central neurons, cells that have limited energy stores and are consequently highly sensitive to changes in oxygen pressure and supply of necessary nutrients. As particular neuronal subpopulations becomes active when specific tasks are performed, the vasculature promotes a highly localized increase in perfusion to prevent local hypoxia and depletion of nutrients 1. This is a form of functional hyperemia known as neurovascular coupling, and is dependent on the proper operation of the neurovascular unit, composed of active neurons, astrocytes, and cerebral arteries 2. Intracerebral parenchymal arterioles, a group of blood vessels encompassing parenchymal, penetrating and pre-capillary arterioles, are centrally important for this response and it is then critical to study them individually in order to investigate neurovascular coupling 3.
Parenchymal arterioles are small (20 - 70 &#181;m internal diameter) high-resistance blood vessels that perfuse distinct neuronal populations within the brain. Branching out from pial arteries on the surface, parenchymal arterioles penetrate into the brain parenchyma at a nearly 90&#7506; angle to feed the subsurface microcirculation (Figure 1). These arterioles play a critical role in maintaining appropriate perfusion pressure as they are the most distal smooth muscle-containing vessels protecting the capillaries. In contrast to the surface pial circulation, parenchymal arterioles lack collateral branches and anastomoses, and consequently are &#34;bottlenecks&#34; of the cerebral circulation 4. As a result, dysfunction of parenchymal arterioles contributes to the development of cerebrovascular diseases such as vascular cognitive impairment and small ischemic strokes (also known as silent or lacunar strokes). Studies indicate that parenchymal arterioles dysfunction can be induced by essential hypertension 5, chronic stress 6, and is an early event in small vessel disease genetic mouse model 7. Further, experimentally-induced occlusion of single penetrating arterioles in rats is sufficient to cause small ischemic strokes that are cylindrical in shape, similar those observed in older humans 8.
In addition to these anatomical distinctions, mechanisms regulating contractile function differ between pial arteries and parenchymal arterioles. Myogenic vasoconstriction is greater in parenchymal arterioles 9, possibly because of the lack of extrinsic innervation 10, distinct modes of mechanotransduction 11, and differences in intracellular Ca2+ signaling 12,13 in vascular smooth muscle cells. Evidence suggests that endothelium-dependent vasodilator mechanisms also differ between these vascular segments, with parenchymal arteries exhibiting greater reliance on mechanisms involving Ca2+-activated K+ channels and electrotonic communication within the vascular wall compared with diffusible factors such as nitric oxide and prostacyclins 14. Therefore, data gathered in experiments using pial arteries may not necessarily apply to parenchymal arterioles, leaving a gap in our knowledge of local control of cerebral perfusion.
Despite their importance, parenchymal arterioles are vastly under-studied, primarily due to the technical challenges with isolation and mounting for ex vivo study. In this manuscript we describe a methodology to isolate and cannulate cerebral parenchymal arterioles, which can be used for pressure myography, or to isolate tissue for immunolabeling, electrophysiology, molecular biology, and biochemical analysis.

<table border="0" cellpadding="0" cellspacing="0" width="629">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">artificial Cerebrospinal Fluid</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:216px;">NaCl</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">S-640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">P217</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MgCl Anhydrous</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">M-8266</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NaHCO3</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">S233</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NaH2PO4</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D-(+)-Glucose</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">G2870</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">CaCl2</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:127px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isolation/ Cannulation</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo Microscope</td>
			<td style="width:127px;">Olympus</td>
			<td style="width:119px;">SZX7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:216px;">Super Fine Forceps</td>
			<td style="width:127px;">Fine Science Tools</td>
			<td style="width:119px;">11252-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:216px;">Vannas Spring Scissors</td>
			<td style="width:127px;">Fine Science Tools</td>
			<td style="width:119px;">15000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Wiretrol 50 &#956;L</td>
			<td style="width:127px;">VWR Scientific</td>
			<td style="width:119px;">5-000-1050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.2 &#956;m Sterile Syringe Filter</td>
			<td style="width:127px;">VWR Scientific</td>
			<td style="width:119px;">28145-477</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropipette Puller</td>
			<td style="width:127px;">Sutter Instruments</td>
			<td style="width:119px;">P-97</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Borosilicate Glass O.D.: 1.2 mm, I.D.: 0.68 mm</td>
			<td style="width:127px;">Sutter Instruments</td>
			<td style="width:119px;">B120-69-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dark Green Nylon Thread</td>
			<td style="width:127px;">Living Systems Instrumentation</td>
			<td style="width:119px;">THR-G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Linear Alignment Single Vessel Chamber</td>
			<td style="width:127px;">Living Systems Instrumentation</td>
			<td style="width:119px;">CH-1-LIN</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pressure Servo Controller with Peristaltic Pump</td>
			<td style="width:127px;">Living Systems Instrumentation</td>
			<td style="width:119px;">PS-200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Video Dimension Analyzer</td>
			<td style="width:127px;">Living Systems Instrumentation</td>
			<td style="width:119px;">VDA-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Four Channel Recorder with LabScribe 3 Recording and Analysis Software</td>
			<td style="width:127px;">Living Systems Instrumentation</td>
			<td style="width:119px;">DAQ-IWORX-404</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heating Unit</td>
			<td style="width:127px;">Warner Instruments</td>
			<td style="width:119px;">64-0102</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Automatic Temperature Controller</td>
			<td style="width:127px;">Warner Instruments</td>
			<td style="width:119px;">TC-324B</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and cannulation of cerebral parenchymal arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

Figure 5A shows a representative constriction of mouse PAs to 60 mM KCl aCSF to evaluate the integrity of the preparation. PAs should constrict between 15 - 30% in the presence of 60 mM KCl. If the constriction is below 15%, discard the PA and cannulate another one, as it suggests that the arteriole was damaged during the isolation and cannulation process.
Figure 5B illustrates PA constriction t...

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Isolation and Cannulation of Cerebral Parenchymal Arterioles

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

isolation-and-cannulation-of-cerebral-parenchymal-arterioles

Research

JoVE Journal

Neuroscience

11.5K Views.  University of Nevada School of Medicine. This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

Video: Isolation and Cannulation of Cerebral Parenchymal Arterioles

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher 
spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis 
and treatment of neurovascular diseases such as stroke, Alzheimer's disease, and head injury.
Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. 
Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.
Click here to view the related article <a href="http://www.jove.com/Details.php?ID=1731">Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.</a>

We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

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 ...

Comprehensive Endovascular and Open Surgical Management of Cerebral Arteriovenous Malformations

Arteriovenious malformations (AVMs) are associated with significant morbidity and mortality, and have a rupture risk of ~3% per year. Treatment of AVMs must be tailored specifically to the lesion, with surgical resection being the gold standard for small, accessible lesions. Pre-operative embolization of AVMs can reduce nidal blood flow and remove high-risk AVM features such as intranidal or venous aneurysms, thereby simplifying a challenging neurosurgical procedure. Herein, we describe our approach for the staged endovascular embolization and open resection of AVMs, and highlight the advantages of having a comprehensively trained neurovascular surgeon leading a multi-disciplinary clinical team. This includes planning the craniotomy and resection to immediately follow the final embolization stage, thereby using a single session of anesthesia for aggressive embolization, and rapid resection. Finally, we provide a representative case of a 22-year-old female with an unruptured right frontal AVM diagnosed during a seizure workup, who was successfully treated via staged embolizations followed by open surgical resection.

Surgery is the gold standard for accessible arteriovenious malformations (AVMs), and pre-operative embolization can simplify this procedure. We describe our approach for staged endovascular embolization and open resection of AVMs, and provide a representative clinical example highlighting the advantages of a comprehensively trained neurovascular surgeon leading a multi-disciplinary clinical team.

Arteriovenious malformations (AVMs) are associated with significant morbidity and mortality, and have a rupture risk of ~3% per year. Treatment of AVMs ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...