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>
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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><tbody><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Dumont #55 Forceps</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>Hardened Fine Iris Scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>14090-11</td><td></td></tr><tr><td>Scissors - Straight / Sharp / Sharp&amp;nbsp;&amp;nbsp; 16.5 cm</td><td>Fine Science Tools</td><td>14002-16</td><td></td></tr><tr><td>Dumont #7b Forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>11270-20</td><td></td></tr><tr><td>Stereoscopic Zoom Microscope</td><td>Nikon</td><td>SMZ745T</td><td></td></tr><tr><td>CellBIND Surface 60mm Culture Dish</td><td>Corning</td><td>#3295</td><td></td></tr><tr><td>Peristaltic Pump - MINIPULS 3</td><td>Gilson</td><td>M312</td><td></td></tr><tr><td>Pentobarbital Sodique</td><td>Ceva Sant&amp;eacute; Animale</td><td>FR/V/2770465 3/1992</td><td></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.bdigitaluss.remotexs.co/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.

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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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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

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Medicine

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Immunology and Infection

Conjunctival Commensal Isolation and Identification in Mice

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an &#8220;in-lab&#8221; made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Hormonal, Metabolic, and Electrical Regulation of Isolated Hypothalamic Neurons

Isolation of Targeted Hypothalamic Neurons for Studies of Hormonal, Metabolic, and Electrical Regulation

The hypothalamus regulates fundamental metabolic processes by controlling functions as varied as food intake, body temperature, and hormone release. As the functions of the hypothalamus are controlled by specific subsets of neuronal populations, the ability to isolate them provides a major tool for studying metabolic mechanisms. In this regard, the neuronal complexity of the hypothalamus poses exceptional challenges. 
For these reasons, new techniques, such as Magnetic-Activated Cell Sorting (MACS), have been explored. This paper describes a new application of magnetic-activated cell sorting (MACS) using microbead technology to isolate a targeted neuronal population from prenatal mice brains. The technique is simple and guarantees a highly pure and viable primary hypothalamic neuron culture with high reproducibility. The hypothalamus is gently dissociated, neurons are selectively isolated and separated from glial cells, and finally, using a specific antibody for a cell surface marker, the population of interest is selected. 
Once isolated, targeted neurons can be used to investigate their morphological, electrical, and endocrine characteristics and their responses in normal or pathological conditions. Furthermore, given the variegated roles of the hypothalamus in regulating feeding, metabolism, stress, sleep, and motivation, a closer look at targeted and region-specific neurons may provide insight into their tasks in this complex environment.

Author Spotlight: Hypothalamic Neural Mechanism Insights 

Here we present a protocol to grow specific hypothalamic cell subtypes in culture. The cells can be selected based on opportune/unique membrane markers and used in many applications, including immunofluorescence, electrophysiological, and biochemical assays.

The hypothalamus regulates fundamental metabolic processes by controlling functions as varied as food intake, body temperature, and hormone release. As ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Biology

En Face Preparation of Mouse Blood Vessels

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the three-dimensional tissue morphology is from such sections. In addition, the sections of tissues examined may not contain the region within the tissue that is necessary for the purpose of the ongoing study. This latter limitation hinders histopathological studies of blood vessels since vascular lesions develop in a focalized manner. This requires a method that enables us to survey a wide area of the blood vessel wall, from its surface to deeper regions. A whole mount en face preparation of blood vessels fulfills this requirement. In this article, we will demonstrate how to make en face preparations of the mouse aorta and carotid artery and to immunofluorescently stain them for confocal microscopy and other types of fluorescence-based imaging.

En Face Preparation of Mouse Blood Vessels

A procedure for making en face preparations of the mouse carotid artery and aorta is described. Such preparations, when immunofluorescently stained with specific antibodies, enable us to study localization of proteins and identification of cell types within the entire vascular wall by confocal microscopy.

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Isolating Endothelial Cells from Murine Dermal Lymphatic Capillaries

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Author Spotlight: Magnetic Bead-Based Isolation of Murine Dermal Lymphatic Endothelial Cells

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...

Protocol for Isolating the Mouse Circle of Willis

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Chapters in this video

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Conjunctival Commensal Isolation and Identification in Mice

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Isolation of Targeted Hypothalamic Neurons for Studies of Hormonal, Metabolic, and Electrical Regulation

Isolation of Murine Coronary Vascular Smooth Muscle Cells

En Face Preparation of Mouse Blood Vessels

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Murine Dermal Lymphatic Endothelial Cell Isolation