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Effects of viscosity and oxygen content on cerebral blood flow in ischemic and normal rat brain
JOURNAL OF THE
NEUROLOGICAL SCIENCES ELSEVIER
Journal of the Neurological Sciences 124 (1994) 15-20
Basic Section Effects of viscosity and oxygen content on cerebral blood flow in ischemic and normal rat brain D a n i e l J. C o l e a,., J o h n C. D r u m m o n d
b,c, P i y u s h M. P a t e l b,c, S u z z a n n e M a r c a n t o n i o
a
Departments of Anesthesiology, a Loma Linda Unicersity, Loma Linda, CA 92354, USA, b VA Medical Center, 3350 La Jolla Village Drire, San Diego, CA 92162, USA, r Unicersity of California at San Diego, La Joila, CA 92161, USA
(Received 8 October 1993; revised 10 December 1993; accepted 20 December 1993)
Abstract The mechanism of hemodilution-induced increases in cerebral blood flow (CBF) was investigated. Hemodilution was achieved with a molecular hemoglobin solution (DCLHb) and albumin which have similar viscosities but different oxygen carrying capacities. Part A: CBF was assessed in rats after one of the following regimens: (1) control-hematocrit not manipulated, (2) 30/Alb-hematocrit decreased to 30% with albumin, (3) 30/DCLHb-hematocrit decreased to 30% with DCLHb, or (4) 16/Alb/DCLHb-hematocrit decreased to 30% with albumin and then 16% with DCLHb. For viscosity matched groups (30/Alb and 30/DCLHb), CBF was greater in animals with decreased oxygen content (30/Alb); while in oxygen content matched groups (30/Alb and 16/Alb/DCLHb), CBF was greater in animals with decreased viscosity (16/AIb/DCLHb) (p < 0.05). Part B: Middle cerebral artery occlusion was performed, hemodilution achieved as in Part A, and CBF determined. For viscosity matched groups (30/Alb and 30/DCLHb), CBF was less in rats with decreased oxygen content (30/Alb); while in oxygen content matched groups (30/Alb and 16/Alb/DCLHb), CBF was greater in animals with decreased viscosity ( 1 6 / A l b / D C L H b ) (p < 0.05). This data supports the premise, that in normal brain, both viscosity and oxygen content effect CBF; while in ischemic brain, a decrease in viscosity but not oxygen content increases CBF. Key words: Albumin; Brain; Cerebral blood flow; Focal cerebral ischemia; Hemodilution; Hemoglobin
1. Introduction Hemodilution is known to increase cerebral blood flow (CBF) in both normal and ischemic brain (Cole et al. 1992b; Wood et al. 1983). Two mechanisms which may account for this CBF response include a decrease in viscosity, or a vasodilatory response to reduced oxygen content. Previous reports are inconsistent as to whether viscosity (Korosue and Heros 1992; Muizelaar et al. 1992; Paulson et al. 1973) or oxygen content (Back and yon Kummer 1991; H[iggendal et al. 1966) effect the CBF response to hemodilution. One reason for this controversy may be whether normal or ischemic brain is assessed. In normal brain CBF should be an inverse function of viscosity (Wood and Kee 1985); and with intact myogenic/neurogenic vascular
* Corresponding author. Tel.: (909) 824-4475, Fax: (909) 824-4143. 0022-510X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-510X(94)00014-F
responses, a vasodilatory response to decreases in oxygen content is teleologically plausible. Conversely, in ischemic brain with impaired myogenic/neurogenic vascular function, any inherent vascular response to oxygen content is likely to be attenuated or abolished. Moreover, during ischemia, when cerebral vessels are maximally dilated (Symon et al. 1976; Meyer et al. 1973; Waltz 1968), a reduction in oxygen content should not induce a vasodilatory response. In addition, in ischemic brain, with low flow and shear rates, a given decrease in viscosity causes a greater increase in CBF than in normal brain (Harrison 1989; Mirhashemi et al. 1987). Accordingly, in normal brain both viscosity and oxygen content should effect CBF; while during ischemia viscosity, but not oxygen content, should have a meaningful influence on CBF. Previously, in a model of middle cerebral artery occlusion (MCAo) in rats, we observed a dose-depend-
16
D,Z Coleet al. /Journal of the Neurological Sciences 124 (1994) 15-20
ent increase in CBF when hemodilution was employed with a molecular hemoglobin solution (Cole et al. 1992b). ,accordingly, in the present study, we assessed the extent to which viscosity and oxygen content effect the CBF response to hemodilution in normal and ischemic rat brain. Hemodilution was achieved with 10~ diaspirin cross-linked hemoglobin (DCLHb) and 10% albumin which have similar viscosities but different oxygen carrying capacities.
2. Materials and methods
The preparation and assay of DCLHb has been described (Cole et al. 1992b). In brief, outdated human red blood cells were processed to molecular hemoglobin which was cross-linked at the a chain by bis(3,5-dibromosalicyl)fumarate (final concentration 10.2 g. dl-I). The viscosity of DCLHb (1.3 centistokes) is similar to albumin (Usami et al. 1971) and less than whole blood (> 4.0 cS; DeVenuto et al. 1981). After approval by the Animal Research Committee of Loma Linda University, fasted male spontaneously hypertensive rats (SHR) of similar weights and ages (350-400 g, 16-20 weeks) were anesthetized with isoflurane (1.44%, end-tidal). Following orotracheal intubation the lungs were ventilated with a Harvard rodent respirator (Harvard, Boston, MA). The femoral vessels were cannulated for continuous blood pressure monitoring (Full Scale Transducer/TA 2000 Recorder, Gould, Cerritos, CA), blood sampling, and isotope and fluid administration. Maintenance fluids (0.9% NaCl) were infused at 4 ml. kg- ~. h-~. Other than protocol manipulation, physiologic variables were maintained within normal limits and validated immediately before CBF assessment (PaCO2, PaO2, pH, glucose, mean arterial blood pressure, and hematocrit). Cranial temperature was controlled at 37°C with a heating blanket.
PartA Each rat was randomized to one of the following groups (blood volume increased by 8.0 ml and the target hematocrit achieved over a 30-min period). Control (n --9). Blood volume was increased by giving 8.0 ml of fresh donor blood. 3 0 / A l b (n = 9). Blood volume and hematocrit were manipulated by a 5.0 ml exchange transfusion and an additional 8.0 ml of 10% albumin (Travenol Laboratories; Glendale, CA). 3 0 / D C L H b (n =9). Blood volume and hematocrit were manipulated by a 5.0 ml exchange transfusion and an additional 8.0 ml of 10% DCLHb (Baxter Healthcare Corporation; Deerfield, IL; lot 2905T008).
1 6 / A I b / D C L H b (n = 9). Blood volume and hematocrit (16%) were manipulated in two steps. First a 5.0 ml exchange transfusion and an additional 8.0 ml of 10% albumin was given for a hematocrit of 30%. In the second step, 12.0 ml of DCLHb was given as an exchange transfusion for a final hematoerit of 16%. After a 30-min equilibration period, 100/zCi. k g - i of 14C-iodoantipyrine (New England Nuclear, Boston, MA) was given over 46 s. Arterial blood was collected and ~4C activity determined with a quench correction (Beckman 8000 Liquid Scintillation Spectrometer; Beckman, Brea, CA). After ~4C infusion, the brains were removed in < 60 s and placed in 2-methylbutane (-35°C). The brains were sectioned in 20-/~m increments, and 10 sections surrounding each of five anatomically predetermined coronal planes placed on Kodak OM-1 film (Kodak, Rochester, NY) for 21 days. The five anatomical planes were in 2.0-mm increments that spanned middle cerebral artery distribution. Section 1 was at the anterior midline extent of the corpus callosum, and section 5 was 1.0 mm posterior to the posterior midline extent of the corpus callosum (Cole et al. 1992a). After film processing, CBF was assessed with a D r e x e l / D U M A S Image Analysis System (Drexel University, Philadelphia, PA) according to Sakurada et al. (1978). Global CBF was determined in each anatomic section and reported as an average for the five sections. All image analysis was performed by an independent observer who was blinded to study protocol. Part B In different SHR rats, via a subtemporal craniectomy, MCAo was achieved with 10-0 monofilament nylon suture ia two locations to achieve consistent ischemia to both cortical and sub-cortical tissue (Cole et al. 1990a,b). 30 rain after MCAo, each rat was randomly assigned to one of four hemodilution groups as in Part A ( n - 8 in each group). After 60 min of MCAo, CBF was assessed as in Part A with the following exception. In the center of middle cerebral artery distribution, CBF was determined only in an area of consistent ischemia (defined by previous studies; Cole et al. 1990a,b, 1992). This area corresponds to the lateral 40% of the left hemisphere (see Fig. 1). Part C Different SHR rats were prepared as in Part A (n = 6 for each group). Arterial blood was collected and analyzed for hematocrit, total hemoglobin, and oxygen content (IL-282 Co-oximeter; Instrumentation Laboratory, Lexington, MA) (Dennis and Valeri 1980); and viscosity was measured at shear rates of 45, 90, 225, and 450 s -1 by a Wells-Brookfield Micro Viscometer (Model LVT-C/P; Brookfield Engineering Laboratories, Stoughton, MA) (DeVenuto et al. 1981).
D.J. Cole et al. ~Journal of the Neurological Sciences 124 (1994) 15-20
17
creased oxygen content) versus the 3 0 / D C L H b group ( p < 0.05). For oxygen content matched groups ( 3 0 / A l b and 1 6 / A l b / D C L H b ) , CBF was greater in the 1 6 / A l b / DCLHb group (decreased viscosity) versus the 3 0 / A l b group ( p < 0.05) (see Fig. 2).
\
Fig. 1. Area of consistent ischemia as determined by previous studies in this model of MCAo (Cole et al. 1990a,b).
The data were compared by A N O V A with t-tests and a Bonferroni correction factor as indicated (Wilkinson 1987). P < 0.05 was considered significant.
3. R e s u l t s
All data are reported as mean +_ SD. Except for expected differences in hematocrit there were no between groups differences in the physiologic values for either Part A or B. The hematocrits for Part A were 44 + 1, 30 + 1, 29 + 1, and 16 + 1 for the control, 3 0 / Alb, 3 0 / D C L H b , and 1 6 / A I b / D C L H b groups, respectively; while for Part B, the hematocrits were 45 + 1, 30 + 1, 30 + 1, and 16 + 1, respectively.
Part A (normal brain) (see Table 1) For viscosity matched groups ( 3 0 / A l b and 3 0 / DCLHb), CBF was greater in the 3 0 / A l b group (decreased oxygen content) versus the 3 0 / D C L H b group ( p < 0.05). For oxygen content matched groups ( 3 0 / A l b and 1 6 / A I b / D C L H b ) , CBF was greater in the 1 6 / A l b / DCLHb group (decreased viscosity) versus the 3 0 / A l b group ( p < 0.05). Part B (ischemic brain) (see Table 1) For viscosity matched groups ( 3 0 / A l b and 3 0 / DCLHb), CBF was less in the 3 0 / A l b group (de-
Part C There were expected differences in hematocrit, total hemoglobin, and oxygen content, and a direct correlation between hematocrit and viscosity (see Table 2). The viscosity data at shear rates of 45, 90, 225, and 450 s-~ are reported in Table 2.
4. D i s c u s s i o n
The data presented here support the premise that, in normal brain, both viscosity and oxygen content effect the CBF response to hemodilution. In addition, the data suggest that during ischemia viscosity and not vasodilation secondary to decreased oxygen content is the mechanism of hemodilution-induced increases in CBF. These results are consistent with recent data which infer that viscosity is the predominant mechanism of CBF increases during hemodilution (Korosue and Heros 1992; Muizelaar et al. 1992). Two mechanisms which are postulated to regulate the CBF response to hemodilution are explained in terms of the Hagen-Poiseuille equation.
Q = ( ei - e o ) r 4 / 8 L r l where Q = flow, L = length of conduit, Pi = inflow pressure, 77 = viscosity, Po = outflow pressure, r = vessel radius. The relative contribution of each mechanism may depend on whether normal or ischemic brain is evaluated. The first mechanism by which hemodilution increases CBF is a direct vasodilatory response due to decreased oxyger content and delivery to the brain (Back and von Kummer 1991). According to the Hagen-Poiseuille equation, a small change in the vessel radius can profoundly effect CBF. For normal brain,
Table 1
CBF for Part A (normal brain) and Part B (ischemic brain). Values are mean + SD. See text for definition of group abbreviations Control 30/Alb 30/DCLHb 16/AIb/DCLH b Part A
CBF (ml. 100 g- i. min- !) Part B
CBF(ml.100g-t.min -l)
137.6+ 14.2 12.4+ 5.4 #
* p < 0.05 versus the control and 30/DCLHb groups. #p < 0.05 versus the other three groups.
165.2+ 16.4* 27.3+_ 7.9 #
142.3+ 16.8 55.5+- 8.0 #
199.7+ 22.5 * 77.7+- 7.3 #
18
D.J. Cole et al. /Journal of the Neurological Sciences 124 (1994) 15-20
Fig. 2. A representative autoradiograph (section 3) for each group in Part B. A: Control group, B: 30/AIb group, C: 30/DCLHb group, and D: 16/AIb/DCLHb group.
when myogenic and neurogenic vascular responses are active, it is plausible that this mechanism has a meaningful influence on CBF. In addition, the HagenPoiseuille equation predicts a second mechanism, viscosity, as effecting an increase in CBF during hemodilution (Paulson et al. 1973). The data from this study support both viscosity and vasodilation (secondary to decreased oxygen content) as operative mechanisms which effect CBF in normal brain. The present data are consistent with recent observations in which viscos-
ity (Muizelaar et ai. 1992) and oxygen content (Korosue and Heros 1992) were proposed to effect the hemodilution-induced increase in CBF in normal brain. Conversely, during ischemia, with maximal vasodilation and vasoparalysis (Symon et al. 1976; Meyer et al. 1973; Waltz 1968), it is difficult to conceive that reductions in oxygen content would induce further vasodilatation. In addition, in an ischemic vasculature with low flow and stasis, a decrease in systemic oxygen content would not be expected to effect an additional
Table 2 Physiologic data (mean + / ± SD) for Part C which was measured after stabilization at the target hematocrit. See text for definition of group abbreviations
Hematocrit (%) Total hemoglobin (g. dl- i) Oxygen content (ml. dl- ~) Viscosity (centistokes) Shear rate 45 s - i Shear rate 90 s-~ Shear rate 225 s- 1 Shear rate 450 s- I
Control
30/AIb
30/DCLHb
16/AIb/DCLHb
44 ± 1 14.3 ± 0.3 19.3 ± 0.4
30 ± 1 " 9.4±0.3 c,, 12.8±0.3 c,~
30 ± 1 " 12.8±0.3 * 17.5±0.3 *
16 ±1 # 9.5±0.3 c,, 12.7±0.3 r,,
4.6±0.1 3.9±0.1 3.3 ± 0.1 3.1 ± 0.1
2.7±0.1" 2.4±0.1" 2.2±0.1" 2.1±0.1"
2.6±0.1" 2.3±0.1" 2.1±0.1" 2.0±0.1"
* p < 0.05 versus the control group. # p < 0.05 versus the other three groups. p < 0.05 versus the control and 30/DCLHb groups.
1.5±0.1 1.5±0.1 1.4±0.1 1.4±0.1
# # # #
D.J. Cole et al. /Journal of the Neurological Sciences 124 (1994) i5-20
reduction in oxygen content beyond that which is already being extracted by the ischemic tissue. Recent data have demonstrated decreased vascular reactivity to hemodilution-induced reductions in oxygen content during impaired cerebral perfusion (Back and von Kummer 1991), and no CBF change in response to hypoxic hypoxia during MCAo in rabbits (Korosue and Heros 1992). Viscosity is the intrinsic resistance of a fluid to flow. Hematocrit, the major determinant of blood viscosity, has a pronounced effect during ischemia (Harrison 1989). During low flow states, with low shear rates, a given reduction in hematocrit effects a greater reduction in viscosity (Stone et al. 1968) and relative increase in CBF than in normal brain (Harrison 1989). Accordingly, it is plausible that during ischemia, viscosity and not oxygen content is the primary determinant of hemodilution-induced increases in CBF. This hypothesis is supported by the present data and that from other investigators (Muizelaar et al. 1986; Korosue and Heros 1992) which have observed viscosity as the predominant mechanism of hemodilution-induced increases in CBF during ischemia. A critique of this study is that DCLHb influences mechanisms of CBF regulation other than viscosity and oxygen content. One possibility is that DCLHb binds nitric oxide, an endothelium derived relaxing factor (Alayash et al. 1993), which would tend to counter a CBF increase due to reduced oxygen content (vasodilation). With the exception of the 3 0 / D C L H b group for Part A, CBF was significantly greater for the DCLHb groups than the Control or 3 0 / A l b groups. Thus, if cerebral nitric oxide were bound by DCLHb, the quantitative results that were statistically increased wt ~uld in fact be underestimations of the real differences if nitric oxide were not a factor. Accordingly, the qualitative conclusions of this study are likely not affected by this possibility. (For the 3 0 / D C L H b group in Part A, the potential binding of nitric oxide by DCLHb may have accounted for the absence of an expected change in CBF between the Control and 3 0 / D C L H b group when viscosity was reduced.) A second critique would question the validity of correlating blood viscosity (determined by a viscometer) to changes in CBF (Gaehtgens and Marx 1987). In a cone plate viscometer there is uniform distribution, throughout the fluid being evaluated, of shear rate. However, in the microcirculation there is a variable distribution of shear rates across the vessel lumen. Moreover, other factors (e.g., red cell aggregation and deformability, plasma viscosity, and protein composition) may have a meaningful influence on viscosity in the cerebral microcirculation. In addition, we acknowledge an uncertain relationship between in vitro measurements of systemic viscosity and cerebral microcirculatory viscosity. However, a quantitative relationship
19
between systemic and cerebral hematocrit has been described with confirmation that hemodilution decreases cerebral hematocrit (Todd et al. 1992). Thus, while the absolute quantitative relationship between the viscometric data and changes in microcirculatory blood flow is controversial, the qualitative correlations and conclusions of the data should be valid. In summary, the effect of viscosity and oxygen content on CBF was determined in normal and ischemic rats. The data supports the premise, that in normal brain, both viscosity and oxygen content effect CBF; while in ischemic brain, a decrease in viscosity but not oxygen content increases CBF.
Acknowledgement The authors gratefully acknowledge the technical assistance of Terrill Osborne.
References Alayash, A.I., Fratantoni, J.C., Bonaventura, C., Bonaventura, J. anu Cashon, R.E. (1993) Nitric oxide binding to human ferrihemoglobins cross-linked either a or/3 subunits. Arch. Biochem. Biophys., 303: 332-338. Back, T. and yon Kummer, R. (1991) Oxygen reactivity of cerebral circulation determined in cats. J. Cereb. Blood Flow. Metab., 11: $458. Cole, D.J., Drummond, LC., Osborne, T.N. and Matsumura, J. (1990a) Hypertension and hemodilution during cerebral ischemia reduce brain injury and edema. Am. J. Physiol., 259:H211-217. Cole, D.J., Drummond, J.C., Ruta, T.S. and Peckham, N.H. (1990b) Hemodilution and hypertension effects on cerebral hemorrhage in cerebral ischemia in rats. Stroke, 21: 1333-1339. Cole, D.J., Matsumura, LS., Drummond, J.C. and Schell, R.M. (1992a) Focal cerebral ischemia in rats: Effects of induced hypertension, during r~perfusion, on CBF.J. Cereb. Blood Flow. Metab., 12: 64-69. Cole, DJ., Schell, R.M., Przybelski, R.J., Drummond, J.C. and Bradley, K. (1992b) Focal cerebral ischemia in rats: effect of hemodilution with a-a cross-linked hemoglobin on CBF.J. Cereb. Blood Flow Metab., 12: 971-976. Dennis, R.C. and Valeri, C.R. (1980) Measuring percent oxygen saturation of hemoglobin, percent carboxyhemoglobin and methemoglobin, and concentrations of total hemoglobin and oxygen in blood of man, dog, and baboon. Clin. Chem., 26: 13041308. DeVenuto, F., Busse, K.R. and Zegna, A.I. (1981) Viscosity of human blood hemodiluted with crystalline hemoglobin solution. Transfusion, 21: 752-756. Gaehtgens, P. and Marx, P. (1987) Hemorrheological aspects of the pathophysiology of cerebral ischemia. J. Cereb. Blood Flow Metab., 7: 259-265. H/iggendal, E., Nilsson, N.J. and Norbiick, B. (1966) Effect of blood corpuscle concentration on cerebral blood flow. Acta Chir. Scand., Suppl. 364: 3-12. Harrison, M.J.G. (1989) Influence of haematocrit in the cerebral circulation. Cerebrovasc. Brain Metab. Rev., !: 55-67. Korosue, K. and Heros, R.C. (1992) Mechanism of cerebral blood flow augmentation by hemodilution in rabbits. Stroke, 23: 1497.
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D.J. Cole et ai. /Journal of the Neurological Sciences 124 (1994) 15-20
Me,x~er, J.S., Shimazu, R., Fukuuchi, Y., Ouchi, T., Okamoto, S., Koto, A. and Ericsson, A.D. (1973) Impaired neurologic cerebrovascular control and dysautoregulation after stroke. Stroke, 4: 169-186. Mirhashemi, S., Ertefai, S., Messmer, K. and Intaglietta, M. (1987) Model analysis of the enhancement of tissue oxygenation by hemodilution due to increased microvascular flow velocity. Microvasc. Res., 34: 290-301. Muizelaar, J.P., Wei, E.P., Kontos, H.A. and Becket, D.P. (1986) Cerebral blood flow is regulated by changes in blood pressure and in blood viscosity alike. Stroke, 17: 44-48. Muizelaar, J.P., Bouma, G.J., Levasseur, J.E. and Kontos, H.A. (1992) Effect of hematocrit variations on cerebral blood flow and basilar artery diameter in vivo. Am. J. Physiol., 262: H949-H954. Paulson, O.B., Parring, H.-H., Olesen, J. and Skinhej, E. ~1973) Influence of carbon monoxide and of hemodilution on cerebral blood flow and blood gases in man. J. Appl. Physiol., 35:111-116. Sakurada, O., Kennedy, C., Jehle, J., Brown, J.D., Carbin, G.L. and Sokoloff, L. (1978) Measurement of local cerebral blood flow with iodo-C-14-antipyrine. Am. J. Physiol., 234: H59-H66. Stone, H.O., Thompson, H.K. and Schmid-Nielson, K. (1968) Influence of erythroc~ytes on blood viscosity. Am. J. Physiol., 214: 913-918.
Symon, L., Branston, N.M. and Strong, A.J. (1976)Autoregulation in acute focal ischemia. An experimental study. Stroke, 7: 547-554. Todd, M.M., Weeks, J.B. and Warner, D.S. (1992) Cerebral bl'-,~d flow, blood volume, and brain tissue hematocrit during isovole ~,ic hemodilution with hetastarch in rats. Am. J. Physiol., 263:I-17582. Usami, S., Chien, S. and Gregersen, M.I. (1971) Hemoglobin solution as a plasma expander: Effects on blood viscosity. Proc. Soc. Exp. Biol. Med., 136: 1232-1235. Waltz, A.G. (1968) Effect of blood pressure on blood flow in ischemic and in nonischemic cerebral cortex. The phenomena of autoregulation and luxury perfusion. Neurology, 18: 613-621. Wilkinson, L. (1987) Multivariate General Linear Hypothesis. In: SYSTAT: The System for Statistics, SYSTAT Inc., Evanston, IL, pp. 22-3O. Wood, J.H., Simeone, F.A., Fink, E.A. and Golden, M.A. (1983) Hypervolemic hemodilution in experimental focal cerebral ischemia. Elevations o~ cardiac output, regional cortical blood flow, and ICP after intras ascula~ ,,olume expansion with low-molecular weight dextran. J. l~Zeurosurg.,59: 500-509, Wood, J.H. and Kee, D.B. (1985) Hemorheology of the cerebral circulation in stroke. Stroke, 16: 765-772.
NEUROLOGICAL SCIENCES ELSEVIER
Journal of the Neurological Sciences 124 (1994) 15-20
Basic Section Effects of viscosity and oxygen content on cerebral blood flow in ischemic and normal rat brain D a n i e l J. C o l e a,., J o h n C. D r u m m o n d
b,c, P i y u s h M. P a t e l b,c, S u z z a n n e M a r c a n t o n i o
a
Departments of Anesthesiology, a Loma Linda Unicersity, Loma Linda, CA 92354, USA, b VA Medical Center, 3350 La Jolla Village Drire, San Diego, CA 92162, USA, r Unicersity of California at San Diego, La Joila, CA 92161, USA
(Received 8 October 1993; revised 10 December 1993; accepted 20 December 1993)
Abstract The mechanism of hemodilution-induced increases in cerebral blood flow (CBF) was investigated. Hemodilution was achieved with a molecular hemoglobin solution (DCLHb) and albumin which have similar viscosities but different oxygen carrying capacities. Part A: CBF was assessed in rats after one of the following regimens: (1) control-hematocrit not manipulated, (2) 30/Alb-hematocrit decreased to 30% with albumin, (3) 30/DCLHb-hematocrit decreased to 30% with DCLHb, or (4) 16/Alb/DCLHb-hematocrit decreased to 30% with albumin and then 16% with DCLHb. For viscosity matched groups (30/Alb and 30/DCLHb), CBF was greater in animals with decreased oxygen content (30/Alb); while in oxygen content matched groups (30/Alb and 16/Alb/DCLHb), CBF was greater in animals with decreased viscosity (16/AIb/DCLHb) (p < 0.05). Part B: Middle cerebral artery occlusion was performed, hemodilution achieved as in Part A, and CBF determined. For viscosity matched groups (30/Alb and 30/DCLHb), CBF was less in rats with decreased oxygen content (30/Alb); while in oxygen content matched groups (30/Alb and 16/Alb/DCLHb), CBF was greater in animals with decreased viscosity ( 1 6 / A l b / D C L H b ) (p < 0.05). This data supports the premise, that in normal brain, both viscosity and oxygen content effect CBF; while in ischemic brain, a decrease in viscosity but not oxygen content increases CBF. Key words: Albumin; Brain; Cerebral blood flow; Focal cerebral ischemia; Hemodilution; Hemoglobin
1. Introduction Hemodilution is known to increase cerebral blood flow (CBF) in both normal and ischemic brain (Cole et al. 1992b; Wood et al. 1983). Two mechanisms which may account for this CBF response include a decrease in viscosity, or a vasodilatory response to reduced oxygen content. Previous reports are inconsistent as to whether viscosity (Korosue and Heros 1992; Muizelaar et al. 1992; Paulson et al. 1973) or oxygen content (Back and yon Kummer 1991; H[iggendal et al. 1966) effect the CBF response to hemodilution. One reason for this controversy may be whether normal or ischemic brain is assessed. In normal brain CBF should be an inverse function of viscosity (Wood and Kee 1985); and with intact myogenic/neurogenic vascular
* Corresponding author. Tel.: (909) 824-4475, Fax: (909) 824-4143. 0022-510X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-510X(94)00014-F
responses, a vasodilatory response to decreases in oxygen content is teleologically plausible. Conversely, in ischemic brain with impaired myogenic/neurogenic vascular function, any inherent vascular response to oxygen content is likely to be attenuated or abolished. Moreover, during ischemia, when cerebral vessels are maximally dilated (Symon et al. 1976; Meyer et al. 1973; Waltz 1968), a reduction in oxygen content should not induce a vasodilatory response. In addition, in ischemic brain, with low flow and shear rates, a given decrease in viscosity causes a greater increase in CBF than in normal brain (Harrison 1989; Mirhashemi et al. 1987). Accordingly, in normal brain both viscosity and oxygen content should effect CBF; while during ischemia viscosity, but not oxygen content, should have a meaningful influence on CBF. Previously, in a model of middle cerebral artery occlusion (MCAo) in rats, we observed a dose-depend-
16
D,Z Coleet al. /Journal of the Neurological Sciences 124 (1994) 15-20
ent increase in CBF when hemodilution was employed with a molecular hemoglobin solution (Cole et al. 1992b). ,accordingly, in the present study, we assessed the extent to which viscosity and oxygen content effect the CBF response to hemodilution in normal and ischemic rat brain. Hemodilution was achieved with 10~ diaspirin cross-linked hemoglobin (DCLHb) and 10% albumin which have similar viscosities but different oxygen carrying capacities.
2. Materials and methods
The preparation and assay of DCLHb has been described (Cole et al. 1992b). In brief, outdated human red blood cells were processed to molecular hemoglobin which was cross-linked at the a chain by bis(3,5-dibromosalicyl)fumarate (final concentration 10.2 g. dl-I). The viscosity of DCLHb (1.3 centistokes) is similar to albumin (Usami et al. 1971) and less than whole blood (> 4.0 cS; DeVenuto et al. 1981). After approval by the Animal Research Committee of Loma Linda University, fasted male spontaneously hypertensive rats (SHR) of similar weights and ages (350-400 g, 16-20 weeks) were anesthetized with isoflurane (1.44%, end-tidal). Following orotracheal intubation the lungs were ventilated with a Harvard rodent respirator (Harvard, Boston, MA). The femoral vessels were cannulated for continuous blood pressure monitoring (Full Scale Transducer/TA 2000 Recorder, Gould, Cerritos, CA), blood sampling, and isotope and fluid administration. Maintenance fluids (0.9% NaCl) were infused at 4 ml. kg- ~. h-~. Other than protocol manipulation, physiologic variables were maintained within normal limits and validated immediately before CBF assessment (PaCO2, PaO2, pH, glucose, mean arterial blood pressure, and hematocrit). Cranial temperature was controlled at 37°C with a heating blanket.
PartA Each rat was randomized to one of the following groups (blood volume increased by 8.0 ml and the target hematocrit achieved over a 30-min period). Control (n --9). Blood volume was increased by giving 8.0 ml of fresh donor blood. 3 0 / A l b (n = 9). Blood volume and hematocrit were manipulated by a 5.0 ml exchange transfusion and an additional 8.0 ml of 10% albumin (Travenol Laboratories; Glendale, CA). 3 0 / D C L H b (n =9). Blood volume and hematocrit were manipulated by a 5.0 ml exchange transfusion and an additional 8.0 ml of 10% DCLHb (Baxter Healthcare Corporation; Deerfield, IL; lot 2905T008).
1 6 / A I b / D C L H b (n = 9). Blood volume and hematocrit (16%) were manipulated in two steps. First a 5.0 ml exchange transfusion and an additional 8.0 ml of 10% albumin was given for a hematocrit of 30%. In the second step, 12.0 ml of DCLHb was given as an exchange transfusion for a final hematoerit of 16%. After a 30-min equilibration period, 100/zCi. k g - i of 14C-iodoantipyrine (New England Nuclear, Boston, MA) was given over 46 s. Arterial blood was collected and ~4C activity determined with a quench correction (Beckman 8000 Liquid Scintillation Spectrometer; Beckman, Brea, CA). After ~4C infusion, the brains were removed in < 60 s and placed in 2-methylbutane (-35°C). The brains were sectioned in 20-/~m increments, and 10 sections surrounding each of five anatomically predetermined coronal planes placed on Kodak OM-1 film (Kodak, Rochester, NY) for 21 days. The five anatomical planes were in 2.0-mm increments that spanned middle cerebral artery distribution. Section 1 was at the anterior midline extent of the corpus callosum, and section 5 was 1.0 mm posterior to the posterior midline extent of the corpus callosum (Cole et al. 1992a). After film processing, CBF was assessed with a D r e x e l / D U M A S Image Analysis System (Drexel University, Philadelphia, PA) according to Sakurada et al. (1978). Global CBF was determined in each anatomic section and reported as an average for the five sections. All image analysis was performed by an independent observer who was blinded to study protocol. Part B In different SHR rats, via a subtemporal craniectomy, MCAo was achieved with 10-0 monofilament nylon suture ia two locations to achieve consistent ischemia to both cortical and sub-cortical tissue (Cole et al. 1990a,b). 30 rain after MCAo, each rat was randomly assigned to one of four hemodilution groups as in Part A ( n - 8 in each group). After 60 min of MCAo, CBF was assessed as in Part A with the following exception. In the center of middle cerebral artery distribution, CBF was determined only in an area of consistent ischemia (defined by previous studies; Cole et al. 1990a,b, 1992). This area corresponds to the lateral 40% of the left hemisphere (see Fig. 1). Part C Different SHR rats were prepared as in Part A (n = 6 for each group). Arterial blood was collected and analyzed for hematocrit, total hemoglobin, and oxygen content (IL-282 Co-oximeter; Instrumentation Laboratory, Lexington, MA) (Dennis and Valeri 1980); and viscosity was measured at shear rates of 45, 90, 225, and 450 s -1 by a Wells-Brookfield Micro Viscometer (Model LVT-C/P; Brookfield Engineering Laboratories, Stoughton, MA) (DeVenuto et al. 1981).
D.J. Cole et al. ~Journal of the Neurological Sciences 124 (1994) 15-20
17
creased oxygen content) versus the 3 0 / D C L H b group ( p < 0.05). For oxygen content matched groups ( 3 0 / A l b and 1 6 / A l b / D C L H b ) , CBF was greater in the 1 6 / A l b / DCLHb group (decreased viscosity) versus the 3 0 / A l b group ( p < 0.05) (see Fig. 2).
\
Fig. 1. Area of consistent ischemia as determined by previous studies in this model of MCAo (Cole et al. 1990a,b).
The data were compared by A N O V A with t-tests and a Bonferroni correction factor as indicated (Wilkinson 1987). P < 0.05 was considered significant.
3. R e s u l t s
All data are reported as mean +_ SD. Except for expected differences in hematocrit there were no between groups differences in the physiologic values for either Part A or B. The hematocrits for Part A were 44 + 1, 30 + 1, 29 + 1, and 16 + 1 for the control, 3 0 / Alb, 3 0 / D C L H b , and 1 6 / A I b / D C L H b groups, respectively; while for Part B, the hematocrits were 45 + 1, 30 + 1, 30 + 1, and 16 + 1, respectively.
Part A (normal brain) (see Table 1) For viscosity matched groups ( 3 0 / A l b and 3 0 / DCLHb), CBF was greater in the 3 0 / A l b group (decreased oxygen content) versus the 3 0 / D C L H b group ( p < 0.05). For oxygen content matched groups ( 3 0 / A l b and 1 6 / A I b / D C L H b ) , CBF was greater in the 1 6 / A l b / DCLHb group (decreased viscosity) versus the 3 0 / A l b group ( p < 0.05). Part B (ischemic brain) (see Table 1) For viscosity matched groups ( 3 0 / A l b and 3 0 / DCLHb), CBF was less in the 3 0 / A l b group (de-
Part C There were expected differences in hematocrit, total hemoglobin, and oxygen content, and a direct correlation between hematocrit and viscosity (see Table 2). The viscosity data at shear rates of 45, 90, 225, and 450 s-~ are reported in Table 2.
4. D i s c u s s i o n
The data presented here support the premise that, in normal brain, both viscosity and oxygen content effect the CBF response to hemodilution. In addition, the data suggest that during ischemia viscosity and not vasodilation secondary to decreased oxygen content is the mechanism of hemodilution-induced increases in CBF. These results are consistent with recent data which infer that viscosity is the predominant mechanism of CBF increases during hemodilution (Korosue and Heros 1992; Muizelaar et al. 1992). Two mechanisms which are postulated to regulate the CBF response to hemodilution are explained in terms of the Hagen-Poiseuille equation.
Q = ( ei - e o ) r 4 / 8 L r l where Q = flow, L = length of conduit, Pi = inflow pressure, 77 = viscosity, Po = outflow pressure, r = vessel radius. The relative contribution of each mechanism may depend on whether normal or ischemic brain is evaluated. The first mechanism by which hemodilution increases CBF is a direct vasodilatory response due to decreased oxyger content and delivery to the brain (Back and von Kummer 1991). According to the Hagen-Poiseuille equation, a small change in the vessel radius can profoundly effect CBF. For normal brain,
Table 1
CBF for Part A (normal brain) and Part B (ischemic brain). Values are mean + SD. See text for definition of group abbreviations Control 30/Alb 30/DCLHb 16/AIb/DCLH b Part A
CBF (ml. 100 g- i. min- !) Part B
CBF(ml.100g-t.min -l)
137.6+ 14.2 12.4+ 5.4 #
* p < 0.05 versus the control and 30/DCLHb groups. #p < 0.05 versus the other three groups.
165.2+ 16.4* 27.3+_ 7.9 #
142.3+ 16.8 55.5+- 8.0 #
199.7+ 22.5 * 77.7+- 7.3 #
18
D.J. Cole et al. /Journal of the Neurological Sciences 124 (1994) 15-20
Fig. 2. A representative autoradiograph (section 3) for each group in Part B. A: Control group, B: 30/AIb group, C: 30/DCLHb group, and D: 16/AIb/DCLHb group.
when myogenic and neurogenic vascular responses are active, it is plausible that this mechanism has a meaningful influence on CBF. In addition, the HagenPoiseuille equation predicts a second mechanism, viscosity, as effecting an increase in CBF during hemodilution (Paulson et al. 1973). The data from this study support both viscosity and vasodilation (secondary to decreased oxygen content) as operative mechanisms which effect CBF in normal brain. The present data are consistent with recent observations in which viscos-
ity (Muizelaar et ai. 1992) and oxygen content (Korosue and Heros 1992) were proposed to effect the hemodilution-induced increase in CBF in normal brain. Conversely, during ischemia, with maximal vasodilation and vasoparalysis (Symon et al. 1976; Meyer et al. 1973; Waltz 1968), it is difficult to conceive that reductions in oxygen content would induce further vasodilatation. In addition, in an ischemic vasculature with low flow and stasis, a decrease in systemic oxygen content would not be expected to effect an additional
Table 2 Physiologic data (mean + / ± SD) for Part C which was measured after stabilization at the target hematocrit. See text for definition of group abbreviations
Hematocrit (%) Total hemoglobin (g. dl- i) Oxygen content (ml. dl- ~) Viscosity (centistokes) Shear rate 45 s - i Shear rate 90 s-~ Shear rate 225 s- 1 Shear rate 450 s- I
Control
30/AIb
30/DCLHb
16/AIb/DCLHb
44 ± 1 14.3 ± 0.3 19.3 ± 0.4
30 ± 1 " 9.4±0.3 c,, 12.8±0.3 c,~
30 ± 1 " 12.8±0.3 * 17.5±0.3 *
16 ±1 # 9.5±0.3 c,, 12.7±0.3 r,,
4.6±0.1 3.9±0.1 3.3 ± 0.1 3.1 ± 0.1
2.7±0.1" 2.4±0.1" 2.2±0.1" 2.1±0.1"
2.6±0.1" 2.3±0.1" 2.1±0.1" 2.0±0.1"
* p < 0.05 versus the control group. # p < 0.05 versus the other three groups. p < 0.05 versus the control and 30/DCLHb groups.
1.5±0.1 1.5±0.1 1.4±0.1 1.4±0.1
# # # #
D.J. Cole et al. /Journal of the Neurological Sciences 124 (1994) i5-20
reduction in oxygen content beyond that which is already being extracted by the ischemic tissue. Recent data have demonstrated decreased vascular reactivity to hemodilution-induced reductions in oxygen content during impaired cerebral perfusion (Back and von Kummer 1991), and no CBF change in response to hypoxic hypoxia during MCAo in rabbits (Korosue and Heros 1992). Viscosity is the intrinsic resistance of a fluid to flow. Hematocrit, the major determinant of blood viscosity, has a pronounced effect during ischemia (Harrison 1989). During low flow states, with low shear rates, a given reduction in hematocrit effects a greater reduction in viscosity (Stone et al. 1968) and relative increase in CBF than in normal brain (Harrison 1989). Accordingly, it is plausible that during ischemia, viscosity and not oxygen content is the primary determinant of hemodilution-induced increases in CBF. This hypothesis is supported by the present data and that from other investigators (Muizelaar et al. 1986; Korosue and Heros 1992) which have observed viscosity as the predominant mechanism of hemodilution-induced increases in CBF during ischemia. A critique of this study is that DCLHb influences mechanisms of CBF regulation other than viscosity and oxygen content. One possibility is that DCLHb binds nitric oxide, an endothelium derived relaxing factor (Alayash et al. 1993), which would tend to counter a CBF increase due to reduced oxygen content (vasodilation). With the exception of the 3 0 / D C L H b group for Part A, CBF was significantly greater for the DCLHb groups than the Control or 3 0 / A l b groups. Thus, if cerebral nitric oxide were bound by DCLHb, the quantitative results that were statistically increased wt ~uld in fact be underestimations of the real differences if nitric oxide were not a factor. Accordingly, the qualitative conclusions of this study are likely not affected by this possibility. (For the 3 0 / D C L H b group in Part A, the potential binding of nitric oxide by DCLHb may have accounted for the absence of an expected change in CBF between the Control and 3 0 / D C L H b group when viscosity was reduced.) A second critique would question the validity of correlating blood viscosity (determined by a viscometer) to changes in CBF (Gaehtgens and Marx 1987). In a cone plate viscometer there is uniform distribution, throughout the fluid being evaluated, of shear rate. However, in the microcirculation there is a variable distribution of shear rates across the vessel lumen. Moreover, other factors (e.g., red cell aggregation and deformability, plasma viscosity, and protein composition) may have a meaningful influence on viscosity in the cerebral microcirculation. In addition, we acknowledge an uncertain relationship between in vitro measurements of systemic viscosity and cerebral microcirculatory viscosity. However, a quantitative relationship
19
between systemic and cerebral hematocrit has been described with confirmation that hemodilution decreases cerebral hematocrit (Todd et al. 1992). Thus, while the absolute quantitative relationship between the viscometric data and changes in microcirculatory blood flow is controversial, the qualitative correlations and conclusions of the data should be valid. In summary, the effect of viscosity and oxygen content on CBF was determined in normal and ischemic rats. The data supports the premise, that in normal brain, both viscosity and oxygen content effect CBF; while in ischemic brain, a decrease in viscosity but not oxygen content increases CBF.
Acknowledgement The authors gratefully acknowledge the technical assistance of Terrill Osborne.
References Alayash, A.I., Fratantoni, J.C., Bonaventura, C., Bonaventura, J. anu Cashon, R.E. (1993) Nitric oxide binding to human ferrihemoglobins cross-linked either a or/3 subunits. Arch. Biochem. Biophys., 303: 332-338. Back, T. and yon Kummer, R. (1991) Oxygen reactivity of cerebral circulation determined in cats. J. Cereb. Blood Flow. Metab., 11: $458. Cole, D.J., Drummond, LC., Osborne, T.N. and Matsumura, J. (1990a) Hypertension and hemodilution during cerebral ischemia reduce brain injury and edema. Am. J. Physiol., 259:H211-217. Cole, D.J., Drummond, J.C., Ruta, T.S. and Peckham, N.H. (1990b) Hemodilution and hypertension effects on cerebral hemorrhage in cerebral ischemia in rats. Stroke, 21: 1333-1339. Cole, D.J., Matsumura, LS., Drummond, J.C. and Schell, R.M. (1992a) Focal cerebral ischemia in rats: Effects of induced hypertension, during r~perfusion, on CBF.J. Cereb. Blood Flow. Metab., 12: 64-69. Cole, DJ., Schell, R.M., Przybelski, R.J., Drummond, J.C. and Bradley, K. (1992b) Focal cerebral ischemia in rats: effect of hemodilution with a-a cross-linked hemoglobin on CBF.J. Cereb. Blood Flow Metab., 12: 971-976. Dennis, R.C. and Valeri, C.R. (1980) Measuring percent oxygen saturation of hemoglobin, percent carboxyhemoglobin and methemoglobin, and concentrations of total hemoglobin and oxygen in blood of man, dog, and baboon. Clin. Chem., 26: 13041308. DeVenuto, F., Busse, K.R. and Zegna, A.I. (1981) Viscosity of human blood hemodiluted with crystalline hemoglobin solution. Transfusion, 21: 752-756. Gaehtgens, P. and Marx, P. (1987) Hemorrheological aspects of the pathophysiology of cerebral ischemia. J. Cereb. Blood Flow Metab., 7: 259-265. H/iggendal, E., Nilsson, N.J. and Norbiick, B. (1966) Effect of blood corpuscle concentration on cerebral blood flow. Acta Chir. Scand., Suppl. 364: 3-12. Harrison, M.J.G. (1989) Influence of haematocrit in the cerebral circulation. Cerebrovasc. Brain Metab. Rev., !: 55-67. Korosue, K. and Heros, R.C. (1992) Mechanism of cerebral blood flow augmentation by hemodilution in rabbits. Stroke, 23: 1497.
20
D.J. Cole et ai. /Journal of the Neurological Sciences 124 (1994) 15-20
Me,x~er, J.S., Shimazu, R., Fukuuchi, Y., Ouchi, T., Okamoto, S., Koto, A. and Ericsson, A.D. (1973) Impaired neurologic cerebrovascular control and dysautoregulation after stroke. Stroke, 4: 169-186. Mirhashemi, S., Ertefai, S., Messmer, K. and Intaglietta, M. (1987) Model analysis of the enhancement of tissue oxygenation by hemodilution due to increased microvascular flow velocity. Microvasc. Res., 34: 290-301. Muizelaar, J.P., Wei, E.P., Kontos, H.A. and Becket, D.P. (1986) Cerebral blood flow is regulated by changes in blood pressure and in blood viscosity alike. Stroke, 17: 44-48. Muizelaar, J.P., Bouma, G.J., Levasseur, J.E. and Kontos, H.A. (1992) Effect of hematocrit variations on cerebral blood flow and basilar artery diameter in vivo. Am. J. Physiol., 262: H949-H954. Paulson, O.B., Parring, H.-H., Olesen, J. and Skinhej, E. ~1973) Influence of carbon monoxide and of hemodilution on cerebral blood flow and blood gases in man. J. Appl. Physiol., 35:111-116. Sakurada, O., Kennedy, C., Jehle, J., Brown, J.D., Carbin, G.L. and Sokoloff, L. (1978) Measurement of local cerebral blood flow with iodo-C-14-antipyrine. Am. J. Physiol., 234: H59-H66. Stone, H.O., Thompson, H.K. and Schmid-Nielson, K. (1968) Influence of erythroc~ytes on blood viscosity. Am. J. Physiol., 214: 913-918.
Symon, L., Branston, N.M. and Strong, A.J. (1976)Autoregulation in acute focal ischemia. An experimental study. Stroke, 7: 547-554. Todd, M.M., Weeks, J.B. and Warner, D.S. (1992) Cerebral bl'-,~d flow, blood volume, and brain tissue hematocrit during isovole ~,ic hemodilution with hetastarch in rats. Am. J. Physiol., 263:I-17582. Usami, S., Chien, S. and Gregersen, M.I. (1971) Hemoglobin solution as a plasma expander: Effects on blood viscosity. Proc. Soc. Exp. Biol. Med., 136: 1232-1235. Waltz, A.G. (1968) Effect of blood pressure on blood flow in ischemic and in nonischemic cerebral cortex. The phenomena of autoregulation and luxury perfusion. Neurology, 18: 613-621. Wilkinson, L. (1987) Multivariate General Linear Hypothesis. In: SYSTAT: The System for Statistics, SYSTAT Inc., Evanston, IL, pp. 22-3O. Wood, J.H., Simeone, F.A., Fink, E.A. and Golden, M.A. (1983) Hypervolemic hemodilution in experimental focal cerebral ischemia. Elevations o~ cardiac output, regional cortical blood flow, and ICP after intras ascula~ ,,olume expansion with low-molecular weight dextran. J. l~Zeurosurg.,59: 500-509, Wood, J.H. and Kee, D.B. (1985) Hemorheology of the cerebral circulation in stroke. Stroke, 16: 765-772.