This work was funded by a grant from the Center for Innovation in Prevention and Treatment of Airway Diseases, NINDS grant (NS052574), and from the Sandler Program for Asthma Research.

1. Equipment
The major components of a contraction measurement device are shown schematically in Figure 2A).
<ol>
 <li>A tissue bath. The tissue bath maintains an oxygenated physiological solution at warm temperature. For mice trachea rings, we use a 10 ml tissue bath that contains a water jacket for circulating a warming solution, a fritted glass inlet to bubble oxygen (95%/5% O2/CO2 mixture) and inlet and outlet ports for changing solutions. A reservoir PSS solution is stored with constant bubbling of 95%/5% O2/CO2 mixture in a 37 &deg;C water bath (not shown). For solution exchanges, PSS solution is pumped from the reservoir to the tissue bath inlet (bottom port) at approximately 100 mls per minute to allow relatively fast solution exchange. The solution outlet is through an overflow port (top port) that allows constant volume (~10 ml) in the tissue bath during solution exchange. We use a Haake heating circulator to pump warm water through the tissue bath jacket (to maintain 37 &deg;C). Tissue baths can be obtained from a number of suppliers and come in a variety of sizes and styles to fit the experimental needs of the investigator. </li>
 <li>A force transducer. To measure isometric tension, the trachea tube is threaded over L-shaped ends of two stainless steel rods (Figure 2A). Care should be taken to use a stainless steel type that is compatible with biological material. The top rod is connected via a clip to an isometric force transducer. The bottom rod holds the trachea at a fixed position, and is mounted on a micrometer for adjustment of passive tension and/or muscle length. Contraction of the trachea creates tension on the force transducer, which is converted to a voltage signal at the preamplifier. The bottom rod may also be configured to include two rectangular platinum plates (4 mm apart) that flank the trachea (Figure 2B). The platinum plates are wired to a Grass S88 stimulator that allows delivery of an electric field across the trachea. Open wires and solder are coated with Sylgard (Sylgard 184 Silicone Elastomer, Dow Corning Corp, Midland, MI), to prevent leaching of metals into the bath solution.</li>
 <li>A/D converter, computer and acquisition software. Signals from the preamplifiers are recorded on a MacLab 8 A/D system. This is an older version of the current ADInstrument Powerlab hardware. We use the program Chart (ADInstruments) that allows continuous recording of tension throughout the experiment. Tension generation of trachea muscle is quite slow, and therefore we find that acquisition of 100 points per second is adequate. The tension measurement is calibrated using known weights (up to 5 grams) before each experiment. Similar systems are available from other venders (e.g., BioPac, GW Instruments).</li>
</ol>
2. Trachea Isolation
<ol>
 <li>Before tissue isolation the force transducer is calibrated with known weights, and the tissue bath is filled with normal PSS (see Table I). The air inlet is adjusted to obtain a light stream of O2/CO2.</li>
 <li>Tracheas from two month or older mice are optimal. Younger animals may be used but tracheas obtained from these require greater skill to mount on the force transducer wires because of their small size. Prior to dissection, mice are deeply sedated with isoflurane. The proper level of sedation is reached when a toe-pinch with forceps is unable to elicit a response. Mice are immediately sacrificed by cervical dislocation. An important note: We have observed that Avertin (Tribromoethanol), a sedative commonly used in mice, has strong relaxant effects on airway smooth muscle and therefore should not be used for trachea contraction studies.</li>
 <li>The skin (and fur) is removed from thorax to throat. Ribs are cut from the base of the sternum, laterally (on both sides) to the top of the heart. The sternum and ribs are then pulled forward to the throat to reveal the heart/lungs, thymus, trachea (ventral) and esophagus (attached to - and dorsal to trachea).</li>
 <li>The trachea is excised by cutting below the bronchial bifurcation and above the pharynx. The trachea is placed into an ice-cold oxygenated (95/5) PSS solution (composition given in Table I).</li>
 <li>The trachea is dissected clean of surrounding tissues. During cleaning, the trachea can be held at the pharynx or below the bifurcation. However, caution should be taken not to directly apply the forceps to the trachea itself. Fine scissors may be used to cut off surrounding tissues, but the cut should always be made parallel to the trachea to avoid damage. This part of the procedure is facilitated if the trachea preparation is pinned below the bifurcation and above the pharynx onto a Sylgard-coated dish (Sylgard 184 Silicone Elastomer, Dow Corning Corp, Midland, MI).</li>
 <li>After removing surrounding tissue, the trachea is cut below the pharynx and above the bronchial bifurcation and gently mounted on the force transducer wires.</li>
 <li>The trachea is threaded over two L-shaped metal prongs (Figure 2A). One prong is connected to a force-displacement transducer for continuous recording of isometric tension. Another prong is connected to a micrometer. The tissue bath is then raised so that the trachea is immersed in PSS. Mounting of the trachea should be done as quickly as possible to minimize the time that trachea are held outside of PSS. With practice, mounting of the trachea can be done within one minute but we generally avoid times longer than 3 minutes to avoid loss of viability.</li>
 <li>The micrometer is adjusted slowly to obtain a passive tension of ~10 mN (~ 1 gram-force). The optimal resting tension was determined empirically and we have found that passive tension of ~ 5 - 10 mN results in an equivalent, maximal response to high potassium stimulation. This is consistent with a number of other studies that utilize a passive tension in this range16,17,18. Over the first 5-10 minutes, trachea passive tension tends to decline somewhat (stress-relaxation phenomenon) and the micrometer is used to adjust the passive tension to ~10 mN during equilibration. The trachea is allowed to equilibrate for at least 1 hour before experimental challenges.</li>
</ol>
3. High Potassium Stimulation
Following equilibration, the trachea is challenged twice with a high potassium PSS solution (67 mM KCl, Table I). The contraction generally requires ~ 5-10 minutes to reach steady-state at which time the tissue bath is rinsed several times with normal PSS to completely relax the trachea. The potassium contraction is repeated a second time, and a third time (if necessary) until reproducible contractions are obtained.
4. Contraction Evoked by Electrical Stimulation
The trachea is flanked by two rectangular platinum plates (electrodes) that allow electric field stimulation (EFS) to the preparation. The contractile response to EFS is a function of frequency and voltage. It is also affected by physical parameters such as the area of the electrodes and the distance between them. The power characteristics of the stimulator also affect responses such that at higher voltages and current outputs the stimulator can reach its maximum. The characteristics of any EFS system should be determined by examining the muscle contractile responses at differing stimulus durations, frequencies, voltages and pulse durations. For our experimental setup, we have found that electrodes separated by ~ 4 mm, and stimulation amplitude of 44 V (0.5 ms pulses) and 30 Hz are optimal to achieve reproducible near-maximal contractile responses.
5. Contraction Evoked by Cholinergic Stimulation
The responsiveness of the trachea to exogenously applied compounds is evaluated either by addition of a single dose of the drug of interest or, by multiple additions of the drug in a cumulative dose fashion. For the trachea, our laboratory routinely used carbachol to activate cholinergic receptors because, unlike acetylcholine, carbachol is not degraded by acetylcholinesterase. A reasonable dose-response range is from 10-8 to 10-5 M carbachol. Fitting of the Log[carbachol]-contractile response curve with a Hill-type equation allows an estimation of EC50 (half-maximal effective concentration) that is a measure of sensitivity of tracheal contraction to the cholinergic agonist 19. It is worth noting that a given dose of carbachol will give a slightly greater response as a single dose than as part of a cumulative dose response curve.
6. Representative Results
An example of a contractile response to high potassium is shown in Figure 3A. The contraction reaches a maximum within approximately 10 minutes but may show a small decline thereafter. During early washout of high potassium, the muscle may show a transient increase in contraction that is due to a drop in temperature as the small volume of unheated PSS solution in the solution lines transiently perfuses the preparation. This can be minimized by having a minimum dead volume in the tubing connecting the heated PSS reservoir and tissue bath, and also by exchanging solution relatively quickly (generally we pump solutions at 100 ml/min). Each preparation will have some differences in contractile response due to differences in muscle mass or damage incurred during the dissection. Figure 3B shows two tracheas of different muscle mass challenged with high potassium and carbachol. Although the cholinergic-evoked contractions differ, they are similar after normalization to the response with high potassium solution (Figure 3C).
Figure 4 shows an example of a carbachol (cholinergic) evoked contraction using single doses (A) and a cumulative increase (B). The carbachol solutions are added directly to the bath and the bubbled gas helps in rapid mixing. It is worth noting that addition of a single dose (i.e. 1 &mu;M, Figure 4A) has a slightly larger response than the equivalent concentration during a cumulative dose-response curve (1 &mu;M, Figure 4B). Figure 4C shows a plot of contraction force as a function of carbachol concentrations using data from Figure 4B. Effects of carbachol saturate at 10-5 M concentration. Although cholinergic agonists initiates contraction through calcium release mechanisms, a substantial component of the contraction is also mediated by depolarization and activation of voltage-dependent calcium channels 20.
Figure 5A shows an example of EFS-evoked contractions. Tracheas are stimulated using 0.5 millisecond duration, 40 volt pulses until contractions reach a plateau (see inset a1). An increase in stimulation frequency causes an increased contractile response (frequency-response curve is plotted in Figure 5B). Electric field stimulation has been shown to evoke contractions predominantly by activating presynaptic nerves. This is evidenced by the effect of botulinum toxin, a blocker of neurotransmitter release that blocks the majority of the EFS-evoked contractions of trachea 21. Moreover, tetrodotoxin, an agent that blocks Na+ channels also inhibits nerve activity and eliminates the response of trachea to EFS.
<img alt="Figure 1" src="/files/ftp_upload/3703/3703fig1.jpg" /> 
Figure 1. Diagram of the major signaling pathways in an isolated trachea preparation. Shown is a cholinergic axon terminal innervating a tracheal smooth muscle cell. The major signaling pathways are M3- and M2- muscarinic acetylcholine receptor activation (mACHR) that cause calcium release through IP3 receptors (M3) and reduction of cAMP (M2). M2 receptors (and some contribution of M3 receptors) also cause cholinergic-evoked depolarization that activates L-type voltage-dependent calcium channels and calcium influx. Common contractile agents and their effectors are 1. high potassium (depolarize smooth muscle cell and cholinergic axon), 2. electric field stimulation (EFS, depolarizes cholinergic axon) and 3. exogenous application of cholinergic agents such as acetylcholine or carbachol (activates muscarinic receptors directly).
<img alt="Figure 2" src="/files/ftp_upload/3703/3703fig2.jpg" /> 
Figure 2. Diagram of equipment used to measure tracheal contractions. A. The force transducer, micrometer and tissue bath are mounted on supporting rods via screw clamps. The tracheal ring is threaded over the top and bottom rods. In the diagram, the tissue bath is positioned below the preparation (i.e. during mounting of the trachea onto the force transducer). During contraction studies, the tissue bath is moved vertically to bathe the preparation. B. For electric field stimulation the bottom rod is modified to include two platinum plates that are mounted laterally to the trachea holding wire. The platinum plates are connected by electrical wires to a stimulator.
<img alt="Figure 3" src="/files/ftp_upload/3703/3703fig3.jpg" /> 
Figure 3. Examples of high potassium (67 mM) contractile response of trachea. (A) shows duplicate, reproducible responses to high potassium. (B) Examples of contractile response to carbachol of two different tracheas. (C) Responses to the tracheas in B are similar when normalized to the high potassium response.
<img alt="Figure 4" src="/files/ftp_upload/3703/3703fig4.jpg" /> 
Figure 4. Examples of carbachol-induced contractions. (A) Carbachol-induced contractions using single doses followed by washout. (B) Example cumulative dose-response curve for trachea in A. (C) Peak contractions from B are plotted as a function of carbachol concentration.
<img alt="Figure 5" src="/files/ftp_upload/3703/3703fig5.jpg" /> 
Figure 5. Examples of contractions evoked by electric field stimulation. (A) Electric field stimulation of trachea contraction using 0.5 ms pulse, 40 volts, and various stimulation frequencies as indicated. Inset is time-expanded contraction at 30 Hz. (B) Peak contractions from A are plotted as a function of stimulation frequency.
Normal PSS
<table border="1">
 <tbody>
 <tr>
 <td>Salt</td>
 <td>Conc.(mM)</td>
 <td>Amount(g/2 L)</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>119</td>
 <td>13.91</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>4.7</td>
 <td>0.7</td>
 </tr>
 <tr>
 <td>KH2PO4</td>
 <td>1.18</td>
 <td>0.32</td>
 </tr>
 <tr>
 <td>MgSO4 x 7H2O</td>
 <td>1.17</td>
 <td>0.58</td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>18</td>
 <td>3.02</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>0.026</td>
 <td>0.1 ml of 0.5 M</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>11</td>
 <td>3.96</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>12.5</td>
 <td>8.56</td>
 </tr>
 <tr>
 <td>CaCl2</td>
 <td>2</td>
 <td>400 ml of 10 mM</td>
 </tr>
 </tbody>
</table>
High K+ PSS (NaCl and KCl adjustments)
<table border="1">
 <tbody>
 <tr>
 <td>Salt </td>
 <td>Conc. (mM) </td>
 <td>Amount (g/2 L)</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>56.7</td>
 <td>6.628</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>67</td>
 <td>9.991</td>
 </tr>
 </tbody>
</table>
Table 1. Recipe for PSS solutions. Note: Solutions are made fresh weekly, with ultrapure quality water, and are stored in a refrigerator for no longer than 5 days to avoid contaminating growth.

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Comp Physiol. 282, R1443-R1449 (2002).</li><li>Trevisani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+in+vitro+expression+of+B1+receptor+in+the+mouse+trachea+and+urinary+bladder.">Evidence for in vitro expression of B1 receptor in the mouse trachea and urinary bladder.</a> Br. J. Pharmacol. 126, 1293-1300 (1038).</li><li>Mehats, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDE4D+plays+a+critical+role+in+the+control+of+airway+smooth+muscle+contraction.">PDE4D plays a critical role in the control of airway smooth muscle contraction.</a> FASEB J. 17, 1831-1841 (2003).</li><li>Kumar, R. K., Herbert, C., Foster, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+"classical"+ovalbumin+challenge+model+of+asthma+in+mice.">The "classical" ovalbumin challenge model of asthma in mice.</a> Curr. Drug Targets. 9, 485-494 (2008).</li></ol>

No conflicts of interest declared.

The protocol presented here provides a physiological preparation to assess airway muscle function. We generally operate 3-4 organ bath preparations simultaneously, however, prepackaged systems are available from a number of suppliers that allow simultaneous measurements of up to 8 preparations (ADInstruments, World Precision Instruments, and Harvard Apparatus). We have utilized a number of force transducers and tissue organ baths with equivalent results. However, we find that electric field stimulation provides som...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Analogue-Digital Converter</td>
 <td>ADInstruments</td>
 <td>PowerLab 4/35</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Carbachol
 (Carbamoylcholine Chloride)</td>
 <td>Sigma-Aldrich</td>
 <td>C4832
 </td>
 <td>10-2 M in water (aliquots can be stored at -20&deg;C)</td>
 </tr>
 <tr>
 <td>Charting Software </td>
 <td>ADInstrtuments</td>
 <td>LabChart</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Heating Circulator</td>
 <td>Haake</td>
 <td>Mixer Mill MM400</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Isometric Force Transducer</td>
 <td>Kent Scientific</td>
 <td>TRN001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Stimulator</td>
 <td>Grass Technologies</td>
 <td>S88 Dual Output Square Pulse Stimulator</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tissue Bath</td>
 <td>WPI</td>
 <td>47264</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

in vitro measurements of tracheal constriction using mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

Watch this Scientific Journal Video about In vitro Measurements of Tracheal Constriction Using Mice at JoVE.com

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

in-vitro-measurements-of-tracheal-constriction-using-mice

Research

JoVE Journal

Medicine

20.7K Views.  UT Health Science Center, San Antonio. Topi transgenici sono stati estremamente utili per attribuire funzione fisiologica ai geni. Come tale, la ricerca in generale, e studi funzionali delle vie aeree, in particolare, hanno subito un notevole spostamento verso modelli murini. Qui forniamo protocolli per In vitro Studi di costrizione trachea per valutare la funzionalità della muscolatura liscia delle vie aeree in murino.

Video: In vitro Measurements of Tracheal Constriction Using Mice

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

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 ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Minimally Invasive Transverse Aortic Constriction in Mice

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

Minimally invasive transverse aortic constriction (MTAC) conserves the essentials of regular transverse aortic constriction (TAC) while eliminating the use of a ventilator with tracheal intubation. It proves to be a highly desirable method for high-throughput studies on left ventricular overload, particularly in translational studies.

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Sleeve Gastrectomy in Mice using Surgical Clips

The number of people who are overweight and obese is continually increasing both in the adult and adolescent populations. This coincides with the increased universal phenomenon of type 2 diabetes (T2D) and other metabolic problems. Bariatric surgery, such as SG, is currently one of the most effective and commonly used long-term treatment for obesity and T2D, but the association between them is not completely explored yet. The mechanisms underlying the outcomes seen after bariatric surgery in humans can be investigated based on preclinical animal studies. The SG reduces body weight, glucose levels and many metabolic parameters, and is easy to perform with a low incidence of complications. The goal of this work is to provide a simple method and an uncomplicated preclinical model of bariatric surgery in animals for researchers.

The prevalence of diabetes and obesity is continuously increasing worldwide. The mechanisms between diabetes, obesity and their associated mortality and co-morbidities need to be further investigated. Here, we present a protocol for sleeve gastrectomy (SG) in animals as an uncomplicated preclinical model of bariatric surgery.