In the cardiopulmonary bypass (CPB) used by an aortic arch surgery, deep hypothermia and selective cerebral perfusion have been performed for the purpose of cerebroprotection, but postoperative brain complications occasionally occur. Improvement of surgical techniques and perfusion methods were carried out for these complications, but the complications still exist.1 In addition, a new method for the cerebroprotection is necessary because in late years an upward trend of the setting temperature is seen. At present, antegrade selective cerebral perfusion for cerebroprotection is widely used in clinical settings as an established procedure to repair thoracic aortic aneurysm. Although there are slight differences in factors such as the period of circulation interruption, position of the transfusion vessel, and blood flow amount, in general, during such procedures blood supply is achieved by nonpulsatile perfusion using a roller or a centrifugal pump.
Cerebral perfusion through nonpulsatile flow is a nonphysiological state in higher vertebrates because supply to various organs is achieved through pulsatile pumping of the heart. Surgical procedures using CPB sometimes cause brain damage, and this is probably linked to low perfusion.2 In fact, CPB creates many problems,3 including loss of pulse pressure, blood dilution, and low body temperature, all of which can cause deterioration of cerebral circulatory function into various nonphysiological states4,5 as a result of nonpulsatile flow.
There have been many studies related to cerebral flow and metabolism during CPB to determine whether low perfusion of brain tissue is caused by CPB,6–9 using a range of CPB systems to highlight changes in cerebral hemodynamics. However, there have only been a few studies to examine the changes in cerebral microcirculation during CPB.10 In comparison with the previous study, our synchrotron imaging can assess microcirculatory function in real time. In addition, our study can evaluate pulsatile flow versus nonpulsatile flow of single animal.
Using the rat CPB system developed by us11,12 and a pulsatile flow generator composed of a simple magnet valve and control device,13 we established pulsatile selective cerebral perfusion in the rat model. Making use of these systems/devices, we performed angiography at one of the world’s largest synchrotron radiation facilities (SPring-8) to evaluate differences in cerebral microcirculation between nonpulsatile flow and pulsatile flow selective cerebral perfusion. We assessed perfusion distribution states through methods based on relative vessel diameter of the internal carotid artery (ICA), blood vessel count in a set region (angiographic score), and brightness (radiopacity).
Although a blood vessel diameter of 200 μm can be measured with conventional medical angiography devices, microvessels of 20 μm can be measured with high-resolution synchrotron radiation microangiography systems making use of monochromatic x-rays at 33.2 keV (above the K-edge of iodine). This improvement in spatial and temporal resolution enables the visualization of microvessels, which autoregulate blood flow to organs, at the 20–200 μm arteriole-small artery level, which enables better comprehension of the physiology of diseases in vivo.14–16
Materials and Methods
All experiments were performed at the 28B2 beamline of the Japan Synchrotron Radiation Research Institute, SPring-8 in Hyogo, Japan. Male Wistar rats (n = 8, 400–500 g) were purchased from a licensed vendor and maintained with free access to food and water in compliance with the guidelines of the Physiological Society of Japan and approved by the regulations for Animal Experiments of Hiroshima International University.
Surgical Procedure and Experimental Conditions
Pentobarbital (Nembutal) was administered intraperitoneally at 60 mg/kg to anesthetize the animals. Thereafter, anesthesia was maintained by administration of 25 mg/h/kg until the end of the experiment. After a cervical midline incision, tracheal intubation was carried out with a 14 G tube and animals artificially ventilated with a rodent ventilator (Respirator Model SN-480-7; Shinano Corp., Tokyo, Japan) at a tidal volume of 10 ml/kg/respiratory at a rate of 50 respiratory/min (before thoracotomy) and 70 respiratory/min (at thoracotomy) at 100% oxygen.
Electrocardiography was used to monitor the rats. An arterial line was inserted in the femoral artery for fluid maintenance and drug infusion. A temperature probe was placed in the larynx, and a heating pad beneath the rat was used to maintain core temperature. A median sternotomy was conducted, and the rats were fully heparinized through the arterial line (100 IU in 1 ml saline). Activated clotting time was maintained at 400 s or more in all cases throughout the study. As a (aortic) transmitting vessel, a 1.1 mm diameter polyethylene catheter was antegradely inserted in the right common carotid artery; this was followed by insertion under direct vision of an 18 gauge indwelling needle into the right atrium as a blood draining vessel. A diagram illustrating the experiment setup at SPring-8 is shown in Figure 1.
Potassium-based cardiac arrest was induced and maintained at normothermia and severe hypothermia (25°C) while rats, secured on an acrylic board, were exposed to the horizontal synchrotron radiation beam. Angiography recordings with high-intensity synchrotron radiation were conducted intermittently in the nonpulsatile selective cerebral perfusion or pulsatile flow selective cerebral perfusion with the aid of a pulsatile flow generator placed in the nonpulsatile flow system. Cerebral perfusion was maintained at 4 ml/min. Heparinized contrast agent Iomeron (Bracco-Eisai, Tokyo, Japan; iodine 350 mg/cm3 containing heparin 13 IU/ml) was introduced at a speed of 0.4 ml/s17 during imaging. Angiography was first performed at a normal/low temperature in the nonpulsatile flow mode, then during pulsatile flow, and after severe hypothermia caused by “surface-induced hypothermia.” Lastly, nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine-methyl ester hydrochloride (L-NAME, 50 mg/kg IV), was administered in the severely hypothermic state because half-life of L-NAME is very long; there is an influence on the experiment under other conditions, and angiography during pulsatile cerebral perfusion was performed again after 30 minutes of equilibration.
Pulsatile flow was generated by activation of a magnetic valve with a 1 Hz signal through the pulsatile flow generator of the rat CPB system created by us. We confirmed that the signal from control unit was the same as the real pulsatile times during the experiment.
Cardiopulmonary Bypass Preparation
The rat CPB system is composed of a circuit, heat exchanger, oxygenator (membrane area: 0.03 m2, fill ration: 2.0 ml; Senkou Ika Kogyo, Tokyo, Japan), and roller pump (MF-01; JMS Co., Hiroshima, Japan) and has a fill ration of 15 ml. Baseline conditions for selective cerebral perfusion included an O2 flow rate of 100 ml/min during a blood delivery volume of 4 ml/min at 36°C for normothermic and 25°C for severe hypothermic recordings. We maintained a pCO2 of approximately 40 mm Hg partial pressure in the arterial blood through the addition of O2 and CO2 in a ratio of 95:5 to the oxygenator. A hematocrit of approximately 35% was maintained with rat blood from donor animals during selective cerebral perfusion through a one-way process of drainage from the right atrium, while blood supply was made through the ICA.
Synchrotron Radiation and Angiographic Analysis
The x-ray detector system has been described previously by us.14–16 Synchrotron radiation has a broad and continuous spectrum. A crystal monochromator selects a single energy of radiation. A rotating-disk x-ray shutter, which is situated between the monochromator and the object of interest, produces pulsed monochromatic x-rays. After transmission through the animal, x-rays are detected using the SATICON image detector (Hamamatsu Photonics, Hamamatsu, Japan).14,15 Images (1,024 × 1,024 pixels) were recorded at a rate of 30 frames per second for 3.3 s (100 frames) and stored in 10 bit format. The input field (field of view) of the x-ray SATICON camera was 9.5 mm × 9.5 mm (1 pixel = ~10 µm). Shutter open time was ~4.0 ms. Monochromatic x-ray energy was adjusted to 33.2 keV, just above the iodine K-edge energy for maximal contrast. The highly collimated x-ray beam, the small x-ray source, and long distance between the source and the tissue of interest does not result in a significant magnification of the blood vessels (i.e., 1.004).17
The diameter of ICA (>200 μm) was measured from individual frames of each scan series. An angiographic score was calculated for each angiograph frame as the ratio of grid (25 mm2) intersections crossed by opacified arteries divided by the total 1,848 grid intersections on printed images (20 cm × 20 cm). The angiographic score was determined as the mean value recorded by three blinded observers.18,19
To establish an index of microvessel perfusion distribution for both perfusion methods at normothermia and severe hypothermia, the change in mean transmitted x-ray intensity of a defined area of each image (63.000 pixels in the center of each image) was compared with the intensity at the start of imaging as the control. Intensity changes were followed until the contrast agent had spread evenly through the capillaries in both nonpulsatile and pulsatile flow states.
All experimental data were expressed as mean ± standard error (SE). Paired Student’s t-tests were used to compare the difference between groups. p < 0.05 was considered to be statistically significant.
At SPring-8, we performed angiography during selective cerebral perfusion of pulsatile and nonpulsatile. Figure 2 shows the pressure in the sending circuit during an angiography.
Diameter of Internal Carotid Artery
Internal carotid artery diameter was manually measured when the contrast agent had evenly spread through the capillaries (Figure 3A). At normothermia, ICA diameters during nonpulsatile and pulsatile flow were 321.1 ± 30.4 μm vs. 420.0 ± 42.9 μm, respectively (p < 0.05). At severe hypothermia, the same diameters were 274.12 ± 13.96 μm vs. 312.50 ± 13.28 μm, respectively (p < 0.05). Irrespective of temperature, ICA diameter was observed to be significantly larger with pulsatile flow (Figure 4A-1 and A-2). The relative increase in ICA diameter under pulsatile flow was especially pronounced at normothermia (Figure 4A-3).
The angiographic scores for nonpulsatile and pulsatile flow during selective cerebral perfusion at normothermia were 0.198 ± 0.013 and 0.258 ± 0.010, respectively (p < 0.001; Figure 3B). Scores for nonpulsatile and pulsatile flow at severe hypothermia were 0.151 ± 0.017 and 0.214 ± 0.015, respectively (p < 0.01). At both temperatures, pulsatile flow showed a significantly higher angiographic score, but especially so at normothermia (Figure 4B).
Perfusion Distribution Index
At each temperature, the image mean transmitted x-ray intensity during pulsatile flow progressively decreased from the initial stage of contrast entry; therefore, radiopacity increased with microvessel perfusion. There were indications that perfusion distribution of the contrast agent reached arterioles and smaller vessels. Compared with the transmitted intensity in the nonpulsatile state at normothermia, mean intensity decreased more rapidly with pulsatile flow. When comparing final intensity, which was arbitrarily determined as the time point at which arteriole–capillary perfusion was achieved, there was a significant difference from the intensity at entry time at normothermia (Figure 5A, nonpulsatile flow 88.63 ± 1.21% vs. pulsatile 81.89 ± 0.90%, p < 0.05). However, during severe hypothermia, there was no appreciable difference in apparent extent of perfusion between the two types of flow in the region of interest (Figure 5B, nonpulsatile 82.22 ± 2.81% vs. pulsatile 80.16 ± 1.79%, p > 0.05).
Pulsatile Flow After L-NAME Treatment
The relative change compared with the maximum ICA diameter during nonpulsatile flow was not different at 98.50 ± 1.7% (vs. 114.96 ± 4.6% pulsatile flow before L-NAME), but the angiographic score was significantly depressed at 0.125 ± 0.019 (vs. 0.214 ± 0.015, p < 0.0235 pulsatile before L-NAME). Mean x-ray intensity after contrast reached the microvessels was also similar at 81.83 ± 2.10% (vs. 80.16 ± 1.79% pulsatile before L-NAME; Figure 6).
We conducted selective cerebral perfusion using rats at normothermia and severe hypothermia and performed an investigation of the cerebral microcirculation in pulsatile and nonpulsatile selective cerebral perfusion states. Dynamic synchrotron angiography under several set conditions allowed us to determine changes in the maximum diameter of the ICA that was the major conduit artery supplying the base of the brain and assess changes over time in microvessel perfusion of a vessel count and average tissue radiopacity.
Using our CBP circuit, we were able to achieve nonpulsatile and pulsatile selective cerebral perfusion at two core temperatures in the same animals. Importantly, we showed that the mean maximum ICA diameter increase during pulsatile flow at normothermia was double that of the increase under hypothermia (32% vs. 15%). Angiographic score, based on the proportion of visible arterial segments (arteriole and artery), was 30% larger under normothermic pulsatile conditions in comparison with the nonpulsatile baseline condition. Lowering the core temperature to 25°C decreased the number of perfused vessels visible by 20% under nonpulsatile flow conditions, but the vessel score was restored by pulsatile flow at the lower temperature. As evident in the angiographs of the brain stem in Figures 4 and 5, during pulsatile perfusion, the number of small artery (>100 μm) and arteriole segments (<100 μm) were increased. In this study, the changes in cerebral perfusion we observed are not attributive to change of a pCO2 in blood gas equilibrium as this was controlled by the oxygenator. Hence, pulsatile perfusion improved the cerebral vasodilation and the number of arterial microvessel segments perfused in a manner directly dependent on core temperature.
Similarly, the rate of decline in angiogram brightness, or the increase in radiopacity, within the brain stem and overlying cerebellum was enhanced by pulsatile selective cerebral perfusion at normothermia, at least. This suggests that the greater fluid energy inherent in pulsatile flow20 increases capillary perfusion by recruiting arteriolar vessels and thus providing enhanced perfusion distribution. It is not clear why the brightness ratio change did not differ between perfusion states under hypothermic conditions. However, increased venous transit of contrast agent might contribute to the similar final radiopacities attained under both pulsatile and nonpulsatile flow states during hypothermia.
Although the merit of pulsatile selective cerebral perfusion was demonstrated at both set temperatures, the improvement in perfusion was markedly reduced with the administration of L-NAME regardless of pulsatile flow. The diameter of ICA and the perfusion indices after L-NAME were roughly equivalent with that under nonpulsatile flow before L-NAME. From this, it is surmised that there is a profound relationship between the efficacy of pulsatile flow and NO production. We believe that NO secretion from endothelium is stimulated by the mechanical shear stress of pulsatile flow on vessel endothelium.21–23
In recent years, an increasing tendency to raise the temperature of setup system has been observed for nonpulsatile selective cerebral perfusion in procedures for thoracic aortic aneurysm conducted in clinical settings. At present, nonpulsatile selective cerebral perfusion is conducted at temperatures ranging from extreme to intermediate hypothermia.24 In this study, we could evaluate the change in body temperature and two flow pattern perfusion of single animal. It is presumed that selective cerebral perfusion will be performed more at normothermia as advancements are made in surgical techniques and medical equipment. Pulsatile selective cerebral perfusion system that we developed supply the brain with physiologic pulsatile conditions, and thus increased cerebroprotective efficacy during CBP surgery might be expected during selective cerebral perfusion based on the findings presented in this study.
In conclusion, the significance of pulsatile flow in selective cerebral perfusion is clear after evaluation of ICA vessel diameter, angiographic score, and perfusion distribution based on angiographs recorded with high-intensity synchrotron radiation. The efficacy of pulsatile flow in enhancing cerebral microvessel perfusion during normothermic conditions was noted over severe hypothermia. Our dynamic angiograph scans also suggest that NO production has a major influence in the mechanisms of pulsatile flow–mediated perfusion.
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