This work was supported by Paris VI University and a Pierre Fabre Innovation grant.

All procedures were performed in accordance with European Community standards for the care and use of laboratory animals, with the approval of the local ethics committee for animal experimentation (Ile de France-Paris-Committee, Authorization 4270).
1. Anesthesia
<ol>
	<li>Infuse a lethal dose of pentobarbital (up to 1 mg/10 g body weight) intraperitoneally (27-gauge needle and 1-ml syringe) into adult mice before surgery.</li>
</ol>
2. Vessel Perfusion
NOTE: There is no need to apply vet ointment to the eyes during vessel perfusion. This procedure is rapid (5-10 minutes) and ends in the death of the animal. Confirm the lack of response with a toe pinch.&#160;
<ol>
	<li>Using iris scissors, make an incision, about 4 cm long, into the abdominal wall and peritoneum, just beneath the rib cage.</li>
	<li>Make a small incision (a few millimeters long) in the diaphragm and then continue the incision of the diaphragm along the entire length of the rib cage to expose the pleural cavity.</li>
	<li>Lift the sternum away and clamp the tip of the sternum with the hemostat; place the hemostat on the neck. Carefully trim the adipose tissue connecting the sternum to the heart.</li>
	<li>Pass the 15-gauge perfusion needle through the left ventricle into the apex of the heart.</li>
	<li>Finally, use scissors to cut one of the liver lobes to create an outlet. 
	NOTE: An alternative outlet can be created by using iris scissors to create an incision to the right atrium.</li>
	<li>Perfuse the animal with 25 to 50 ml of phosphate-buffered saline (PBS) with a pump operating at a rate of 2.5 ml/min. The liver should blanch as the blood is replaced with PBS.</li>
	<li>After approximately five minutes, once the fluid from the liver is completely clear, stop the perfusion.</li>
	<li>If immunostaining or regular staining is planned, perfuse the animal with 50 ml of paraformaldehyde (PFA; 4% in PBS) for 15 min. 
	NOTE: Caution, PFA fumes are toxic. Perfusion of the animal with PFA should be carried out in a ventilated fume hood.</li>
</ol>
3. Isolation of the Brain and the Circle of Willis
<ol>
	<li>Isolation of the brain
	<ol>
		<li>Remove the head with a pair of surgical scissors.</li>
		<li>Make a midline incision with iris scissors, along the skin from the neck to the nose.</li>
		<li>Trim off the skin to expose the skull and remove any residual muscles and adipose tissues with iris scissors.</li>
		<li>Place the sharp end of the iris scissors into the foramen magnum on one side and carefully slide them along the inner surface of the skull to the external auditory meatus (also known as the ear canal).</li>
		<li>Reproduce the incision described in 3.1.4 on the contralateral side and make a midline cut along the inner surface of the inter-parietal bone to the start of the sagittal suture.</li>
		<li>Plant the iris scissors in the frontal bone, right between the eyes, in the sagittal suture and then open them to split the skull in two.</li>
		<li>Lift out the brain, grabbing the olfactory bulbs and using the iris scissors to cut off the nerve connections on its ventral surface.</li>
		<li>Remove the brain and place it in a 60-mm Petri dish containing ice-cold PBS for CoW isolation. Completely immerse the brain in the PBS. If the brain was fixed with 4% PFA (for subsequent sectioning and immunostaining or regular staining), keep it in a bath of 4% PFA at 4 &#176;C for 24 hr.</li>
	</ol>
	</li>
	<li>Isolation of the circle of Willis 
	NOTE: A dissecting microscope is required for CoW isolation. The brain should be kept at 4 &#176;C throughout the entire procedure.
	<ol>
		<li>Put the brain upside down (i.e., on its dorsal surface) to visualize the CoW.</li>
		<li>Use a small forceps to grab the anterior cerebral arteries (ACA) at the base of the olfactory lobes (a, Figure 1) and exert pressure to dissociate them from the vessel continuum. Use the same procedure to cut the middle cerebral arteries (MCA) in b (Figure 1).</li>
		<li>Use the sharp ends of the forceps to lift and remove the major arteries forming the CoW from the cortex.</li>
		<li>Lift up the start of the posterior communicating arteries (PCA) to disconnect them from the brain, by gripping the middle cerebral arteries (MCA) with the forceps. Pick up the anterior arteries (ACA and MCA) and pull them gently over the optic chiasm in an anterior-dorsal direction. To prevent disruption of the CoW, interrupt the the procedure to deal with the other arteries.</li>
		<li>Repeat steps 3.2.2 and 3.2.3 for the superior and posterior cerebellar arteries (SCA)/(PCA) (c, Figure 1) and for the basilar artery (BA) (d, Figure 1), pulling them in a dorsal-anterior direction. Stop at the end of the procedure described in 3.2.4.</li>
		<li>Remove the entire CoW by pulling gently with the forceps. Place the CoW in a 60-mm Petri dish filled with ice-cold PBS and remove any remaining attached brain tissue with two forceps, holding the CoW in place with small pins.</li>
		<li>Keep the harvested CoW at -80 &#176;C for subsequent processing for RNA purification (RNA extraction yields large amounts of RNA &#8212; approximately 500 ng) or protein extraction. 
		Note: The CoW can be maintained ex vivo for 24 hr, by adapting the organ bath system developed for isolated mouse olfactory artery. 7</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54352/54352fig1.jpg" /> 
Figure 1: Schematic Diagram of a Ventral View of the Mouse Brain Highlighting the CoW. The CoW is formed from the two internal carotid arteries (MCA), which are derived from the two anterior cerebral arteries (ACA); the basilar artery (BA) branches into the posterior (PCA) and superior (SCA) cerebral arteries, and two vertebral arteries (VA).

<ol>
	<li>Beckmann, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+cerebrovascular+abnormalities+and+blood+flow+disturbances+in+APP23+mice+modeling+Alzheimer's+disease.">Age-dependent cerebrovascular abnormalities and blood flow disturbances in APP23 mice modeling Alzheimer's disease.</a> J Neurosci. 23 (24), 8453-8459 (2003).</li><li>Sadasivan, C., Fiorella, D. J., Woo, H. H., Lieber, B. 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The authors have nothing to disclose.

We describe here a reproducible protocol for the isolation of the circle of Willis. The most common cerebrovascular disorders involving the CoW are CAA-associated vasculopathies, intracranial atherosclerosis and intracranial aneurysm, all of which affect the walls of arterial vessels. The risk factors are well known, but the molecular pathogenesis of these cerebral disorders remains poorly understood and specific biological markers for predicting their occurrence are lacking. There is considerable interest in methods for isolating the CoW from transgenic mice to link macroscopic observations with molecular changes. For example, by inducing consistent aneurysms in a mouse model in which a particular gene is knocked out(as described by Hosaka et al.8) and analyzing the CoW, it should be possible to determine whether treatments targeting the protein encoded by this gene could prevent intracranial aneurysms and/or subarachnoid hemorrhages. Obviously, this method could also be used to analyze the effect of a particular drug on the progression of CAA-associated vasculopathies in the various transgenic mouse models of AD.
It is easier to isolate mouse arteries than to purify entire mouse microvessels, but such sampling is nevertheless more difficult in mice than in rats. Indeed, the rat CoW is embedded in much stiffer meninges, making it possible to isolate the whole structure in one go. In addition to the problem of the small size of the vessels in mice, this procedure is also tricky due to the transparency of the vessels following their perfusion with PBS before the removal of the brain. We therefore recommend practicing without the infusion, starting from step 3 after anesthesia to make it easier to distinguish the blood vessels. Finally, as mouse cerebral vessels are very fine and break easily, the dissection should be carried out very carefully, without rushing, to ensure that the entire structure is isolated in one piece, by first grabbing the ACA. The purity of the CoW preparation can be evaluated by comparing the levels of expression of the neuronal and vessel genes. With practice, a single CoW provides only sufficient RNA for the study of 5 - 10 gene expressions. For large-scale screening, several mouse CoWs should be pooled.
Gauthier et al.9 described a method for isolating small pieces of microvessels (based on the use of collagenase/dispase to digest fragments of brain and of glass beads to trap them) that yields smooth muscle or endothelial cells that can be maintained in culture when placed in an appropriate medium. The advantage of our method is that it isolates large vessels, without altering vessel structure. The dissection of specific branches of the CoW may also be of interest, to determine the correlation between the gene expression profile of a particular branch and possible susceptibility to cerebrovascular diseases, such as intracranial aneurysm. Such approaches may provide an explanation as to why some ACA anatomical and genetic variations are correlated with a higher prevalence of ACA aneurysms. This dissection requires pressure to be exerted with forceps at the base of each specific branch, to dissociate it from the other branches, at the start of the CoW isolation procedure.
Thus, in addition to making it possible to derive cells from the CoW 10 or to screen for gene expression 11 and protein production 12 or posttranslational protein modifications, this method can be used for ex vivo studies, by simply adapting the organ bath system developed for isolated mouse olfactory arteries. 7 Following incubation of the entire CoW in culture medium containing antibiotics, the secretome released by this specific structure can be obtained and analyzed. An analysis of this secretome in mouse models of CAA would make it possible, for example, to determine the CAA-related inflammatory status of these cerebral arteries. The resulting conditioned medium could also be used to determine the effects of the secretome on each vessel wall cell type phenotype. Pathological CoW preparations could also be used to identify possible differences in biochemical signals, such as cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), or Ca2+ concentrations, detectable with fluorescence resonance energy transfer (FRET)-based biosensors. Virus-delivered biosensors have been used on brain slice preparations containing experimentally accessible mature living neurons with a preserved morphology, leading to the detection of profound differences in D1 response between the pyramidal cortical neurons and striatal medium spiny neurons. 13
The modeling of cerebrovascular diseases in transgenic mice expressing FRET-based second messenger sensor proteins 14 for the imaging of biochemical signals in the isolated CoW should further provide further insight into the biochemistry, pathophysiology, and pharmacotherapy of these diseases.

The cerebral arterial circle (circulus arteriosus cerebri), also known as the circle of Willis (CoW), loop of Willisor Willis polygon) was first described by Thomas Willis in 1664. It is a circulatory anastomosis located around the optic chiasma and hypothalamus that can be considered as a central hub supplying blood to the brain and surrounding structures. Blood enters this structure via the internal carotid and vertebral arteries and it flows out of the circle via the interior middle and posterior cerebral arteries. Each of these arteries has left and right branches on either side of the circle. The basilar, post communicating, and anterior communicating arteries complete the circle (Figure 1 and Figure 2). The risk of impaired blood flow in any of the outflow arteries is minimized by the merging of blood entering the circle from the carotid and cerebral arteries, thereby guaranteeing that sufficient blood is supplied to the brain. This structure also serves as the main route for collateral blood flow in severe occlusive diseases of the internal carotid artery.
Several types of cerebrovascular disorders have their origin in the CoW. The most common are cerebral amyloid angiopathy (CAA)-associated vasculopathies, intracranial atherosclerosis and intracranial aneurysms. 1,2,3 These disorders may lead to hypoperfusion due to vasodilation, and intracerebral and/or subarachnoid hemorrhages ultimately translating into ischemic or hemorrhagic strokes or, at best, a transient ischemic attack. Recent advances in diagnostic procedures, including neuroimaging, possibly combined with angiography, have made it possible to diagnose these major cerebrovascular diseases clinically, without the need for a brain biopsy. Nevertheless, effective and specific treatments (pharmacological or endovascular) are currently lacking and there is therefore a need to define new molecular targets.
The identification of novel drug targets for the prevention of these diseases in humans will require animal models and ways of isolating the cerebro-vasculature including the CoW. Such models should provide evidence of and clues to the specific changes, including inflammatory changes, occurring in the walls of the large vessels in animal models of intracranial artery aneurysm, CAA or intracranial atherosclerosis. 4,5,6
We have established a method for mouse CoW isolation to facilitate studies of vessel inflammation in Alzheimer&#39;s disease (AD) and related diseases, such as CAA. This method for isolating the mouse CoW was developed for the assessment of inflammatory cerebrovascular gene expression during disease progression. Together with the detection of amyloid beta deposition within the walls of the leptomeningeal and pial arteries, this method could make it easier to determine the possible relationship between inflammatory gene expression in the cerebro-vasculature wall and A&#946;-peptide accumulation. The vascular network of the brain, including the leptomeningeal and pial in the subarachnoid space, is an extension of the large arteries forming the circle of Willis. The method described here could be used to isolate the CoW of any mouse lineage and could be used for all types of screening (e.g., gene expression, protein production and posttranslational protein modifications) on the large vessels of the mouse cerebro-vasculature.

<table border="0" cellpadding="0" cellspacing="0" width="692">
	<tbody>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:173px;">D8537</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Dumont #55 Forceps</td>
			<td style="width:173px;">Fine Science Tools</td>
			<td style="width:173px;">11295-51</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Hardened Fine Iris Scissors&#160;</td>
			<td style="width:173px;">Fine Science Tools</td>
			<td style="width:173px;">14090-11</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Scissors - Straight / Sharp / Sharp&#160;&#160; 16.5 cm</td>
			<td style="width:173px;">Fine Science Tools</td>
			<td style="width:173px;">14002-16</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Dumont #7b Forceps&#160;</td>
			<td style="width:173px;">Fine Science Tools</td>
			<td style="width:173px;">11270-20</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Stereoscopic Zoom Microscope</td>
			<td style="width:173px;">Nikon</td>
			<td style="width:173px;">SMZ745T</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">CellBIND Surface 60mm Culture Dish</td>
			<td style="width:173px;">Corning</td>
			<td style="width:173px;">#3295</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Peristaltic Pump - MINIPULS 3</td>
			<td style="width:173px;">Gilson</td>
			<td style="width:173px;">M312</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Pentobarbital Sodique</td>
			<td style="width:173px;">Ceva Sant&#233; Animale</td>
			<td style="width:173px;">FR/V/2770465 3/1992</td>
			<td style="width:173px;"></td>
		</tr>
	</tbody>
</table>

protocol for isolating the mouse circle of willis

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and hypothalamus that supplies blood to the brain and surrounding structures. It has been implicated in several cerebrovascular disorders, including cerebral amyloid angiopathy (CAA)-associated vasculopathies, intracranial atherosclerosis and intracranial aneurysms. Studies of the molecular mechanisms underlying these diseases for the identification of novel drug targets for their prevention require animal models. Some of these models may be transgenic, whereas others will involve isolation of the cerebro-vasculature, including the CoW.The method described here is suitable for CoW isolation in any mouse lineage and has considerable potential for screening (expression of genes, protein production, posttranslational protein modifications, secretome analysis, etc.) studies on the large vessels of the mouse cerebro-vasculature. It can also be used for ex vivo studies, by adapting the organ bath system developed for isolated mouse olfactory arteries.

The PBS-perfused mouse is killed and the CoW is isolated as described in section 3.2 of the protocol. When the dissection is performed correctly, the CoW should come out in one piece and should be slightly transparent due to the absence of residual blood in the vasculature.
<img alt="Figure 2" src="/files/ftp_upload/54352/54352fig2.jpg" /> 
Figure 2: The Mouse CoW after Isolation. (A) Overview of the CoW in a 10-cm Petri dish. (B) Details of the various branches of the CoW. MCA for middle cerebral arteries, ACA for anterior cerebral arteries, BA for basilar artery, PCA for posterior cerebral arteries and SCA for superior cerebral arteries. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54352/54352fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The purity of the CoW preparation can be checked by ensuring that specific vascular genes are strongly expressed whereas the expression of neuronal genes is undetectable. More specifically, the cleaned CoW should specifically express vascular smooth muscle cell markers, such as smooth muscle actin (SMA), smooth muscle-myosin heavy chain (SM-MHC) and smoothelin. These markers are barely expressed in other parts of the brain. An opposite pattern of expression should be obtained for neuronal genes, with neuronal markers (Mog, Map2) only barely detectable in the CoW.
Levels of mRNA can be assessed by RT-qPCR with normalization relative to a reference gene transcript (here, that of GAPDH). Typical results are shown in Figure 3A and B.
<img alt="Figure 3" src="/files/ftp_upload/54352/54352fig3.jpg" /> 
Figure 3: Expression of Neuronal and Contractile Genes in the Mouse Circle of Willis and Brain. The RT-qPCR results were normalized against those for a reference gene (GAPDH). They are expressed as the mean &#177; SD of 6 to 12 independent experiments; Comparison of the CoW with the brain: n.s, not significant, *, P &#60; 0.05, **, P &#60; 0.005, and ***, P &#60; 0.001. (A) Expression of contractile genes: SMA (smooth muscle actin), smoothelin, SM-MHC (smooth muscle-myosin heavy chain 11) and E-selectin. (B) Expression of neuronal genes: Mog (myelin oligodendrocyte glycoprotein) and Map2 (microtubule associated protein 2).
The expression pattern of the endothelial marker E-selectin in brain lacking the CoW is very similar to that obtained for CoW samples, possibly reflecting the existence of a brain capillary network. Table 1 lists the mean Cp numbers for each transcript.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Mean Cp</td>
			<td colspan="3">GAPDH</td>
			<td colspan="3">SMA</td>
			<td colspan="3">Smoothelin</td>
			<td colspan="3">SM-MHC</td>
			<td colspan="3">E-Sel</td>
		</tr>
		<tr>
			<td>Mean</td>
			<td>SD</td>
			<td>N</td>
			<td>Mean</td>
			<td>SD</td>
			<td>N</td>
			<td>Mean</td>
			<td>SD</td>
			<td>N</td>
			<td>Mean</td>
			<td>SD</td>
			<td>N</td>
			<td>Mean</td>
			<td>SD</td>
			<td>N</td>
		</tr>
		<tr>
			<td>CoW&#160;</td>
			<td>20.86</td>
			<td>0.93</td>
			<td>12</td>
			<td>22.02</td>
			<td>0.67</td>
			<td>12</td>
			<td>25.29</td>
			<td>1.89</td>
			<td>12</td>
			<td>23.37</td>
			<td>0.80</td>
			<td>12</td>
			<td>30.56</td>
			<td>1.66</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Brain</td>
			<td>18.71</td>
			<td>0.72</td>
			<td>6</td>
			<td>33.91</td>
			<td>2.06</td>
			<td>6</td>
			<td>31.08</td>
			<td>3.17</td>
			<td>6</td>
			<td>26.05</td>
			<td>0.73</td>
			<td>6</td>
			<td>28.45</td>
			<td>0.63</td>
			<td>6</td>
		</tr>
		<tr>
			<td>B.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Mean Cp</td>
			<td colspan="3">GAPDH</td>
			<td colspan="3">Mog</td>
			<td colspan="3">Map2</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mean</td>
			<td>SD</td>
			<td>N</td>
			<td>Mean</td>
			<td>SD</td>
			<td>N</td>
			<td>Mean</td>
			<td>SD</td>
			<td>N</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CoW&#160;</td>
			<td>21.04</td>
			<td>0.41</td>
			<td>6</td>
			<td>32.13</td>
			<td>1.63</td>
			<td>6</td>
			<td>30.21</td>
			<td>0.85</td>
			<td>6</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brain</td>
			<td>19.09</td>
			<td>0.49</td>
			<td>6</td>
			<td>26.43</td>
			<td>0.88</td>
			<td>6</td>
			<td>23.30</td>
			<td>0.99</td>
			<td>6</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Mean Crossing Point for the Expression of Neuronal and Contractile Genes in the Mouse Circle of Willis and Brain. Calculations are based on comparison of the precise cycle determined by crossing points (Cp) at a constant level of fluorescence. The crossing point is the cycle number corresponding to the maximum second derivative of the amplification curve. Higher Cp values are associated with lower levels of expression. For a given gene, expression is considered to be undetectable if the Cp exceeds 35. (A) Cp of the reference gene: GAPDH; and of contractile genes: SMA, smoothelin, SM-MHC and E-selectin. (B) Cp of the reference gene: GAPDH; and neuronal genes: Mog and Map2.

Watch this Scientific Journal Video about Protocol for Isolating the Mouse Circle of Willis at JoVE.com

Protocol for Isolating the Mouse Circle of Willis

We describe here a reproducible protocol for isolating the mouse circle of Willis.

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

13.0K Views.  Sorbonne Universités. We describe here a reproducible protocol for isolating the mouse circle of Willis.

Video: Protocol for Isolating the Mouse Circle of Willis

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...