This study was supported in part by the Deutsche Forschungsgemeinschaft (DFG), project GZ PI 771/1-1; SFB 656 "Cardiovascular Molecular Imaging," M&uuml;nster, Germany (projects C6, Z2, B3, and PM3); EU NoE "Diagnostic Molecular Imaging&mdash;DIMI" (WP 11.1 and 11.2). The study was also funded in part by the British Heart Foundation, UK.

1. Preparing the shear stress modifier (cuff)
<ol>
	<li>The shear stress modifier consists of two longitudinal halves of a cylinder with a cone shaped lumen. The half shells are made of thermoplastic polyetherketone produced by a plastic casting procedure. The cast elements are sent out while still connected to the runner. Hence, the half shells have to be cut off before usage. Each cast contains half shells of different sizes ranging from 150 &mu;m - 300 &mu;m (lowest inner diameter at downstream end).</li>
	<li>The cuff preparation should be performed under a surgical microscope.</li>
	<li>Hold the cuff half shell with blunt forceps and gently cut it off from the cast by using a sharp scalpel. This procedure will lead to half shell precursors which have to be further processed.</li>
	<li>Place the half shell precursor on an even non-slippery plate; fix it with blunt forceps and cut it exactly along the preformed incision in order to remove the closed end. This will yield the cuff half shell ready for use.</li>
	<li>Control the half shells under the microscope and gently remove any remaining sharp edges. The conical inner lumen is essential for inducing different qualities of shear stress within, upstream and downstream of the implanted cuff. A groove on the outer surface of the half shells running perpendicular to the inner lumen serves as a guiding for the thread which finally sticks the half shells together to form the functional cuff.</li>
	<li>Store the cuff half shells, classified according to size, in 70% ethanol.</li>
</ol>
2. Implanting the cuff around the right carotid artery
<ol>
	<li>ApoE knockout mice should be around 10 weeks of age and at a weight of at least 20 grams. If plaque development should be investigated under high cholesterol diet, starting such a western type diet 4 weeks prior the cuff implantation is recommended.</li>
	<li>The whole surgical procedure should be performed under at least semi-sterile conditions: Wear a surgical gown, mask and cap and sterile gloves. Sterilize the instruments for 30 seconds in a bead instrument sterilizer and place them on a sterile opaque sheet. Beware! Instruments have to cool down before use!</li>
	<li>Place the mouse in an anesthesia induction chamber filled with 3% isoflurane until it is completely anesthetized. Verify depth of anesthesia with response to toe pinch. Alternatively, anesthesia can be performed with intraperitoneal (i.p.) injection of Ketamine (80 mg/kg) and Xylazine (10 mg/kg) or alternate anesthetics approved by the IACUC. For i.p. injection, hold the mouse in supine position and inject the anesthetic in the left lower quadrant of the abdomen.</li>
	<li>Place the mouse on a heated surgical plate, in supine position. In order to prevent the eyes of running dry moisten them with eye salve (e.g. Bepanthen eye and nose lotion). Spread the fore- and hind-paws and tape them down to the plate. If performing isoflurane anaesthesia, the narcotic gas flow (2% isoflurane) has to be supplied via a small rodent mask.</li>
	<li>Remove the hair between the mandible and the sternum by either applying depilatory agent (e.g. Pilca Med) or by using a fine electric shaver (e.g. Wella Contura). Shaving has to be performed with caution in order to avoid irritating the skin. If using a depilatory agent, give it 1-2 min for penetrating the hair, thereafter, gently rub until all hair and depilatory agent is removed.</li>
	<li>Have ready previously prepared cuff half shells of different sizes (two of each size) and a 2.5 cm-long piece of 6-0 silk suture for interconnecting the half shells at the site of carotid occlusion.</li>
	<li>Disinfect the operating field with liberal amount of Betadine. Use sharp small scissors to open the skin and the underlying fascia of the neck by a 4-5mm medial incision starting from the top of the sternum (manubrium).</li>
	<li>Expand the opening, shift the right parotid gland aside and insert a surgical spreader. Then bluntly dissect into the deep, just left of the trachea (from the surgeons view), where the right sternomastoideus muscle crosses the right omohyoid muscle, until you are able to identify the pulsating right common carotid.</li>
	<li>Using very fine angled or curved forceps (e.g. Dumont #5/45), dissect the right common carotid artery by gently removing the surrounding connective tissue. Separate the carotid from the vagus nerve - the white, stringy object running directly along the carotid - as this step is necessary to completely expose the vessel. Be careful neither to harm the vagus nerve nor a branch of the left internal jugular vein, which is also in close connection to the carotid.</li>
	<li>In order to choose the right cuff size, compare the diameter of the exposed carotid with the inner-diameter of the cuff half shells: The largest width of the cuffs lumen should meet the outer diameter of the carotid.</li>
	<li>Now, carefully put the tip of the forceps under the carotid, open the forceps, thread the piece of 6-0 silk suture under the carotid and form a loop. Between the loop and the carotid place one cuff half shell beneath the carotid. The side of largest narrowing has to be downstream.</li>
	<li>Place the second cuff half shell within the loop on top of the carotid.</li>
	<li>Gently tighten the suture loop using suturing forceps and node the thread. By doing this the functioning shear stress modifier is formed. For the precise fitting of the cuff it is essential that the suture is running exactly within the preformed groove on the outer surface of the cuff.</li>
	<li>Move the right parotid gland back in its original position, approximate and close the skin either using a small amount of 6-0 prolene suture or, alternatively, you can use wound clips.</li>
	<li>Inject a single dose of 5 mg/kg Carprofen (e.g. Rimadyl) subcutaneously to provide prophylactic pain treatment and place the mouse in a warming chamber until it recovers. Normally, this takes 30-60 min when using Ketamine/Xylazine anesthesia. When using isoflurane inhalation anaesthesia the recovery period is substantially shorter (10-20 min).</li>
	<li>In our experience, animals regain normal unstressed activity within the first 24 hours after cuff implantation with the intervention being performed by an experienced surgeon. However, if the animal appears to be distressed even one day post-surgery, repeat the analgesic treatment and consult with veterinary staff.</li>
</ol>
3. Explanting the cuff and the carotid arteries
<ol>
	<li>For histological analysis the carotid arteries have to be harvested at the end of the observation period. Before starting explantation, the animal has to be killed according to IACUC guidelines.</li>
	<li>If the cuff half shells are intended for reuse, carefully remove all tissue fragments and the connecting suture from the plastic elements, dissecting from the carotid arteries, wash and store the half shells in 70% alcohol solution. One should keep in mind that the cuff is embedded in connective tissue adhesions after several weeks of implantation and that one has to dissect the cuff with caution in order not to harm the vessel walls.</li>
	<li>Alternatively, the cuff can be explanted and embedded together with the carotid artery. The plastic material of the cuff is resistant to normal fixation solutions and solvents used for paraffin embedding. Also, the embedded samples can be cut using a normal microtome equipped with normal blades.</li>
</ol>
4. Representative Results
The cuff has to be placed always around one of the two common carotid arteries of an animal (Fig. 1A, 1B) - the contralateral side serves as a control. On the image presenting the two half shells of the cuff (Fig. 1B) the conical shape of the inner lumen is visible. This conical shape is essential for establishing the three regions with distinct flow dynamics. Typical shear stress patterns induced by the cast calculated from Doppler measurements 11 and the corresponding flow velocities based on phase-contrast velocity MR imaging 12 are given in Figure 2.
When the cuff is implanted in ApoE knockout mice fed a western type diet the altered flow dynamics provoke shear stress induced atherosclerotic plaque deposition (Fig. 3): Upstream of the conical constriction low laminar shear stress leads to massive development of atherosclerotic plaques of a more vulnerable phenotype, characterized by lipid cores close to the central lumen covered only by a thin fibrous cap (Fig. 3A1). The conical inner lumen of the cuff leads to an increase in flow velocity. Nearly no plaque deposition is observed in this area. Directly downstream of the site of the bottleneck the immediate broadening of the artery results in an area of vortices and oscillatory flow parameters, which causes less extended plaque development of a more stable phenotype (i.e. less lipid cores which are localized more close to the media).
<img alt="Figure 1" src="/files/ftp_upload/3308/3308fig1.jpg" /> 
Figure 1. The shear stress modifier - assembly and placement. (A) Schematic drawing of the implanted cuff: The cuff is placed around the right common carotid artery (RCCA) - the contralateral side (LCCA = left common carotid artery) serves as control. (B) In vivo MRI angiography in a mouse: In the maximum intensity projection of a three-dimensional time-of-flight MR angiography the conical constriction induced by the cast (white arrow) is visible (for details see 12). (H=head, F=feet, R=right, L=left). In the upper right corner a macroscopic view on the inner lumen and the outer surface of the cuff half shells is given. When assembled, two half shells form a conical cylinder which modifies the flow dynamics within the vessel in a defined way. To ensure the right fit, a groove on the outer surface of the cuff (yellow arrow) serves as a guiding for the linking suture.
 
<img alt="Figure 2" src="/files/ftp_upload/3308/3308fig2.jpg" /> 
Figure 2. Regions of different flow dynamics and shear stress. Implantation of the cuff alters flow dynamics and subsequently shear stress in the constricted carotid artery in a defined manner. In the upper schematic illustration the different flow properties are indicated and the local approximate values for shear stress are given (based on Doppler measurements 11). Below, corresponding flow velocities for a single animal measured by MRI upstream of the implanted cuff are shown in a T1-weighted cross-sectional MRI-image of the neck (phase-contrast velocity imaging, for details see 12).
<img alt="Figure 3" src="/files/ftp_upload/3308/3308fig3.jpg" /> 
Figure 3. Shear stress induced plaque development in ApoE knockout mice. The upper left corner shows the macroscopic view on the opened neck and exposed carotid arteries of an ApoE knockout mouse under high fat (western type) diet 8 weeks after implantation of a shear stress modifier around the right common carotid artery (RCCA). The opaque white region of the vessel (*) upstream of the cuff corresponds to a site of extensive atherosclerotic plaque deposition. The yellow arrow indicates the thread which combines the two cuff halves. In contrast, there are no signs of plaque deposition in the left common coronary artery (LCCA). 
(A)-(C) Representative HE stained cross-sections of the right common carotid artery of an ApoE knockout mouse under western type diet 8 weeks after implantation of the cuff. The schematic illustration gives the corresponding planes (dotted lines) where the sections were located. (A) Upstream of the cuff atherosclerosis leads to massive plaque deposition. Large lipid cores (red arrows), which in part are located close to the central lumen (detail A1) characterize the vulnerable character of these plaques. (B) At the plane of the cuff bottleneck nearly no plaque development is obvious, whereas directly downstream of the cuff (C) oscillatory flow leads to moderate plaque development of a more stable type (i.e. less or no lipid cores). (C1, C2 = half shells of the cuff, P = plaques, asterisk = vessel lumen)

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The authors have nothing to disclose.

In order to minimize experimental variation it is recommended to work with animals of nearly the same age and with the same diet history. A recently published investigation demonstrates that the shear stress modifier applied in wildtype mice might be a good model for the investigation of endothelial dysfunction and early inflammatory responses induced by altered flow dynamics 13. However, for the enquiry of atherosclerotic plaque development transgenic hyperlipidemic mouse models (e.g. the ApoE knockout mouse)...

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Shear stress modifier (polyetherketone)</td>
 <td>Promolding BV
 <a href="http://promolding.nl">http://promolding.nl</a></td>
 <td>The casts are sent out while still connected to the runner. Thus, the single elements have to be cut off before usage.</td>
 </tr>
</tbody>
</table>

implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

Watch this Scientific Journal Video about Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice at JoVE.com

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

implantation-carotid-cuff-for-triggering-shear-stress-induced

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20.1K 조회수.  Westfälische Wilhelms-University Münster. 이 문서에서 제시 constricting 커프스는 murine 공통 경동맥에서 죽상 경화증을 유발하기 위해 설계되었습니다. 이식 커프스가 다른 염증성 phenotypes의 atherosclerotic 병변의 발전을 게재 낮은 높이 oscillatory 전단 응력의 잘 정의된 영역을 생성하는 자사의 내부 루멘의 원뿔 모양으로 인해.

비디오: Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models&#160;&#8211;&#160;both resulting in artery stenosis but each underlying a different pathway of development&#160;&#8211; are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked.

This video shows two models of intimal plaque development in murine arteries and emphasizes the differences in myointimal hyperplasia and atherosclerosis.

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.