Cardiovascular Research Laboratory

 

Our state of the art systems are well suited to screen vascular drug effects at early phases of drug developments. The facilities are available on a contractual basis.

Methods:
-Cannulated microvascular measurements (arteriolar diameter: 60-350 micrometer)
-Simultaneous measurement of vascular contractile responses (isometric or isobaric conditions) and changes in intracellular Ca-concentrations
-Experties in vessels isolated from various species including human. In detail, large vessels: human saphenous veins, rat basillar arteries, carotid arteries, mesenteric arteries and aorta; small arteries: human and rat coronary arteries, rat and mouse skeletal muscle (m. gracilis) arterioles


Contact us:
E-mail: Attila Toth, PhD.
Telephone: +36 52 414-928
Fax: +36 52 414-928



1. Isometric contractile measurement system (vascular rings)

The isometric contractile system is suitable to measure vascular contractile responses simultaneously with continous recording of intracellular Ca-concentrations (Fura-2 based Ca-imaging). The measurements are performed on segments of arteries (e.g. rings from basilar artery of the rat). The experiments usually include the assessment of arteriolar viability by the application of KCl (Fig. 1 and 2).

 

Fig. 1 Representative recording of contractile force and intracellular Ca-concentration changes in isolated basilar artery segments of the rat.

 

 

Fig. 2 Changes in intracellular Ca-concentration and contractile force in isolated basilar artery segments of the rat upon KCl stimuli. The animated picture gives insight into the detection of intracellular Ca-concentrations. A summary of the individual data is shown, bars are mean+/-SEM.

 

An example for a vasoactive agent is sertotonin (Fig. 1 and Fig. 3). Serotonin evokes a significantly higher vasoconstriction than KCl with similar changes in intracellular Ca-concentrations (Fig. 2 and Fig. 3). This fact suggests that serotonin evoked contraction is mediated by both an increase in intracellular Ca-concentration and an increase in Ca-sensitivity of vascular smooth muscle cells.

Fig. 3 Changes in intracellular Ca-concentration and contractile force in isolated basilar artery segments of the rat upon serotonin (5-HT) stimuli. The animated picture gives insight into the detection of intracellular Ca-concentrations. A summary of the individual data is shown, bars are mean+/-SEM.

 

 


2. Cannulated microarteriolar system

Cannulated systems have many advantages compared to the conventional isometric (ring) systems: smaller (resistance) arteries can be tested; presence of a spontanaeous myogenic tone; direct data about changes in the vascular diameter; more physiological conditions. These advantages makes this system suitable to test drug effects at the resistance arterioles; to measure endothelial function (not only NO but also EDHF mediated dilations); and to use small tissue samples as a sorce of arterioles.

The system may be used to screen potential vascular side effects upon drug development. For example, the capsaicin receptor (TRPV1) has been considered to be specific to sensory neurons and considerable pharmacological effort was exerted to develop analgesic agents by blocking this receptor. However, a functional TRPV1 expression has been found in arteriolar smooth muscle cells as well, suggesting potential side effects (Kark et al.).

 

2.1. Measurement of vascular diameter of cannulated resistance arterioles

Changes in arteriolar diameter of cannulated microarterioles can be recorded by videomicroscopy. Agonist/antagonist effects can be monitored. Important to note, that these arterioles have a spontaneous myogenic tone usually developing at 60 mmHg or at higher intraluminal pressures. Antagonism of this spontanaeus tone results in dilation without preconstriction. Acetylcholine is one of the well characterized endothel dependent vasodilatative agents acting mainly through EDHF in microarterioles (Fig. 4).

Dose-response of acetylchokine to evoke endothel mediated dilations in skeletal muscle arterioles

Fig. 4 Changes in diameter of an arteriole isolated from m. gracilis of the rat upon acetylcholine stimuli. The animated picture gives insight into the dose-response characteristics; a summary of the individual data is also shown, bars are mean+/-SEM.

 

Cannulated microarterioles can also be stimulated by agonists to evoke contraction. One of the best characterized agonists is norepinephrine, which can be used to assess smooth muscle function (Fig. 5).

Dose-response of norepinephrine on skeletal muscle arteries

Fig. 5 Changes in diameter of an arteriole isolated from m. gracilis of the rat upon norepinephrine stimuli. The animated picture gives insight into the dose-response characteristics; a summary of the individual data is also shown, bars are mean+/-SEM.

Arterioles with (i) spontanaeus myogenic tone, (ii) intact endothelium and/or (iii) smooth muscle mediated functions can be pharmacologically tested.

 

2.1. Simultaneous measurement of vascular diameter and intracelluar Ca-concentration in cannulated resistance arterioles

The fluorescent imaging technique makes it possible to record changes in vascular diameter and intracellular Ca-concentrations simultaneously. The cannulated resistance arterioles are loaded with the fluorescent Ca-indicator dye Fura-2AM and images of the arterioles (filter >510 nm) is recorded upon illumination with alternating excitation wavelengths of 340 nm and 380 nm. The intensity ratio for these excitation wavelengths are calculated on every pixel and the resultant image is recorded. Average of ratios (within a region of interest) and the outer diameter of the arteriole can be calculated for every frames. Representative experiments are shown below. Acetylcholine evoked dose-dependent dilation (also shown on Fig. 4) with a decrease in intracellular Ca-concentration (Fig. 6).

Dose-response for acetylcholine

Fig. 6 Changes in intracellular Ca-concentration and arteriolar diameter in an isolated cannulated skeletal muscle arteriole upon acetylcholine stimuli. A representative recording of the cumulative dose-response is shown on the animated figure, the graph depicts the mean+/- SEM data of the individual measurements.

 

Smooth muscle function is usually tested by norepinephrine. Norepinephrine evokes robust contractions together with significant increases in intracellular Ca-concentrations (Fig. 7).

Dose-response for norepinephrine

Fig. 7 Changes in intracellular Ca-concentration and arteriolar diameter in an isolated cannulated skeletal muscle arteriole upon norepinephrine stimuli. A representative recording of the cumulative dose-response is shown on the animated figure, the graph depicts the mean+/- SEM data of the individual measurements.