This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.
This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered "designer" rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.
The earliest studies of vascular function in arteries utilized conduit arteries, and in many cases the aorta. Force generation in large arteries was generally studied by attaching a ring segment of the artery to a force transducer in a tissue bath; in the case of the aorta, by cutting helical strips of the vessel so that the smooth muscle fibers were oriented in a longitudinal direction between the point of attachment and the force transducer, to provide the best estimate of the force generated by contraction of the smooth muscle along its longitudinal axis. The standard technique for cutting helical strips of aortas was to place a glass rod in the lumen of the vessel, make a cut in the vessel wall at the desired angle, and hold on to the end of the exposed edge of the vessel wall as the cut was extended to produce an entire helical strip of the vessel. At that point, the endothelial side of the vessel was generally blotted to remove debris before attaching the vessel strip to the force transducer and submerging the preparation in an oxygenated and temperature controlled tissue bath. Eventually, that approach led to one of the most famous and important discoveries in the history of physiology by Furchgott and Zawadski1, namely the role of endothelium derived relaxing factor (EDRF), subsequently identified as nitric oxide, in regulating vascular function. The crucial event leading to that discovery was a situation in which the investigators maintained an intact endothelium by avoiding contact of the endothelial side of the artery with foreign surfaces, and noticed that the aortic strip did not exhibit the expected contraction to acetylcholine (ACh), but instead relaxed in response to ACh. Based on that observation, the investigators developed a "sandwich" preparation in which they attached an aortic segment with an intact endothelium (but unable to generate contractile force) to a standard helical strip of aorta and converted the ACh-induced contraction into a relaxation.
Two major advances in this area that are extensively used today are the development of preparations to measure active contractile force in small resistance arteries2,3 (such as those in the intestinal mesentery3) and cannulated resistance artery preparations4,5,6. In one of the earliest reports, Mulvany and Halpern3 described the use of the wire myograph preparation to study active contractile force in isolated resistance arteries from the intestinal mesentery of spontaneously hypertensive rats (SHR) and normotensive WKY controls. Subsequent to the development of the wire myograph system, cannulated resistance artery preparations were developed to permit studies of vessels closer to in vivo conditions4,5,6. While both approaches provide valuable results, the cannulated artery preparation has the added advantages of more effectively preserving intrinsic active tone in the arteries; and allowing the investigators to study active myogenic responses to changes in transmural pressure and vessel responses to changes in flow rate and endothelial shear stress (see review by Halpern and Kelley6).
A major goal of the present paper is to describe how to employ the time-honored technique of video microscopy using isolated, cannulated resistance arteries in order to gain precise information regarding the mechanisms that regulate active tone in these crucial vessels, independent of neural, humoral, or parenchymal cell influences. This basic information, employing a standard rat model and examples from our studies of new genetically engineered rat strains, will provide the reader with an idea of the types of the insights regarding vascular function that can be gained with television microscopy approaches, and which can be employed in studies involving any control and experimental group(s) of the investigator's choosing, including powerful new experimental rat models produced by selective inbreeding and newly developed genetic engineering techniques.
Thanks to the precision of television microscopy approaches, measurement of diameter changes in cannulated artery preparations can provide highly valuable information regarding endothelium-dependent and endothelium-independent mechanisms of vascular relaxation, as well as important (and sometimes unexpected) alterations in vascular control mechanisms occurring with hypertension, high salt diet, and other experimental interventions. In addition, measurement of pressure-diameter relationships in isolated and cannulated resistance arteries that are maximally relaxed by treatment with Ca2+-free solution or a pharmacological vasodilator drug, allows the investigator to assess structural changes in arteries due to vascular remodeling and to calculate passive stress-strain relationships7 that can provide important insight into changes in the passive mechanical properties of the arteries that can affect arterial function independent of (or in addition to) changes in active control mechanisms. It is also important to note that information gained from studies of isolated resistance arteries can be supplemented by information obtained utilizing LDF, a practical method for evaluating tissue perfusion at the whole animal level8,9,10, and by information gained from assessing microvessel density using fluorescently labelled GS1 lectin, which specifically binds to glycoprotein moieties in the basement membrane of small arterioles and capillaries11,12. The latter method provides a highly accurate estimate of microvessel density that is not subject to the classic difficulties encountered in estimating microvessel density by counting vessels in vivo, for example missing non-perfused vessels where blood flow is stopped due to active closure of arterioles. When used together, these approaches can provide important insight to correlate functional alterations in isolated resistance arteries to changes in tissue perfusion at the microcirculatory level; and some examples of the use of those valuable approaches in conjunction with cannulated artery techniques will also be provided in the present manuscript.
The present paper focuses on the use of video microscopy techniques to evaluate vascular changes in arteries of outbred Sprague-Dawley rats. However, it is important to note that these techniques have proven to be highly valuable in elucidating phenotypic alterations in highly specialized genetically engineered rat strains created by selective breeding or gene editing using techniques. In this manuscript, we provide examples of how video microscopy techniques have provided important information regarding vascular function in a number of valuable rat models, including the Dahl salt-sensitive (SS) rat-an inbred rat strain that is the most widely used experimental model to study the mechanisms of salt sensitive hypertenson18,19,20,21,22,23; and consomic rats created via selective breeding of SS rats with the salt-insensitive Brown Norway (BN) rat strain. In the consomic rat panels, every chromosome from the Brown Norway rat has been introgressed individually into the Dahl SS24,25,26 genetic background. Use of consomic rat panels has provided valuable clues regarding specific chromosomes that contribute to salt sensitivity of blood pressure and other phenotypes, including vascular reactivity24,25,26,27,28.
Selective breeding strategies utilizing SS rats and consomic rats carrying individual BN chromosomes have also enabled the generation of narrowed congenic strains with small segments of individual Brown Norway chromosomes introgressed into the Dahl SS genetic background22,29. These can provide extremely valuable input on specific genes or narrow regions of chromosomes that can affect crucial physiological variables, such as blood pressure, renal damage, and vascular reactivity22,29. Another powerful addition to the rat genetic toolbox is the development of rat gene knockout models utilizing advanced gene editing techniques including ZFNs, transcriptional activator-like-effector nucleases (TALENS), and most recently CRISPR-Cas913,14,15,16,17. The advent of these powerful techniques that enable genes to be knocked out in the rat is an immensely important development because gene knockout studies to date have used (and continue to use) mice almost exclusively. Another experimental component in the present paper demonstrates the value of cannulated artery techniques and video microscopy to evaluate physiological control mechanisms in knockout rats lacking the master antioxidant and cell protective transcription factor, nuclear factor (erythroid-derived 2)-like-2 (NRF2)30,31, which were developed using TALEN technology in the Sprague-Dawley genetic background17. In those experiments, in vitro video microscopy techniques were used to provide functional verification of loss of the NRF2 gene and to test a potentially valuable therapeutic approach based on direct upregulation of NRF2-mediated antioxidant defenses. NRF-2 is of substantial therapeutic importance in combatting vascular oxidative stress in humans, in light of the disappointing results of clinical trials involving direct administration of antioxidants such as Vitamins C and E32.
The Medical College of Wisconsin Institutional Animal Care and Use Committee (IACUC) approved all protocols described in this paper and all procedures are in compliance with the National Institutes of Health (NIH) Office of Laboratory Animal Welfare (OLAW) regulations.
1. Preparation of Solutions and Vessel Chamber
2. Cannulated Artery Preparation
3. Evaluation of Cerebral Blood Flow Responses with LDF
4. Evaluation of Skeletal Muscle Microvessel Density with GS1 Lectin
In vitro microscopy of cannulated resistance arteries allows for the study of factors influencing active tone in small resistance arteries (and larger arterioles) at normal in vivo transmural pressures and in the absence of parenchymal cell influences. In addition to assessing the reactivity of the vessels to various vasodilator and vasoconstrictor stimuli and myogenic responses to transmural pressure elevation in normal PSS, the Ca2+-free PSS can be added to the perfusate and superfusate at the end of the experiment to determine the maximum vessel diameter and wall thickness. The latter measurements are highly valuable in assessing vascular remodeling, i.e., changes in the wall/lumen ratio that may occur in response to changes in blood pressure or administration of pharmacological agents. Measurements of the diameter of maximally dilated vessels in Ca2+-free PSS are useful to calculate intrinsic tone as ((Dmax-Drest)/Dmax) x 100, where Dmax and Drest are the maximum (Ca2+-free PSS) and resting diameter (PSS), respectively, at the control equilibration pressure (usually 80 mmHg for MCA). In our experience, active tone in the MCA averages around 40% at a transmural pressure of 80 mmHg. Measuring the diameter of maximally dilated arteries is also a practical way to evaluate the passive mechanical properties of the vessels by measuring the diameter of the arteries at different levels of transmural distending pressure and using the results to calculate stress-strain relationships and other mechanical properties of the vessels7,56.
Figure 3 is a schematic representation of three narrowed congenic strains derived from the SS.13BN rat utilizing additional selective breeding approaches. In these rat strains, the chromosomal segments either include the Brown Norway renin allele (Ren1-BN) or are cut off just above (Ren1-SSA) or just below (Ren1-SSB) the renin gene locus (thus retaining the SS renin allele). In that study29, the chromosomal segment containing the Brown Norway renin allele contained only 25 genes.
Figure 4 illustrates the response to the endothelium-dependent vasodilator ACh (1 µM) in isolated MCAs from male Dahl SS rats and the three narrowed congenic strains shown in Figure 3. Combined use of these three congenic strains that either include (Ren1-BN) or are cut off just above (Ren1-SSA) or below (Ren1-SSB) the renin gene limited the region of interest (i.e., the area surrounding the renin gene locus) to approximately 25 genes. In those experiments, the animals were normotensive and fed a low salt (0.4% NaCl) diet. In that study29 endothelium-dependent dilation to ACh was absent in the SS parental strain and in both the congenic strains retaining the SS renin allele, but was restored in the congenic strain carrying the BN renin allele in the SS genetic background. The results of that study supported the findings of earlier studies utilizing SS.13BN consomic rats57,58 and provided strong evidence that endothelial dysfunction is present in SS rats, even when they are normotensive and fed a low salt diet. Taken together, the results of those studies29,57,58 support the hypothesis that endothelial dysfunction in SS rats is due to impaired regulation of the renin gene, resulting in chronic exposure to low levels of angiotensin II in the blood.
Another study59, summarized in Figure 5, compared the response to the endothelium-dependent vasodilator ACh in isolated MCAs (MCA) from male Dahl SS rats and SS.5BN consomic rats carrying chromosome 5 (containing Brown Norway genes for various isoforms of cytochrome P450-4A ω-hydroxylase). In that study, animals were fed either a low salt (0.4% NaCl) or high salt (4% NaCl) diet. In contrast to the MCA from SS rats, endothelium dependent dilation to ACh was maintained in MCA from the SS.5BN consomic rats fed low salt diet. In addition, (and in contrast to results from SS.13BN consomic rats57,58, Sprague-Dawley rats33,39,40,60, and golden hamsters61), a high salt diet failed to eliminate ACh-induced dilation in the SS.5BN rats, indicating that CYP450-4A ω-hydroxylase and 20-hydroxyeicosatetraenoic acid (20-HETE) are important contributors to vascular oxidant stress and endothelial dysfunction in SS rats and during elevations in dietary salt intake in other strains. From an experimental perspective, the failure of the high salt diet to eliminate endothelium-dependent dilation to ACh provides an example of how studies of cannulated resistance arteries can produce unexpected findings that depart from conventional wisdom and could lead to a new understanding of complex control mechanisms affecting the function of these crucial vessels.
Figure 6 shows the response of isolated MCA to ACh (1 µM) in wild type and Nrf2(-/-) knockout rats fed a low salt diet (0.4% NaCl), high salt diet (4% NaCl), or a high salt diet containing a known NRF2 activator30,62. In the absence of salt-induced oxidative stress, ACh-induced dilation was similar in MCA from wild type and Nrf2(-/-) knockout rats. Consistent with the results of earlier studies of Sprague-Dawley rats, the high salt diet eliminated ACh-induced dilation in wild type and in Nrf2(-/-) knockout rats. Addition of the NRF2 inducer to the high salt diet restored endothelium-dependent dilation to ACh in the wild type rats, but not in the Nrf2(-/-) rats, demonstrating successful elimination of the Nrf2 gene in the knockout rats, and supporting the use of NRF2 up-regulators as a therapeutic approach to ameliorate vascular oxidative stress.
Figure 7 summarizes the results of LDF measurements comparing the responses of the pial microcirculation to progressive reductions in arterial blood pressure produced by successive blood volume withdrawals in Sprague-Dawley rats maintained on either a normal salt (0.4% NaCl) diet or a high salt (4% NaCl) diet for four weeks. In contrast to rats fed a low salt diet, rats fed the high salt diet exhibit an impaired ability to maintain a constant blood flow during reductions in perfusion pressure, indicating that the mechanisms responsible for cerebral blood flow autoregulation are compromised by long term exposure to a high salt diet. The results of those experiments are consistent with the presence of impaired vascular relaxation mechanisms in isolated cerebral arteries of salt-fed rats60 and provide a good example of how LDF measurements can be employed to support and extend the findings of studies employing isolated resistance arteries to the level of the whole vascular bed.
In addition to evaluating resistance artery function using isolated arteries and tissue perfusion using laser-Doppler flowmetry, the microvessel density and angiogenic responses to various stimuli such as chronic muscle stimulation54,55 can be evaluated in the same animals utilizing fluorescently-labeled GS1 lectin, which selectively binds to glycoprotein moieties in the basement membrane of arterioles and capillaries. Figure 8 shows typical lectin staining in the cremaster muscle of Sprague-Dawley rats fed a low salt diet or high salt diet for 2 weeks (note the lower density of microvessels in the high salt fed rats). Microvessel density is assessed by counting the intersections of lectin-labeled microvessels with a computer-generated reference grid52, and provides an excellent method to evaluate angiogenic responses54,55 or to detect and quantify the loss of microvessels in conditions such as hypertension and elevated dietary salt intake12,17,53.
Figure 1: In vitro video microscopy setup employed to study isolated, cannulated resistance arteries (A). Cannulated resistance artery tied to glass micropipettes (B) and video micrometer display showing measurement of lumen diameter in cannulated resistance artery (C). Scale bar in Panel B represents the internal diameter of the cannulated artery (µm), and 125 µm in Panel C is the video display of the distance between the moveable reference lines placed manually on the inner walls of the artery by the observer conducting the experiment. Please click here to view a larger version of this figure.
Figure 2: Schematic diagram of the procedure for isolating the middle cerebral artery from the brain and preparing it for cannulation with the micropipettes. Please click here to view a larger version of this figure.
Figure 3: Schematic diagram of narrowed congenic rat strains having small segments of Brown Norway chromosome 13 introgressed into the Dahl salt sensitive genetic background. Chromosomal segments either contained the Brown Norway renin allele (Ren1-BN) or are cut off just above (Ren1-SSA) or just below (Ren1-SSB) the renin gene locus and thus retain the SS renin allele. This figure has been modified and reprinted from Durand et al.29 with permission of the American Physiological Society. Please click here to view a larger version of this figure.
Figure 4: Response of isolated, cannulated MCAs of SS rats and congenic rat strains (illustrated in Figure 2) to 1 µM acetylcholine. Data are expressed as mean diameter change (Δ µm) ±SEM from resting control diameter prior to acetylcholine, and are replotted from an original study by Durand et al.29 Please click here to view a larger version of this figure.
Figure 5: Response of isolated, cannulated MCAs of SS rats and SS.5BN consomic rats to 1 µM acetylcholine in animals fed low salt (0.4% NaCl) or high salt (4% NaCl) diet. Data are expressed as mean diameter change (Δ µm) ±SEM from resting control diameter prior to acetylcholine, and are replotted from an original study by Lukaszewicz et al.64 Please click here to view a larger version of this figure.
Figure 6: Response to 1 µM acetylcholine in isolated, cannulated MCAs of Nrf2(-/-) knockout rats and wild type controls fed low salt diet (0.4% NaCl), high salt diet (4% NaCl), or high salt diet containing a known NRF2 activator30,62. Data are expressed as mean diameter change (Δ µm) ±SEM from resting control diameter prior to acetylcholine. Please click here to view a larger version of this figure.
Figure 7: Cerebral blood flow assessed by LDF in the pial microcirculation. Sprague-Dawley rats maintained on a high salt diet for four weeks exhibited a significant impairment in their ability to maintain blood flow constant as arterial blood pressure was reduced in response to successive blood volume withdrawals. Data are plotted as mean percent of control ±SEM *p <0.05 vs. low salt diet at same mean arterial pressure. Please click here to view a larger version of this figure.
Figure 8: Micrograph of rat cremaster muscles labeled with rhodamine-labeled GS1 lectin to identify arterioles and capillaries for assessment of microvessel density. Cremaster muscles were obtained from Sprague-Dawley rats fed low salt (LS; 0.4% NaCl) or high salt (HS:4% NaCl) diet for 2 weeks, and demonstrate microvascular rarefaction in the HS-fed animals compared to LS controls. Scale bar = 100 µm. Please click here to view a larger version of this figure.
As noted in the introduction, this paper describes the use of television microscopy and isolated resistance artery approaches to evaluate vascular function not only in standard rat models (as employed in the video), but also in highly specialized genetically engineered rat strains, which show the novel and powerful insights that can be gained utilizing these approaches. The use of these powerful techniques to evaluate active tone and passive mechanical properties of small resistance arteries can provide important information regarding a wide spectrum of vascular control mechanisms including endothelium-dependent regulation of active tone in resistance arteries, vascular smooth muscle function under normal and pathophysiological conditions, and passive properties of the arteries related to vascular remodeling and changes in vascular wall mechanics. Endothelial dysfunction has been shown to be a powerful prognostic indicator of multiple adverse cardiovascular events in humans including cardiovascular-related death63, and cannulated resistance artery preparations are especially valuable in detecting endothelial dysfunction and increasing our understanding of the mechanisms of endothelial dysfunction.
To illustrate the effectiveness of in vitro video microscopy techniques employing isolated resistance arteries, we provided examples of the use of these techniques in Dahl salt sensitive (SS) rats and in novel consomic rat strains that exhibit a reduced salt sensitivity of blood pressure compared to the SS parental strain24. Those studies investigated vascular control mechanisms related to genes on two chromosomes that are of particular interest in contributing to salt sensitivity of blood pressure and vascular alterations in the SS rat. Those chromosomes are chromosome 13, carrying the renin gene18,22,29,57,58, and chromosome 5, carrying genes for isoforms of CYP450-4A ω-hydroxylase64,65—the enzyme that synthesizes 20-HETE, which has major effects on renal function and on vascular reactivity66,67,68. Another recent and powerful addition to the rat genetic toolbox is the development of rat gene knockout models utilizing advanced gene editing techniques including: ZFNs; transcriptional activator-like-effector nucleases (TALENS), and most recently CRISPR-Cas913,14,15,16,17. In vitro video microscopy utilizing these techniques to study isolated MCAs from a Nrf2(-/-) knockout rat model that lacks the crucial antioxidant and cell protective transcription factor NRF2, has provided important and previously unknown insight into the mechanisms of salt-induced endothelial dysfunction in the absence of an elevated blood pressure17. Specific results of experiments utilizing these specialized rat models are described in a number of previous reports17,29,57,58,59,60,64,65.
While it is clear that studies of isolated, cannulated resistance arteries are extremely valuable in understanding the mechanisms regulating the function of these critically important vessels under a variety of conditions, it is very important to exercise a number of precautions in order to ensure that accurate and reliable results are obtained. While cerebral arteries and resistance arteries of many other vascular beds exhibit intrinsic tone, some arteries (notably mesenteric resistance arteries) need to be pre-contracted with vasoconstrictors such as norepinephrine in order to evaluate responses to vasodilator stimuli, and to more accurately simulate in vivo conditions, where the vessels are under the influence of neural and humoral vasoconstrictor stimuli, such as norepinephrine released from adrenergic nerve terminals. As such, it is important to be familiar with the basic properties of the arteries to be studied, either from the literature or from carefully conducted preliminary experiments. Because the endothelium plays a major role in regulating large arteries, small arteries, and arterioles in the microcirculation, it is essential to exercise caution to avoid damaging the endothelium during isolation and cannulation of the artery. The classic test for endothelium integrity is the demonstration that ACh causes vessel dilation. One caveat is that, under conditions of oxidative stress, the vessel endothelium may be intact, but vasodilatation to ACh is absent because excessive levels of superoxide scavenge the nitric oxide preventing the vasodilator response. In those cases, endothelial integrity can be verified by repeating the ACh application in the presence of a superoxide scavenger such as tempol, which should restore vasodilation in response to ACh and other endothelium-dependent vasodilator stimuli. Also, in a number of pathological conditions, the endothelium can release substances that cause contraction of the vascular smooth muscle cells and constriction of the artery; and in some cases, endothelium-dependent dilation (or constriction) is mediated via other substances such as cyclooxygenase metabolites, H2O2, epoxygenase metabolites, etc. The classic test for an endothelium-dependent vasodilator or vasoconstrictor substance is to demonstrate that the dilation or constriction of the artery is eliminated by endothelial removal. Finally, the identity of various endothelium-dependent vasodilator and vasoconstrictor substances can generally be ascertained by administration of specific inhibitors or scavengers, such as L-NAME to inhibit nitric oxide synthase, indomethacin to inhibit the formation of cyclooxygenase metabolites, catalase to scavenge H2O2, thromboxane synthase inhibitors, epoxygenase inhibitors, and/or antagonists of the CYP450-4A/20-HETE pathway.
It is also important (and instructive) to quantify the amount of active tone at the end of the experiment by perfusing and superfusing the artery with a Ca2+-free PSS, or administering a maximal dose of a powerful vasodilator agent such as papaverine. A typical level of active resting tone in the MCA, calculated as ((Dmax-Drest)/Dmax) x 100, is approximately 40%, where Dmax and Drest are the maximum (Ca2+-free PSS) and resting diameter (PSS), respectively, at the control equilibration pressure (usually 80 mmHg for MCA). Vessels with substantially less active tone or arteries showing segmental constrictions or dilations are excluded from analysis, as those signs are indicative of trauma to the vessels. Measurement of the diameters of maximally dilated arteries in Ca2+-free solution also allows the investigator to evaluate the passive mechanical properties of the vessels by measuring arterial diameter and calculating stress-strain relationships during sequential elevations in transmural pressure in the maximally dilated vessels7,56. These passive stress-strain relationships are easily obtained, and provide a valuable indication of any changes in mechanical characteristics of the vessels.
Cleanliness of the pipettes, connectors, and tubing supplying the reservoirs is absolutely critical for successful experiments. In this regard, it is important to flush all solutions out of the tubing after the experiment is completed, and to rinse and clean the tissue bath, the delivery tubing, and all reservoirs used to store, warm, and gas-equilibrate the PSS prior to reaching the vessel chamber. Stopcocks and valves in the delivery system should also be cleaned and changed periodically, as should any tubing carrying PSS. A classic sign of contaminated tubing is a gray haze generated by mold and bacteria; and those changes are accompanied by loss of normal reactivity of the blood vessels due to substances produced by the bacterial contamination. However, contamination by bacteria and other microorganisms can still be present in the absence of any visible evidence.
We believe that the present paper provides a helpful example for the use of time-honored techniques that are exceptionally well-suited for studies of the all-important small resistance arteries of different vascular beds. When combined with standard approaches for evaluating tissue perfusion, such as LDF and the GS1 lectin method for evaluating microvessel density, in vitro video microscopy of cannulated resistance arteries provides extremely valuable insight into the factors that control tissue perfusion and how these may be altered in disease states. In addition to providing a powerful means to study fundamental mechanisms of vascular smooth muscle and endothelial function in standard rat models, the use of video microscopy to study individual resistance arteries can be applied to other animal models and to human resistance arteries. The application of video microscopy of isolated resistance arteries of novel genetically engineered rat models opens new doors to understanding phenotypic changes that occur in response to altered function of a multitude (and ever-growing list) of genes.
The authors have nothing to disclose.
The authors express their sincere thanks to Katie Fink and Lynn Dondlinger for their invaluable assistance in the preparation of this manuscript.
Grant Support: NIH #R21-OD018309; #R56-HL065289; and #R01-HL128242.
SS Rat | Medical College of Wisconsin | SS/JHsd/Mcwi strain | Contact Dr. Aron Geurts (ageurts@mcw.edu) |
SS.5BN Consomic Rat | Medical College of Wisconsin | SS-Chr 5BN/Mcwi strain | Contact Dr. Aron Geurts (ageurts@mcw.edu) |
SS.13BN Consomic Rat | Medical College of Wisconsin | SS-Chr 13BN/Mcwi strain | Contact Dr. Aron Geurts (ageurts@mcw.edu) |
Ren1-BN Congenic Rat | Medical College of Wisconsin | SS.BN-(D13hmgc41-D13)hmgc23/Mcwi strain | Contact Dr. Aron Geurts (ageurts@mcw.edu) |
Ren1-SSA Congenic Rat | Medical College of Wisconsin | SS.BN-(D13rat77-D13rat105/Mcwi strain | Contact Dr. Aron Geurts (ageurts@mcw.edu) |
Ren1-SSB Congenic Rat | Medical College of Wisconsin | SS.BN-(D13rat124-D13rat101/Mcwi strain | Contact Dr. Aron Geurts (ageurts@mcw.edu) |
Nrf2(-/-) Knockout Rat and Wild Type Littermates | Medical College of Wisconsin | SD-Nfe212em1Mcwi strain | Contact Dr. Aron Geurts (ageurts@mcw.edu) |
Low Salt Rat Chow (0.4% NaCl)-AIN-76A | Dyets, Inc. | 113755 | |
High Salt Rat Chow (4% NaCl)-AIN-76A | Dyets, Inc. | 113756 | |
Colorado Video Caliper | Colorado Video, Inc. | Model 308 | |
Video Camera | Hitachi | KPM1AN | |
Microscope | Olympus Life Science | CKX41 | |
Television Monitor | Panasonic | WVBM1410 | |
Pressure Transducers | Stoelting | 56360 | |
Blood Pressure Display Unit | Stoelting | 50115 | |
Cannulated Artery Chamber | Living Systems Instrumentation | CH-1 | Single vessel chamber for general use |
Temperature Controller for Single Chamber | Living Systems Instrumentation | TC-09S | |
Gas Dispersion Tube, Miniature,Straight | Living Systems Instrumentation | GD-MS | Provides aeration in the vessel bath |
Gas Exchange Oxygenator, Miniature | Living Systems Instrumentation | OX | Allows gas exchange with perfusate |
Laser-Doppler Flowmeter | Perimed | PeriFlux 5000 LDPM | |
GS1 Lectin | Vector Labs | RL-1102 | |
Glass Capillary Tubes for Micropipettes | Fredrich Haer Co. | 27-33-1 | 2 mm ODX1 mm ID |
Verticle Pipette Puller | David Kopf Instruments | Model 700C | |
Nylon suture material (10/0)-3 PLY | Ashaway Line and Twine Manufacturing Co. | 114-ANM-10 | Single strands of 3 ply nylon suture teased out for use on vessels |
Dumont #5 Forceps-Inox | Fine Science Tools | 11254-20 | |
Vannas Scissors | Fine Science Tools | 15003-08 | |
Protandim | Protandim | NRF2 Inducer: Contact Dr. Joe McCord (JOE.MCCORD@UCDENVER.EDU) | |
Sodium Chloride | Fisher Bioreagents | BP358-212 | |
Sodium Bicarbonate | Fisher Chemical | S233-3 | |
Dextrose (d-glucose) anhydrous | Fisher Chemical | D16-500 | |
Magnesium Sulfate (MgSO4-7H2O) | Sigma Aldrich | M1880-500 G | |
Calcium Chloride (CaCl2-2 H2O) | Sigma | C5080-500G | |
Sodium Phosphate-Monobasic (NaH2PO4) | Sigma | S0751-500G | |
Potassium Chloride (KCl) | Fisher Chemical | P217-500G | |
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) | Sigma | ED255-500G |