There is increasing evidence that neuroprotection can be induced by haemodilution using low-molecular-weight dextran , albumin [2,3], gelatin , alpha-alpha diaspirin cross-linked haemoglobin [3,5] and hydroxyethyl starch (HES) (200/0.5) [6,7]. After haemodilution, cerebral blood flow increases because of the decreased haematocrit (Hct) and the increased cardiac output, which can produce direct physical effects in lowering blood viscosity and reducing erythrocyte aggravation [6,7].
A novel HES (130/0.4) (Voluven®; Fresenius AG, Bad Homburg, Germany) with an average molecular weight of 130 kD and a substitution degree of 0.4 has been developed recently. This HES specification has already been approved in several countries for routine volume replacement. HES 130/0.4 has been reported to have pharmacokinetic and pharmacodynamic advantages such as decreased tissue storage, rapid plasma elimination and low impact on coagulation [8-11]. There are no reports available whether haemodilution with the HES 130/0.4 confers a neuroprotective effect. The present study was conducted to determine whether acute normovolaemic haemodilution (ANH) with 6% HES 130/0.4 could attenuate focal cerebral ischaemia-reperfusion injury in a rat stroke model and to determine the optimum Hct value to be diluted for induction of brain protection.
The experimental protocol was approved by the Ethics Committee for Animal Experimentation and carried out according to the Guidelines for Animal Experimentation of the Fourth Military Medical University.
The animals were provided by the Experimental Animal Center of the Fourth Military Medical University. Seventy male Sprague-Dawley rats weighing 300 ± 20 g were used in this study. In Experiment 1, 30 rats were randomly divided into three groups (n = 10 each): HES, saline and control groups. Rats in HES and saline groups were haemodiluted with 6% HES 130/0.4 or 0.9% saline, respectively, until their Hct decreased to 30% while animals from the control group were not haemodiluted. In Experiment 2, 40 male Sprague-Dawley rats were randomly assigned to four groups (n = 10 each): HES30, HES25, HES30 and control groups. Rats in HES30, HES25 and HES20 groups were haemodiluted with 6% HES 130/0.4 until the Hct decreased to 30%, 25% and 20%, respectively. Animals from the control group were not haemodiluted. At 15 min after haemodilution, all animals were subjected to a 120 min right middle cerebral artery occlusion (MCAO) followed by a 24 h reperfusion period. The neurologic deficit scores were assessed at the end of reperfusion and infarct volumes were compared among groups.
Acute normovolaemic haemodilution
Animals were fasted with access to water ad libitum for 12 h before surgery. The rats were anaesthetized with 4% isoflurane in 96% oxygen by facemask. Anaesthesia was maintained with 2% isoflurane in 98% oxygen. All animals breathed spontaneously during the procedure. Femoral artery was cannulated and connected to a monitor (Model 90602A, Spacelabs Medical Inc., Redmond, WA, USA) via a pressure transducer for monitoring the mean arterial pressure (MAP) and heart rate (HR) while the femoral vein was cannulated for fluid administration. Prior to haemodilution, arterial blood was sampled from the catheter for determination of pH, arterial oxygen (PaO2), arterial carbon dioxide (PaCO2) by using the OMNI Modular System (Roche Diagnostics, Basel, Switzerland) and for determination of haemoglobin (Hb) and Hct by centrifugation as baseline data. ANH was accomplished by continuous withdrawal of blood from the arterial catheter. Blood loss was instantly replaced via the femoral vein catheter with same volume of 6% HES 130/0.4 (Beijing Fresenius Kabi Pharmaceutical Co., Ltd, Beijing, China) or with the threefold volume of 0.9% saline maintaining stable MAP and HR. The blood volume to be exchanged was calculated from the following equation:
Approximately 15 min were required to complete the haemodilution protocol. MAP, HR, Hb, Hct and blood gases were re-evaluated at the end of haemodilution.
Focal cerebral ischaemia
After haemodilution, the animals were allowed to stabilize for 15 min before focal cerebral ischaemia was induced. All the rats were re-anaesthetized with 4% isoflurane. Animals were breathing spontaneously and anaesthesia was maintained with 2% isoflurane delivered by facemask. Rectal temperature was monitored (Model 90651A, Spacelabs Medical Inc.) and kept constant between 37.0°C and 37.5°C by overhead lamps during the procedure. Focal cerebral ischaemia was induced by MCAO using intraluminal filament technique described previously [12,13]. Briefly, the right common carotid artery and the right external carotid artery were exposed through a ventral midline neck incision, and were ligated proximally. A blunt-tip 3-0 nylon monofilament suture (Ethicon Nylon Suture, Ethicon Inc., Osaka, Japan) was inserted through an arteriectomy in the common carotid artery just below the carotid bifurcation, and positioned into the internal carotid artery to a point approximately 17 to 18 mm distal to the carotid bifurcation until a mild resistance was felt when the origins of the anterior cerebral artery, the middle cerebral artery and the posterior communicating artery were occluded. After the intraluminal filament had been fixed in position, the neck incision was closed with suture. The animals were allowed to recover from anaesthesia and reperfusion was accomplished under the same anaesthetic condition by carefully withdrawing the intraluminal suture 120 min after ischaemia. The incision sites were infiltrated with 0.25% bupivacaine hydrochloride for postoperative analgesia.
Recovery and neurological evaluation
After withdrawing the suture and recovery from anaesthesia, the rats were returned to their cages with free access to food and water. At 24 h after reperfusion, the animals were assessed neurologically by an investigator who was unaware to the animal grouping. As described previously [12,13], a six-point scale modified from Longa and colleagues  was used for neurological assessment: 0 = no deficit; 1 = failure to extend left forepaw fully; 2 = circling to the left; 3 = falling to the left; 4 = no spontaneous walking with a depressed level of consciousness; 5 = dead. If the neurological deficit observed was better than the upper grade but worse than the lower grade, the score was expressed as the arithmetic mean of the two grades.
Infarct volume assessment
At the end of neurological assessment, the rats were re-anaesthetized with overdose ketamine and decapitated. The brains were rapidly removed and cooled in iced saline for 10 min. Six coronal sections at 2 mm in thickness were cut with the aid of a brain matrix. Sections were immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma, St Louis, MO, USA) at 37°C for 30 min and then transferred to 10% buffered formalin solution for fixation. Twenty-four hours later, the brain slices were photographed with a digital camera (Kodak DC240, Eastman Kodak Co., Rochester, NY, USA) connected to a computer. Unstained areas were defined as infarct and were measured by using image analysis software (Adobe Photoshop 7.0 CS for Windows, San Jose, CA, USA).
An investigator blinded to the experimental grouping then outlined the zones of infarction (which were clearly demarcated) as well as the outlines of the left and right hemisphere on photographs of each section on the computer screen. Infarct volume was calculated as the integrated product of cross-sectional area and inter-section distance. The infarct volume of each rat was corrected for swelling of the ischaemic hemisphere by applying the formula:
The total infarct volume was calculated as the sum of the infarct areas of the six sections and was presented as percentage of the volume of the contralateral hemisphere [15,16].
Physiological parameters and the infarct volumes were expressed as mean ± SD. NDS were expressed as median (range). Repeated measures analysis of variances (ANOVA) was performed for physiological data [7,17]. The infarct volumes were analysed by one-factor ANOVA followed by posthoc LSD test. NDS were analysed with Kruskal–Wallis test followed by the U-test with Bonferroni correction. P < 0.05 was considered statistically significant.
There were no significant differences in the physiological parameters within or among groups before and after ANH except for Hct and Hb in ANH groups that were decreased to the target values. Also the CaO2 decreased significantly after ANH, as shown in Tables 1 and 2.
The results of NDS and infarct volume of three groups are shown in Table 3 and Figure 1, respectively. The NDS was lower in HES group than in the control and saline groups (P = 0.002 for both comparisons). Infarct volume in the HES group (20.0 ± 4.0%) was significantly smaller compared with controls (38.8 ± 9.6%; P = 0.000) and saline (49.0 ± 10.5%; P = 0.000) groups. Infarct volume the in saline group was also larger compared with controls (P = 0.011).
Physiological data are summarized in Tables 4 and 5. There were no significant differences between groups before and after ANH except for Hct and Hb in ANH groups that were decreased to the target values. Also the CaO2 decreased significantly after ANH.
The results of NDS and infarct volume of four groups are shown in Table 6 and Figure 2. NDS in the HES30 group was significantly lower compared with controls (P = 0.001) and with the HES20 group (P = 0.003). NDS in HES25 group was significantly lower compared with controls (P = 0.004).
The infarct volume were significantly smaller in HES30 (19.3 ± 5.0%) and HES25 (26.9 ± 9.2%) compared with controls (39.0 ± 10.7%) (P = 0.000 and P = 0.005, respectively) and with HES20 (47.5 ± 10.2%) (P = 0.000 for both groups). The infarct volume in HES20 group was larger compared with controls (P = 0.044). No statistical difference was detected between the infarct volumes of the HES30 and HES25 groups.
ANH with 6% HES (130/0.4) to Hct 30% significantly improved neurological outcome and reduced infarct volume in the MCAO rat model. This neuroprotective effect was still detectable when Hct was diluted to 25% while the infarct-limiting effect of ANH disappeared as the Hct reached 20%.
Normovolaemic haemodilution has proven to be beneficial to ischaemic organs. After ANH, the resulting decrease in blood viscosity results in an acceleration of erythrocyte velocity and improvement of tissue oxygen delivery. In a prospective, randomized, controlled animal study Licker and colleagues  showed that pre-ischaemic moderate ANH with 6% HES (200/0.5) conferred cardioprotective effects and improved survival rate in a rat model of myocardial infarct. In addition, benefits of haemodilution with HES on renal function  and cerebral tissues [6,7] were also reported. In contrast, some clinical trials and experimental studies gave negative results [20,21]. The reason for the disparity of results might be attributed to the degrees of haemodilution used in different settings. It must be emphasized that the optimum Hct is pivotal for the protective effect of haemodilution. Excessive haemodilution may lead to organ ischaemia due to a reduced oxygen-carrying capacity induced by decompensation in autoregulatory or rheologic increases of organ blood flow. Homi and colleagues  haemodiluted Wistar rats with 6% HES solution (Hextend) in an experimental model of cardio-pulmonary bypass (CPB) with reversible MCAO-induced focal cerebral ischaemia. They showed that haemodilution to a target haemoglobin level of 6 g dL−1 led to worsening of the neurological functions and to an increase of cerebral infarct volume. In the present study, we confirmed that ANH until Hct 30% and 25%, using 6% HES (130/0.4), was neuroprotective while excessive haemodilution until Hct 20% worsened cerebral ischaemic injury in transient focal cerebral ischaemia in rats.
In addition, the choice of the haemodiluting agents may also be important. For instance, low-molecular-weight dextran has been found to increase plasma viscosity when given for more than 3 days. Furthermore, it may aggravate brain oedema formation and conveys the risk of anaphylaxis . HES, a derivative of amylopectin, apparently lacks these negative properties and could decrease the aggregations of erythrocytes and platelets. HES is characterized by its average molecular weight, its molar substitution (i.e. the ratio of substituted to total anhydroglucose residues on the polymer chain) and the substitution pattern (i.e. the ratio of substitution on residues C2/C6). The newly developed HES (130/0.4) with a mean molecular weight of 130 ± 20 kD, a molar substitution of 0.4, and a C2/C6 ratio of about 9 is metabolized by α-amylase in serum and eliminated via the kidneys. It has no significant effect on plasma glucose concentrations . Phase I studies showed that HES (130/0.4) is more rapidly eliminated from plasma through renal route compared with HES (200/0.5). HES (130/0.4) plasma concentrations return to baseline levels 24 h after infusion of 500 mL of HES (130/0.4). Furthermore, HES (130/0.4) has the same volume efficacy as HES (200/0.5) but is associated with lesser impairment to the coagulation and has no reported adverse effects on kidney function [8,9]. In a randomized, placebo-controlled phase 2 safety study , Rudolf and colleagues demonstrated that compared to saline, there was no significant trend towards a better functional outcome for acute ischaemic stroke patients treated with hypervolaemic haemodilution of 10% HES (130/0.4). However, the therapy was safe and well tolerated. In the present study, we demonstrated that ANH with 6% HES (130/0.4) reduced infarct volume and improved neurological outcome induced by focal cerebral ischaemia in a rat model. We speculate that for patients with mild to moderate cerebral ischaemia, ANH with 6% HES (130/0.4) may be an effective therapeutic approach. But more substantial information must be obtained before this procedure can be advocated for use in humans.
In the present study, haemodilution with saline augmented infarct volume instead of attenuating it. To maintain blood volume, animals in these groups received the threefold volume of saline to compensate for the blood loss. Focal cerebral ischaemia might damage the blood–brain barrier (BBB), which may worsen the original injury by exacerbating vasogenic oedema. For rats haemodiluted with saline, fluid may have transfused through the disrupted BBB thus aggravating brain oedema during reperfusion. Gow and colleagues  showed that saline administration impairs tissue oxygen extraction by increasing interstitial oedema or increasing heterogeneity of microvascular erythrocyte transit times. Wisselink and colleagues  normovolaemically haemodiluted New Zealand White rabbits subjected to spinal cord ischaemia-reperfusion injury with saline in volumes three times to that of the withdrawn blood. They demonstrated that ischaemic spinal cord injury worsened compared with ischaemic animals without normovolaemic haemodilution. These findings are consistent with our present results.
The proposed mechanism underlying neuroprotective effects of haemodilution lies in the acute increase of cerebral blood flow in surrounding regions of ischaemic core and prevention of the unfavourable viscosity as well as erythrocyte aggregation [6,7,24]. Hitomi and colleagues  showed that cerebral ischaemia induces a potent, systemic and long-lasting hyperviscosity of the blood. The decreased blood fluidity, due to either increase of Hct (polycythaemic hyperviscosity), fibrinogen concentration (plasmatic hyperviscosity) or erythrocyte rigidity (sclerocythaemic hyperviscosity) further aggravates brain injury . Consequently, haemodilution does not aim at normal cortex or at the ischaemia core, where the oxygen delivery is no longer an issue, but at the border of the ischaemic lesion (penumbra) surrounding the core where viscosity reduction could be vital for achieving an increase in blood flow. In the penumbra, haemodilution may increase blood flow through its effect on viscosity. Viscosity is increased disproportionately at low flow rates. As the shear rate falls, viscosity may increase substantially leading to further decrease in blood flow. This could initiate a vicious cycle preventing the border zone from recovering [6,7]. Since haemodilution reduces viscosity and erythrocyte aggravation, it can break this vicious cycle. As long as Hct is maintained at a suitable level, blood flow and oxygen delivery of brain will be sufficient for cerebral metabolism. In the present study, when the Hct was maintained between 25% and 30%, an encouraging neuroprotective effect could be achieved. We speculate that the reason for this improvement might be that the regional cerebral oxygen supply is improved. Chang and colleagues  showed that neuroprotective effects of haemodilution are associated with less pronounced increases of glutamate, glycerol, lactate and free radicals in the brain exposed to experimental heatstroke-induced cerebral ischaemia/hypoxia injury. Haemodilution also blocks the pathophysiological cascades of ischaemic stroke, which can alleviate the injury of excitotoxicity, peri-infarct depolarizations and inflammation on ischaemic cells. Kaplan and colleagues  found that HES reduced inflammatory reaction by causing a reduction in leukocyte adherence and migration. Tian and colleagues  reported that haemodilution with 6% HES (200/0.5) significantly reduces the increase of intercellular adhesion molecule-1, tumour necrosis factor-α and interleukin-1 expression induced by ischaemia-reperfusion at the early period. Other studies have suggested that HES may modify the basement membrane or have an anatomic sealant action at separated endothelial junctions, thereby reducing microvascular permeability and reperfusion injury. In summary, besides the direct physical effect of reducing blood viscosity, the mechanisms underlying the protective effect of HES on focal cerebral ischaemic injury are associated with microvascular permeability reduction, c-fos expression reduction in neuron , inflammatory reaction alleviation, anti-oxidation improvement, quick recovery from acidosis  and endothelial nitric oxide synthase increase.
In conclusion, the results of present study strongly support the protective efficacy of ANH with 6% HES 130/0.4 on transient focal cerebral ischaemia when the Hct is decreased to values between 25% and 30% of baseline. However, the detailed mechanism needs to be elucidated with more studies.
This work was supported in part by a research grant from Beijing Fresenius Kabi Pharmaceutical Co., Ltd to LX. The authors declare no potential conflict of interest also including the above-mentioned research grant to LX.
1. Tu YK, Heros RC, Karacostas D et al.
Isovolemic hemodilution in experimental focal cerebral ischemia. Part 2: effect on regional cerebral blood flow and size of infarction. J Neurosurg
2. Chang CK, Chien CH, Chou HL, Lin MT. The protective effect of hypervolemic hemodilution in experimental heat stroke. Shock
3. Cole DJ, Reynolds LW, Nary JC, Drummond JC, Patel PM, Jacobsen WK. Subarachnoid hemorrhage in rats: effect of singular or sustained hemodilution with alpha-alpha diaspirin crosslinked hemoglobin on cerebral hypoperfusion. Crit Care Med
4. Lin SZ, Chiou TL, Song WS, Chiang YH. Isovolemic hemodilution normalizes the prolonged passage of red cells and plasma through cerebral microvessels in the partially ischemic forebrain of rats. J Cereb Blood Flow Metab
5. Rebel A, Ulatowski JA, Joung K, Bucci E, Traystman RJ, Koehler RC. Regional cerebral blood flow in cats with cross-linked hemoglobin transfusion during focal cerebral ischemia. Am J Physiol Heart Circ Physiol
6. Schell RM, Cole DJ, Schultz RL, Osborne TN. Temporary cerebral ischemia effects of pentastarch or albumin on reperfusion injury. Anesthesiology
7. Perez-Trepichio AD, Furlan AJ, Little JR, Jones SC. Hydroxyethyl starch 200/0.5 reduces infarct volume after embolic stroke in rats. Stroke
8. Langeron O, Doelberg M, Ang ET, Bonnet F, Capdevila X, Coriat P. Voluven, a lower substituted novel hydroxyethyl starch (HES 130/0.4), causes fewer effects on coagulation in major orthopedic surgery than HES 200/0.5. Anesth Analg
9. Jungheinrich C, Neff TA. Pharmacokinetics of hydroxyethyl starch. Clin Pharmacokinet
10. Ickx BE, Bepperling F, Melot C, Schulman C, Van der Linden PJ. Plasma substitution effects of a new hydroxyethyl starch HES 130/0.4 compared with HES 200/0.5 during and after extended acute normovolemic haemodilution
. Br J Anaesth
11. Gallandat Huet RC, Siemons AW, Baus D et al.
A novel hydroxyethyl starch (Voluven®
) for effective perioperative plasma volume substitution in cardiac surgery. Can J Anesth
12. Xiong L, Zheng Y, Wu M et al.
Preconditioning with isoflurane produces dose-dependent neuroprotection
via activation of adenosine triphosphate-regulated potassium channels after focal cerebral ischemia in rats. Anesth Analg
13. Wang Q, Xiong L, Chen S, Liu Y, Zhu X. Rapid tolerance to focal cerebral ischemia in rats is induced by preconditioning with electroacupuncture: window of protection and the role of adenosine. Neurosci Lett
14. Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke
15. Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C, Sharp FR. A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab
16. Shichinohe H, Kuroda S, Abumiya T et al.
FK506 reduces infarct volume due to permanent focal cerebral ischemia by maintaining BAD turnover and inhibiting cytochrome c release. Brain Res
17. Belayev L, Khoutorova L, Belayev A et al.
Delayed post-ischemic albumin treatment neither improves nor worsens the outcome of transient focal cerebral ischemia in rats. Brain Res
18. Licker M, Mariethoz E, Costa MJ, Morel D. Cardioprotective effects of acute isovolemic hemodilution in a rat
model of transient coronary occlusion. Crit Care Med
19. Fenger-Eriksen C, Hartig Rasmussen C, Kappel Jensen T et al.
Renal effects of hypotensive anaesthesia in combination with acute normovolaemic haemodilution
with hydroxyethyl starch 130/0.4 or isotonic saline. Acta Anaesthesiol Scand
20. Homi HM, Yang H, Pearlstein RD, Grocott HP. Hemodilution during cardiopulmonary bypass increases cerebral infarct volume after middle cerebral artery occlusion in rats. Anesth Analg
21. Rudolf J. HES in Acute Stroke Study Group. Hydroxyethyl starch for hypervolemic hemodilution in patients with acute ischemic stroke: a randomized, placebo-controlled phase II safety study. Cerebrovasc Dis
22. Gow KW, Phang PT, Tebbutt-Speirs SM et al.
Effect of crystalloid administration on oxygen extraction in endotoxemic pigs. J Appl Physiol
23. Wisselink W, Nguyen JH, Becker MO, Money SR, Hollier LH. Ischemia-reperfusion injury of the spinal cord: the influence of normovolemic hemodilution and gradual reperfusion. Cardiovasc Surg
24. Jones SB, Whitten CW, Monk TG. Influence of crystalloid and colloid replacement solutions on hemodynamic variables during acute normovolemic hemodilution. J Clin Anesth
25. Hitomi A, Satoh S, Ikegaki I, Suzuki Y, Shibuya M, Asano T. Hemorheological abnormalities in experimental cerebral ischemia and effects of protein kinase inhibitor on blood fluidity. Life Sci
26. Forconi S, Turchetti V, Cappelli R, Guerrini M, Bicchi M. Haemorheological disturbances and possibility of their correction in cerebrovascular diseases. J Mal Vasc
27. Chang CK, Chiu WT, Chang CP, Lin MT. Effect of hypervolaemic haemodilution
on cerebral glutamate, glycerol, lactate and free radicals in heatstroke rats. Clin Sci
28. Kaplan SS, Park TS, Gonzales ER, Gidday JM. Hydroxyethyl starch reduces leukocyte adherence and vascular injury in the newborn pig cerebral circulation after asphyxia. Stroke
29. Wu Z, Tian YK, Zhang CH, Wang P. Effect of hemodilution with different plasma substitutes on expression of tumor necrosis factor-α and intertenkinL-1 in brain after global cerebral ischemia-reperfusion in rats. Chin J Anesthesiol
30. Miroslava N, Burda J, Danielisova V, Marala J. The effect of normovolemic hemodilution on c-Fos protein immunoreactivity in the postischemic rat
brain cortex. Int J Neurosci
31. Oda T, Nakajima Y, Kimura T, Ogata Y, Fujise Y. Hemodilution with liposome-encapsulated low-oxygen-affinity hemoglobin facilitates rapid recovery from ischemic acidosis after cerebral ischemia in rats. J Artif Organs