Exp. Eye Res. (1992) 55. 59-63

The Effects

of lsovolumic

Hemodilution STEVEN

Department

of Anesthesia

(Received

Chicago

Blood

Flow

ROTH”

and Critical Care, University 12 July

on Ocular

1997 and accepted

of Chicago, in revised form

Chicago,

IL 60637,

16 September

U.S.A

1997)

Techniques by which retinal blood flow may be increased safely are potentially important in the treatment of retinal vascular disease. It was hypothesized that hemodilution, which increases cerebral blood flow, would also increase retinal blood flow. To investigate the physiological effects of hemodilution in the eye, ocular blood flow was measured in 14 cats using the radioactively labeled microsphere method. After the animals were anesthetized with halothane and oxygen, intraocular and systemic arterial pressure were recorded : blood flows were measured before and after isovolumic hemodilution to a hematocrit of 20-22 y0 using 6% hydroxyethyl starch (a synthetic plasma expander with a molecular weight of 450 in 0.9 y0 saline). In hemodiluted cats, retinal blood flow increased 71 y0 from its baseline value (36.7 If: 6.4 ml 100 g-l min-l to 62.9 + 6.4 ml 100 g-l min-‘, mean f S.E.M., P < 00001). Calculated retinal 0, delivery remained approximately constant, as the increased blood flow countered a significant decrease in arterial 0, content. Choroidal blood flow decreased (129 7 + 140 ml 100 g-l min-’ to 1051 + 144 ml 100 g-’ min-‘) but the change was not statistically significant. Blood flows in the iris and sclera were not significantly altered. Hemodilution increased retinal blood flow without causing a redistribution in ocular blood flow. Key words: blood flow, choroid, iris, retina, sclera, measurement techniques ; microspheres ; hemodilution. 1. Introduction

Although rheological factors play a significant role in regional blood flow in many areas of the microcirculation

(Fan et al., 1980),

available regarding the the regulation of blood the retina and choroid. preretinal 0, tension (Neely et al., 1989). hemodilution

little information

is

significance of these factors in flow in the eye, specifically in Preliminary data indicate that increases after hemodilution It has been suggested that

(HDIL) may play a role in the treatment

of retinal vascular diseases such as retinal artery occlusion (Wiederholt, 1981). Experimental studies in cerebral ischemia have demonstrated significant increases in cerebral blood flow after HDIL (Wood et al., 1981, 1982a). Evidence that retinal and cerebral blood flow are regulated in a similar fashion (Alm and Bill, 1972) suggests that these techniques may be appropriate for restoring flow to the ischemic retina. As a preliminary step in evaluating the usefulness of HDIL in retinal vascular disease, this study was performed to investigate the effects of HDIL on ocular blood flow and retinal 0, delivery in the normal (nonischemic) eye. 2. Materials

and Methods

General Preparation

Fourteen adult cats of either sex weighing 2.0-3.5 kg were used in these experiments. Procedures * For correspondenceat: Department of Anesthesia and Critical Care, University of Chicago Medical Center, 5841 South Maryland Avenue, Box MC-4028, Chicago, IL 60637, U.S.A. 0014483

5/92/070059

+05

$08.00/O

conformed to the ARVO Resolution on the Use of Animals in Research and were approved by the Animal Research Committee of the University of Chicago. Anesthesia was induced with halothane and 0,. Pancuronium, 0.3 mg kg-‘, was administered intravenously after the animal was unconscious and continued as a constant infusion at a rate of 0.2 mg kg-’ hr-I. Halothane concentration was adjusted during surgical preparation in response to changes in heart rate and blood pressure. Following oral endotracheal intubation, the lungs were mechanically ventilated (Harvard animal respirator) to maintain arterial PCO, at 28-30 mmHg and PO, at 100-200 mm.Hg with a 25-30x O,-air mixture. Intraocular pressure (IOP) was measured as described previously using a 21-gauge, 3/4” butterfly needle inserted under direct vision into the anterior chambers of both eyes (Ernest, Goldstick and Engerman, 1983). Catheters were placed via cutdowns into bilateral femoral arteries and veins. One of the arterial catheters was advanced into the abdominal aorta, and the catheter to measure central venous pressure was advanced into the right atrium. The following were continually measured and recorded : arterial pressure (systolic and diastolic in the femoral artery), central venous and, subsequently, left atria1 pressure (CVP and LAP), IOP, temperature, end-tidal CO, concentration (Engstrom Emma capnometer), and end-tidal anesthetic concentration (Puritan-Bennett anesthetic agent analyzer). All pressures (referenced to the level of the right atrium) were measured with Statham P2 3 transducers and recorded on a Gould 2800 S recorder. Mean arterial pressure (MAP) was calculated from the arterial pressure tracing as MAP = 2 / 3 diastolic + 0 1992 Academic Press Limited

60

1/ 3 systolic pressure. Body temperature, measured with an esophageal probe, was maintained at 37°C using a heating blanket and radiant heat lamp. Upon completion of surgical preparation, animals were anticoagulated with heparin. 200 U kg-’ intravenously.

S. ROTH

A left thoracotomy was performed and an 18.5-g plastic catheter was inserted into the left atrium via the left atria1 appendage and secured using purse string sutures. Carbonized microspheres (New England Nuclear), 15.5 & 0.1 ym diameter, labeled with Nb 95, Sn 113, and Ce 141 (specific activity S-10 ,&i g-l), were used as a 1% suspension in saline, with a small amount of Tween 80 added to prevent aggregation. After they were vortexed, shaken vigorously, and sonicated in a warm water bath for at least 20 min, approximately 1.5-1.75 million were injected into the left atria1 catheter over a 20-set period. During this period, no significant changes in arterial blood pressure or heart rate were noted. Following the injection of microspheres, the catheter was flushed with 3 ml of warm normal saline. For 15 set before the start of the injection and 2 min after, blood was sampled from the abdominal aorta using a Harvard pump (Model 22), which functioned as a surrogate organ (Heymann et al., 19 77) collecting blood at a constant rate of 1.36 f 0.01 ml min’.

The average number of cpm per microsphere was calculated by streaking drops of microspheres from the stock solutions onto graph paper three times and then counting the number of microspheres on the paper. Radioactivity on the graph paper was determined with the gamma counter. PO,, PCO,, and pH were determined in arterial blood using an ABL II blood gas analyser (Radiometer). In nine animals, arterial blood was analysed using an IL 482 Co-oximeter and arterial oxygen content was calculated as: [(% arterial saturation/lOO) x hemoglobin concentration (g 100 ml-l) x 1.34 ml g-‘1 + 0.003 ml 100 ml-l mmHg-’ x PO, (mmHg). (The units of oxygen content are thus ml 100 ml-‘, i.e. ~01%). Oxygen delivery in the retina and brain was calculated as the product of blood flow and arterial 0, content. Hematocrit (HCT) is reported as the spun HCT of whole arterial blood. 0, content and HCT were measured three times before and after HDIL, and the average value is reported. Ocular perfusion pressure (OPP, mmHg) was defined as MAP -1OP ; in the supine position this may have resulted in OPP being overestimated by lo-20 mmHg. Since a comparison of OPP before vs. following HDIL was made, only the uncorrected values are reported. Values before and after HDLL were compared by means of ANOVA for repeated measures with a Bonferroni correction for multiple comparisons ; a value of P < 0.003 (0*05/l 6) was the limit for statistical significance. Data are reported as mean f S.E.M.

Assay and Calculations

Studies

After completion of the experiment, the animals were killed by an injection of potassium chloride and sodium thiamylal into the left atrium. The eyes, cerebral cortex and kidneys were removed, dissected, and weighed. The retina, choroid, iris/ciliary body and sclera were separated as described previously (Alm and Bill, 1972) and placed in individual counting tubes, as were the brain and kidneys. Radioactivity in reference blood samples and tissues was counted (Packard Minaxi Autogamma 5000 Series gamma counter). Precautions were taken to minimize geometric error during sample counting (Katz and Blantz, 1972). The linear matrix method (Heymann et al., 19 77) was used for spectral resolution of counts from the different isotopes. Blood flow (ml min-‘) was calculated as : cpm in organ x sample withdrawal rate (ml min-l)/cpm in reference sample (Heymann et al., 1977). Blood flow in ml 100 g-l min-l was determined by dividing blood flow in ml mine1 by the wet weight of the tissues. There was no significant difference in right- vs. leftsided blood flow in the eyes, brain, or kidneys. Thus counts from both eyes were combined for the ocular blood flow determinations, The number of microspheres impacted was calculated by dividing the cpm in the organs by the number of cpm per microsphere.

After baseline blood flows were measured, cats underwent a 3045 min period of isovolumic HDIL. With an infusion/withdrawal pump (Harvard Model 944). blood was withdrawn from an arterial catheter at a rate of approximately 2 ml min-l and simultaneously replaced with an equal volume of 6% hydroxyethyl starch (HespanTM, DuPont, Wilmington, DE). (Hydroxyethyl starch is a synthetic plasma substitute with a molecular weight of 450 and substitution ratio of 0.7. Its osmolality is 3 13 mOsm l-l, similar to that for normal serum osmolality (285-29 5 mOsm 1-l.) Blood withdrawal was continued until the target HCT value of 20-22% was reached. The volume of blood withdrawn and replaced with hydroxyethyl starch was approximately 8090 ml. At least 45-60 min were allowed to elapse after the completion of HDIL for the animal to stabilize before further measurements were taken.

Blood Flow Measurement

3. Results Systemic Effects of &mod&&ion

HCT decreased from 36 f 1 to 20 f 1% (an average of 43x), accompanied by a decrease in MAP from 117+4 to 101+4 mmHg (P < 0.001). IOPdecreased from 19 + 1 to 17 f 1 mmHg (P < @04, N.s.). With

HEMOOILUTION

AND

OCULAR TABLE

BLOOD

I

Efjects of hemodilution on systemic hemodynamics (n = 14 except as noted)

Pre-dilution Hemodilution (ml 100 g-l min-‘)(ml 100 g-l min-‘) I-XT (%I MAP (mmHg) IOP (mmHg) OPP (mmHg) LAP (mmHg) PH PCO, (=Hg) PO, (mm&d Arterial Oz content (vol%)

36+1 117f4 19+1 98+4 1151 7.39 kO.02 29.4 f0.5 180+9 15.8 f 1.0

20*1* 101+4* 17*1

85k4 9+1 739 kO.01 29.4f0.5 179+10

8.8 k 0.5*

(n = 9)

* P < 0,003 vs. pre-dilution. Values are reported as mean + S.E.M. Abbreviations : HCT. hematocrit (%); MAP, mean arterial pressure (mmHg): IOP, intraocular pressure (mmHg) : OPP, ocular perfusion pressure (MAP-IOP. mmHg): LAP, left arterial pressure (mmHg).

TABLE

II

EfSectsof hemodilution on ocular and cerebral bloodJlow and retinal oxygen delivery (n = 14 except as noted)

Pre-dilution Hemodilution (ml 100 g-l min-‘)(ml 100 g-’ min-I) Retina Retinal 0, delivery

36.7k6.4 6.511.4

62.9 Ifr 6.4* 6.3 + 1.0

1297* 140 246$21 6.1kO.9 53.9 + 14.6 8.0 + 1.0

1051 f 144 296+27 6.9 f 0.8 77.4 Ifr 3.6* 6.9 + 0.5

(n = 9)

Choroid Iris/ciliary body Sclera Cerebral cortex Cerebral 0, delivery (n = 9)

* P < 0.003 vs. pre-dilution. Values are reported as mean &

61

FLOW

S.E.M.

the decrease in MAP, OPP decreased from 98 +4 to 8 5 + 4 mmHg (P < 0.004, N.S.) and arterial 0, content decreased from 15.8 f 1.0 to 8.8 f0.5 ~01% 0, (P < 0.003) (Table I). LAP was not significantly altered (11 _+1 mmHg vs. 911 mmHg, P < 0.025, N-S.). Ocular Blood Flow Changes

The most important changes in ocular blood flow occurred in the retina (Table II) where blood flow increased from 36.7 f 6.4 to 62.9 + 6.4 ml 100 g-l min-’ (P < 0.0001). The numbers of microspheres impacted in the combined retinas before and after HDIL were 107 i 16 and 17 7 + 24, respectively. Due to the significant decrease in arterial 0, content, calculated retinal 0, delivery was unchanged (6.5 + 1.4 vs. 6.3 If 1.0 ml 100 g-l min-‘). Choroidal blood flow decreased, but this change was not statistically significant. Microspheres impacted

before HAIL numbered 5967 + 1229 ; after HDIL, 4117 f 1018. There was no significant change in blood flow in the iris/ciliary body and the sclera (Table II). Cerebral cortical blood flow (CBF) also increased significantly (53.9 + 14.6 to 77.4 &- 3.6 ml 100 g-l min-‘, P < 0.0001). CBF increased less (43%) than did retinal blood flow (71%) and also resulted in decreased 0, delivery ; the change in cerebral 0, delivery from baseline during HDIL did not reach statistical significance (Table II). 4. Discussion

A dramatic increase in retinal blood flow occurs during HDIL. This increase in blood flow was not the result of a shift in distribution of ocular blood flow, as only small alterations were produced in the choroidal circulation, and no significant changes occurred in the sclera or iris/ciliary body. The findings of this study are consistent with previous observations by Neely et al. (1989), who demonstrated a 63 Y0 increase in preretinal 0, tension in cats when HCT was reduced 3 7% and blood volume was also replaced with hydroxyethyl starch. In the present study, a 71% increase in retinal blood flow accompanied a 44% reduction in HCT. This study confirms that the increase in preretinal 0, tension was due, at least in part, to an increase in retinal blood flow. Three possible mechanisms may be responsible for the increase in retinal blood flow: (1) alterations in blood viscosity with a redistribution of systemic blood flow favoring the vital organs (heart, brain, etc) ; (2) a physiological response in the retina to lowered 0, delivery ; and (3) a physiological response to the decreased oxygen content of choroidal blood. The first two mechanisms are active in the cerebral cortex (Fan et al., 1980) and most probably in the retina as well. Possibly due to the third mechanism, increases in retinal blood flow were even more dramatic than those occurring in the brain. During HDIL, the HCT is reduced by means of an isovolumic exchange of blood for crystalloid or colloid solution. HDIL lowers blood viscosity, which is determined by several factors, including HCT, erythrocyte aggregation and flexibility, platelet aggregation, and plasma viscosity. Of these, HCT is the most important. Decrease in blood viscosity is followed by a secondary reduction in peripheral vascular resistance, as expressed by the formula R = nZ, where R = total peripheral resistance ; n = viscosity ; Z = total vascular hindrance. Decreased peripheral resistance increases cardiac output due to reduced left ventricular afterload and an increase in venous return (Fan et al., 1980). Distribution of blood flow to the vital organs also changes significantly during HDIL. Blood flows in the liver, intestine, spleen, and kidney decrease (vasoconstriction) while cerebral and myocardial blood flow increase. The increases in blood flow in the heart and brain are disproportionate to the increase in cardiac

62

output, suggesting an important adaptive response by which limited 0, supply is diverted to vital organs (Chapler and Cain, 1981). Attempts have been made to augment CBF with HDIL during or following ischemic injury. Cerebral ischemia caused by reductions in perfusion pressure results in alterations in neuronal activity and a progressive neurological deficit. As decreased perfusion persists, cells die when membrane ion pumps fail. Blood flow slows distal to a narrowed or occluded cerebral artery. In this way, a vicious cycle is established wherein increases in blood viscosity further compromise perfusion and ischemia worsens (Grotta et al., 1982). Animal studies have demonstrated an increase in regional CBF in focal cerebral ischemia following both hypervolemic and isovolumic HDIL with dextran solutions (Wood et al., 1983). In contrast, hyperexpansion of the intravascular volume without HDIL did not increase CBF (Wood, Simeon and Snyder, 1982b). Results of clinical studies have varied. In a multicenter trial, Strand et al. (1984) demonstrated the efficacy of HDIL on neurological deficit in patients with acute cerebral ischemia less than 48 hr in duration. Two other recent large trials failed to show efficacy in acute stroke patients hemodiluted within 2448 hr of the ischemic event (Hemodilution in Stroke Study Group, 19 8 9 ; Asplund, 1989). Nevertheless, it is possible that very early intervention (within hours) may be of benefit. These results in patients with cerebral ischemia suggest a possible value for HDIL in retinal vascular disease. In retinal vascular occlusion, the development of anastomatic connections in the retina could bypass occluded vessels and thus limit the extent of injury from occlusion (Ashton and Henkind, 1965). In a manner analogous to that in the brain, HDIL may increase microcirculatory flow through the collateral vessels and thereby increase blood flow to the ischemic tissue. In this study, calculated retinal 0, delivery did not increase significantly ; therefore, the significant decrease in arterial 0, content was offset by the increase in retinal blood flow. Similar changes were found in the cerebral cortex. Lack of increase in retinal 0, delivery does not necessarily imply decreased 0, delivery at the tissue level. It is known that 0, extraction increases during HDIL (Chapler and Cain, 1986), which may also account for the findings of Neely et al. that HDIL resulted in an increase in 0, availability, probably at the microcirculatory level. The data analysis used a Bonferroni correction (Miller, 1981) to eliminate any possibility that differences in variables between pre- and posthemodilution were not due to random variation. This resulted in a strict level of significance being applied. Nevertheless, even with use of the less stringent P < 0.05 as the limit for statistical significance, the significance changes slightly in that differences in IOP, OPP. and LAP (P < O-04, 0.004. and 0.025,

S. ROTH

respectively) become significant. Changes in major variables of interest, e.g. retinal blood flow, remain highly significant. In conclusion, the present study shows that retinal blood flow increases dramatically during HDIL, and constitutes one mechanism enhancing oxygen availability. It remains to be determined if HDIL may actually be more effective in increasing blood flow in the ischemic, as opposed to the normal, retina. Possibly, other rheological agents should be evaluated to determine if their effects are more or less significant than those with hydroxyethyl starch. This study provides the basis for further evaluation of the effect of HDIL in the ischemic retina.

Acknowledgements The author is grateful to Dr J. Terry Ernest for advice during these studies and to Anne P. Crittenden, who provided technical assistance. Financial support was provided by the Foundation for Anesthesia Education and Research, Baltimore, MD.

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Miller, R. G. Jr. (1981). Simultaneous Statistical Inference, 2nd ed. Pp. 67-70. Springer-Verlag:New York. Neely. K. A., Ernest,J. T.. Goldstick.T. K. and Klein, H. 2.

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AND

OCULAR

BLOOD

FLOW

(1989). Systemic hemodilution increases preretinal oxygen tension. (ARVO Abstracts) Invest. Ophthalmol. Vis. Sci. 30, 66.

Strand, T.. Asplund, K., Ericksson,S., Hagg, E., Lithner, F. and Wester,P.-O.(1984). A randomizedcontrolledtrial of hemodilution in acute ischemicstroke. Stroke 15, 980-9. Wiederholt, M. (1981). Hgmodilution bei okularen Durchblutungsstiirungen.Verh. Dtsch. Ges.Inn. Med. 87, 1354-7. Wood, J. H., Simeone,F. A., Kron, R. E., Snyder, L. L. and Litt, M. (1981). Relationshipof cortical bloodflow and cardiovascularresponsesto alterationsin fresh blood viscosity as determined by capillary step-response viscometry. 1. Crreb. Blood Flow Metab. 1 (Suppl. 1). S2334.

63 Wood, J. H.. Simeone,F. A., Kron, R. E.and Litt, M. (1982a). Rheological aspects of experimental hypervolemic hemodilution with low molecular weight dextran. Relations of cortical blood flow, cardiac output and intracranial pressureto freshbloodviscosityandplasma volume. iVeurosurgerg 11, 739-53. Wood, J. H., Simeone, F. A. and Snyder, L. L. (1982b). Failure of intravascular volume expansion without hemodilutionto elevatecortical blood flow in region of experimentalfocal ischemia.1. Neurosurg.56, 80-91. Wood, J. H., Simeone,F. A., Fink, E.A. and Golden, M. A. ( 1983). Hypervolemic hemodiiution in experimental focal cerebral ischemia:elevationsof cardiac output, regionalcortical bloodflow and ICP after intravascular volume expansionwith low molecularweight dextran. 1. Neurosurg.59. 500-q.