Intravital investigation of rat mesenteric small artery tone and blood flow

Abstract

Key points: Substantial information on rat mesenteric small artery physiology and pharmacology based on in vitro experiments is available. Little is known about the relevance of this for artery function in vivo. We here present an intravital model where rat mesenteric small artery diameters are studied under isolated and controlled conditions in situ with simultaneous measurement of blood flow. The responses of the isolated arteries vary with the anaesthetic used, and they are quantitatively but not qualitatively different from the responses seen in vitro.

Abstract: Functional characteristics of rat mesenteric small arteries (internal diameter ∼150-200 μm) have been extensively studied in vitro using isometric and isobaric myographs. In vivo, precapillary arterioles (internal diameter < 50 μm) have been studied, but only a few studies have investigated the function of mesenteric small arteries. We here present a novel approach for intravital studies of rat mesenteric small artery segments (∼5 mm long) isolated in a chamber. The agonist-induced changes in arterial diameter and blood flow were studied using video imaging and laser speckle analysis in rats anaesthetized by isoflurane, pentobarbital, ketamine-xylazine, or by a combination of fentanyl, fluanison and midazolam (rodent mixture). The arteries had spontaneous tone. Noradrenaline added to the chamber constricted the artery in the chamber but not the downstream arteries in the intestinal wall. The constriction was smaller when rats were anaesthetized by rodent mixture in comparison with other anaesthetics, where responses were qualitatively similar to those reported in vitro. The contraction was associated with reduction of blood flow, but no flow reduction was seen in the downstream arteries in the intestinal wall. The magnitude of different endothelium-dependent relaxation pathways was dependent on the anaesthesia. Vasomotion was present under all forms of anaesthesia with characteristics similar to in vitro. We have established an intravital method for studying the tone and flow in rat mesenteric arteries. The reactivity of the arteries was qualitatively similar to the responses previously obtained under in vitro conditions, but the choice of anaesthetic affects the magnitude of responses.

Keywords: anaesthesia; in situ; in vivo; resistance artery.

Figure 1. The experimental setup for functional intravital studies of rat mesenteric small artery

A, schematic drawing of the experimental set‐up. An anaesthetized rat was placed on a heating plate and a small segment of the intestine was exteriorized. A segment of a branch of the mesenteric artery with the corresponding vein was placed into the reservoir filled with a physiological salt solution (PSS); the other exteriorized tissue was kept moist using bandages soaked in PSS. The arterial diameter was recorded using video microscopy. B, a small segment of exteriorized intestine and the experimental chamber with the arterial and vein braches of mesenteric artery. Parafilm was placed on top of the exteriorized intestine and mesentery to reduce evaporation. i, ii and iii indicate the areas where diameter and blood flow index were measured for the artery in the chamber, for the artery downstream for the chamber and for the artery in the intestinal wall downstream of the chamber, respectively. C, artery and vein passing through the wall of the experimental chamber.

Figure 2. Telemetric recordings of systolic blood pressure (filled circles), diastolic blood pressure (open circles) and heart rate (grey squares) 2 h before anaesthesia and 3 h after injection of rodent mixture (A; n = 4), ketamine–xylazine (B; n = 5), pentobarbital (C; n = 6) and isoflurane inhalation (D; n = 4)

Arrows indicate the beginning of anaesthesia. There was no effect on blood pressure and heart rate during application of drugs in the chamber.

Figure 3. Vasoconstriction to noradrenaline (NA) of rat mesenteric small arteries in situ

A–C, example traces from the experiments where rats were anaesthetized with rodent mixture (A), ketamine–xylazine (B) and isoflurane (C). D, the normalized concentration–response curves from the experiments where rats were anaesthetized with ketamine–xylazine (n = 8), isoflurane inhalation (n = 8), pentobarbital (n = 7) and rodent mixture (n = 5). ^* P < 0.05 for ketamine–xylazine anaesthesia in comparison with isoflurane and pentobarbital anaesthesia; concentration–response curves were compared by extra sum‐of‐squares F test.

Figure 4. Dilatation to increasing concentrations of acetylcholine of arteries preconstricted with noradrenaline (NA)

A, a representative trace from a rat anaesthetized with ketamine–xylazine. B, the concentration–response curves from the experiments where rats were anaesthetized with ketamine–xylazine (n = 8), isoflurane inhalation (n = 7) and pentobarbital (n = 6).

Figure 5. Constriction to arginine vasopressin (AVP) of mesenteric small arteries in situ

A–C, representative traces from the experiments where rats were anaesthetized with rodent mixture (A, n = 3), ketamine–xylazine (B, n = 3) and isoflurane inhalation (C, n = 3). D, normalized concentration–response curves for the experiments shown in A–C and for experiments where rats were anaesthetized with pentobarbital (n = 6). ^* P < 0.05 vs. rodent mixture anaesthesia; concentration–response curves were compared by extra sum‐of‐squares F test.

Figure 6. The rhythmic oscillations in arterial diameter, vasomotion, were induced by preconstriction with either noradrenaline (A and B) or arginine vasopressin (AVP) (C and D) and varied under different forms of anaesthesia

Noradrenaline‐induced vasomotion was analysed in rats anaesthetized by ketamine–xylazine, isoflurane and pentobarbital (n = 15). When arteries were AVP preconstricted, vasomotion was compared between rodent mixture, ketamine–xylazine, isoflurane and pentobarbital anaesthesia. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 (one‐way ANOVA followed by Bonferroni post hoc test).

Figure 7. Spectrum analysis of oscillation frequencies obtained in situ in the presence of 0.2–0.8 μm noradrenaline

Oscillations obtained under control conditions and after incubation with inhibitors. A and B, vasomotion was recorded under control conditions (n = 15) and after incubation with 1 μm TRAM‐34 and 50 nm apamin (n = 8), or after incubation with 100 μm l‐NAME and 3 μm indomethacin (n = 7). A, representative traces from such experiments; B, their averaged spectrum analyses. C and D, in another experiment (representative trace in C), vasomotion was recorded under control conditions, in the presence of 10 μm cyclopiazonic acid (CPA), and in the presence of a combination of 10 μm CPA, 1 μm TRAM‐34 and 50 nm apamin (averaged spectrum analyses in D). n = 5. ^* P < 0.05, ^** P < 0.001 vs. control conditions (two‐way ANOVA followed by Bonferroni post hoc test).

Figure 8. LSI analysis of vascular responses in different parts of the mesenteric circulation

A and B, examples of BFI recorded in the chamber (A; a and v indicate the artery and the vein, respectively) and in the intestinal wall downstream from the chamber (B). Arrows indicate an artery subjected to the segmentation algorithm. C, normalized changes in diameter (left panel) and BFI (right panel) for the artery segment in the chamber to 10 μm noradrenaline (NA) added to the chamber, n = 4. D, normalized changes in diameter (left panel) and BFI (right panel) in response to application of 10 μm NA and 10 μm acetylcholine (ACh) to the chamber for arteries in the intestinal wall downstream of the chamber. Measurements are made under control conditions (n = 8) and in the presence of 1 μm phentolamine (n = 7). E, blood flow through the artery in the chamber measured with Transonic flow probe. Blood flow was significantly suppressed by 10 μm NA application to the chamber. Left panel shows representative recording from the experiment and right panel demonstrates averaged changes in normalized blood flow (n = 11). Blood flow is normalized to an averaged ‘baseline’ flow prior NA application.