Regional cerebral blood flow (rcbf) decreases

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1 1072 Effect f Hemdilutin n Reginal Cerebral Bld Flw During Chrnic Hyperglycemia in Rats Rbert B. Duckrw, MD Reginal cerebral bld flw decreases during chrnic hyperglycemia, a cnditin frequently assciated with elevated hematcrit. T test the hypthesis that an elevated hematcrit is respnsible fr the reduced reginal cerebral bld flw during chrnic hyperglycemia, I used isvlemic hemdilutin t nrmalize the hematcrit in seven nrmglycemic and seven streptztcin-treated (hyperglycemic) rats. Reginal cerebral bld flw was measured in 28 awake, restrained rats (14 nrmglycemic and 14 hyperglycemic) using [ 14 C]idantipyrine and dissectin f 17 brain regins. Hemdilutin lwered the hematcrit by 6 units (13%) and increased the average cerebral bld flw by 14%. Chrnic hyperglycemia did nt elevate the hematcrit, but it decreased the average cerebral bld flw by 12% and that in nine nntelencephalic brain regins by 17%. This effect was independent f changes in hematcrit caused by hemdilutin. The reduced reginal cerebral bld flw during chrnic hyperglycemia is nt caused by elevated hematcrit. (Strke 1990;21: ) Reginal cerebral bld flw (rcbf) decreases during acute 1-2 and chrnic 3 " 5 hyperglyce- «. mia. The cause f this reductin is nt knwn, but a number f pssible mechanisms have been tested. Glucse culd decrease cerebral bld flw (CBF) by increasing plasma smlality. Acute infusin f hypersmtic slutins can affect determinants f CBF such as perfusin pressure, vascular smth muscle tne, and bld viscsity. Hwever, measurements f rcbf during acute infusins f glucse and mannitl indicate that the smtic lad prduced by acute hyperglycemia cannt explain the bserved decrease in rcbf. 2 Alternatively, because f the clse cupling f rcbf t the reginal rate f energy metablism, hyperglycemia culd indirectly decrease CBF by decreasing the metablic rate f the brain. Again, direct measurements f glucse utilizatin during acute and chrnic hyperglycemia d nt supprt this mechanism. 6 Mre recently, measurements f the anatmic density f brain capillaries and the fractin f perfused capillaries indicate that they d nt change during chrnic and acute hyperglycemia, 7 Frm the Department f Medicine (Divisin f Neurlgy), The Miltn S. Hershey Medical Center, The Pennsylvania State University, Hershey, Penn. Supprted by Public Health Service grant and by an American Heart Assciatin Established Investigatr Award. Address fr crrespndence: R.B. Duckrw, Divisin f Neurlgy, The Miltn S. Hershey Medical Center, PO Bx 850, Hershey, PA Received Octber 13, 1989; accepted March 15, excluding these factrs as pssible causes fr the reductin f rcbf during hyperglycemia. Clse inspectin f reprts f reduced rcbf during acute and chrnic hyperglycemia suggest that elevated hematcrit may explain this effect. Acute hyperglycemia induced by the intraperitneal injectin f glucse and chrnic hyperglycemia induced by treatment with streptztcin are frequently assciated with increased hematcrit. 2 ' 3 ' 67 Hematcrit is a majr determinant f whle bld viscsity 8 and, alng with hemglbin cncentratin and xyhemglbin affinity, determines the xygen carrying capacity f the bld. These factrs cntribute t an inverse relatin between hematcrit and CBF. 9 Using hemdilutin t nrmalize the hematcrit in nrmglycemic and streptztcin-treated rats, I tested the hypthesis that an elevated hematcrit is respnsible fr the reduced rcbf during chrnic hyperglycemia. Materials and Methds I used 28 male Sprague-Dawley rats (Charles River Labratries, Inc., Wilmingtn, Mass.) weighing g. The rats had free access t fd and water befre surgery. Chrnic hyperglycemia was induced in 14 rats by the injectin f 60 mg/kg i.v. streptztcin days befre rcbf was measured. The 14 cncurrently prepared nrmglycemic cntrl rats were hused fr similar intervals. The rcbf was measured in awake, restrained rats using prcedures in accrdance with the guidelines f the Miltn S. Hershey Medical Center fr the care f

experimental animals. Surgery was perfrmed using halthane/n 2 O anesthesia. Anesthesia was induced with 3% halthane in 70% N 2 O/30% O 2 and maintained with 0.6% halthane.

2 experimental animals. Surgery was perfrmed using halthane/n 2 O anesthesia. Anesthesia was induced with 3% halthane in 70% N 2 O/30% O 2 and maintained with 0.6% halthane. Skin incisins were infiltrated with 1% prcaine and swabbed with 5% xylcaine intment. Plyethylene catheters were placed in bth femral arteries and ne femral vein. A rectal thermistr prbe was placed, and a heating lamp was used t maintain bdy temperature. Each rat was restrained by a plaster cast placed arund the hip girdle and hind legs. The head was placed in a guilltine, and the apparatus was draped t prevent visual distractin f the rat. One hur was allwed fr recvery frm anesthesia, at which time the rats were awake, alert, and calm. Pulse rate and arterial bld pressure were cntinuusly measured frm ne arterial catheter. Bld samples were btained frm the secnd arterial catheter t measure respiratry gas tensin, ph, hematcrit, plasma smlality, and plasma electrlyte cncentratin just befre rcbf was measured. Bld frm this catheter was als used t determine the appearance functin f radilabeled tracer after injectin. After 1 hur f recvery, the hematcrit was measured and a vlume (V) f arterial bld was withdrawn int a heparinized syringe accrding t the frmula: V=(BVxWT)[l-(HCT d /HCTj)], where BV is the rat bld vlume fractin in milliliters per gram, WT is the bdy weight in grams, and HCT is the initial (i) and desired (d) hematcrit. The value 0.05 was used fr BV and the value 40% was used fr HCT d. The whle bld was centrifuged. T lwer the hematcrit, the plasma was diluted with nrmal saline t achieve the riginal vlume withdrawn and then reinjected. If the rat was hyperglycemic, the nrmal saline used t dilute the plasma cntained 30 mml/1 D-glucse. If the rat was nrmglycemic, the nrmal saline cntained 10 mml/1 D-glucse. Rats nt hemdiluted had bld withdrawn accrding t the same frmula. Hwever, the plasma was discarded and the erythrcytes were mixed with nrmal saline cntaining glucse and then reinjected (sham hemdilutin). The average vlume f bld withdrawn was 2.9 ml fr nrmglycemic rats and 2.2 ml fr hyperglycemic rats. The interval between bld withdrawal and reinfusin was 10 minutes. Mean arterial bld pressure decreased during this interval, but nt t < 100 mm Hg, and returned t baseline upn reinfusin. rcbf was measured 30 minutes later. The rcbf was measured using the diffusible indicatr [ 14 C]idantipyrine (60 mci/mml) and methds previusly described. 3 Briefly, 40 /i.ci f tracer in nrmal saline was injected intravenusly at 17 /il/sec. Freelyflwingdrps f arterial bld were pled at 2-secnd intervals, and aliquts were prepared fr scintillatin cunting. After 20 secnds f tracer expsure, the rat was decapitated and the brain was quickly dissected. Tissue samples frm 17 brain regins (nine nntelencephalic and eight telencephalic) were transprted in a humidified chamber, rapidly Duckrw Hematcrit, Hyperglycemia, and rcbf 1073 weighed, and slubilized fr scintillatin cunting. rcbf was calculated using equatins presented by Sakurada et al. 10 Pa 2, Pac 2, and arterial ph were measured at 37 C (BMS 3 Mk 2, Radimeter America, Inc., Westlake, Ohi). Plasma glucse cncentratin was measured by an autmated glucse xidase methd (Glucse Analyzer 2, Beckman Instruments, Inc., Fullertn, Calif.). Plasma smlality was measured by dew-pint temperature depressin (mdel 550, Wescr Inc., Lgan, Utah). Plasma Na + and K + cncentratins were measured by flame phtmetry (mdel 143, Instrumentatin Labratry, Lexingtn, Mass.). Hematcrit was measured using bld in 20 -/il capillary tubes centrifuged at 13,500g fr 3 minutes. Rats were allcated in an alternating fashin t the streptztcin-treated r the nrmglycemic cntrl grup frm lts f littermates received frm the supplier. Rats frm these tw grups alternately underwent hemdilutin r sham hemdilutin. The intent f the experimental design was t cmpare rcbf between subgrups f nrmglycemic and hyperglycemic rats with equal hematcrits. Because reginal variatins in rcbf have been measured during hyperglycemia, 3 rcbf was cmpared in the 17 brain regins cmbined and in the nine nntelencephalic brain regins cmbined and the eight telencephalic brain regins cmbined. Physilgical parameters were cmpared using analysis f variance (ANOVA) and t tests. rcbf was cmpared using a repeatedmeasures ANOVA 11 (GLM prcedure, SAS Institute, Inc., Cary, N.C.) using the rat as the unit f analysis. Rats were gruped by treatment in a 2x2 factrial design cntrasting nrmal and high plasma glucse cncentratins r nrmal and lw hematcrits. The replicatin factr was individual brain regins, rather than time, t accunt fr the pssible interdependency f rcbf in different regins f the same brain. Results Treatment with streptztcin prduced a threefld elevatin in plasma glucse cncentratin, a failure t gain weight, and a lwer resting mean arterial bld pressure. Mean±SD bdy temperature was 36.7±0.2 C in all subgrups. Befre hemdilutin mean±sd hematcrit was 45 ±2% in the nrmglycemic cntrl grup and 46±3% in the streptztcin-treated grup. Nrmglycemic and hyperglycemic rats with sham hemdilutin had equal mean hematcrits f 47% befre rcbf measurement. Hemdilutin reduced hematcrit by 6 units, prducing a mean hematcrit f 41% in bth nrmglycemic and hyperglycemic rats. The physilgical parameters f all subgrups are shwn in Table 1. The intent f the experimental prcedure was t nrmalize an expected difference in hematcrit between streptztcin-treated and nrmglycemic cntrl rats. Hwever, streptztcin treatment did znt prduce the expected increase in baseline

3 1074 Strke Vl 21, N 7, July 1990 TABLE 1. Physilgical Parameters in Rats Befre Measurement f Reginal Cerebral Bld Flw Subgrup Weight " (g) MABP (mm Hg) Pulse (beats/ min) Arterial PH Cntrl hematcrit Nrmglycemic 7 395±23 132±5 405 ± ±0.03 Hyperglycemic 7 278±26* 116± ± ±0.04* Hemdilutin Nrmglycemic 7409±24 128± ±0.02 Hyperglycemic 7 309±39* 117±16 366± ±0.04 Pa 2 Pac 2 (mm Hg) (mm Hg) 91±4 92±7 94±6 91±7 37±3 36±1 39±2 39±4 [Na + ] (meq/1) 136±4 136±4 136±2 136±3 L J (meq/1) 3.9± ± ± ±0.2 Values are mean±sd. MABP, mean arterial bld pressure. Hct, hematcrit. */> different frm nrmglycemic rats with same hematcrit using analysis f variance and t test. Plasma glucse Plasma Hct cncentratin smlality (%) (mml/1) (mml/kg) 47±3 47±1 41± * * 289±7 317±9* ±9* hematcrit. Nrmalizatin f the hematcrit by hemdilutin did nt eliminate a reductin in rcbf measured during chrnic hyperglycemia (Figure 1). On average, rcbf decreased by 12% in the hyperglycemic rats regardless f hematcrit. Repeatedmeasures ANOVA indicated that rcbf in the grups f rats with high and lw plasma glucse cncentratins differed at/?<0.02. The nine nntelencephalic brain regins in the hyperglycemic rats had a mean rcbf 17% lwer than that in the nrmglycemic grup (Table 2). Hemdilutin increased rcbf by 14%. Repeatedmeasures ANOVA indicated that rcbf in subgrups f rats with high and lw hematcrits differed at /><0.02. The interactin between plasma glucse cncentratin and hematcrit was nt significant (/?=0.56). Discussin The rcbf has been shwn t decrease during chrnic hyperglycemia, 3-5 a cnditin accmpanied by increased hematcrit in rats. 3 If the decrease in E en c "E I c± CD "6 c 0 0== 8 ex Nrm Hyper Nrm Hyper Sham Dilute c E I O O CO "5 c a> a> 0 rcbf is due t an elevatin in hematcrit, nrmalizatin f the hematcrit by hemdilutin wuld prevent the decrease. My data indicate that rcbf is reduced in chrnically hyperglycemic rats despite nrmalizatin f the hematcrit by hemdilutin. Unexpectedly, chrnic hyperglycemia was nt accmpanied by increased hematcrit in this study. Hwever, rcbf was als reduced in hyperglycemic rats underging sham hemdilutin. Bth bservatins indicate that the decreased rcbf during hyperglycemia is nt explained by an elevated hematcrit. Reginal variatins in the effect f chrnic hyperglycemia n rcbf have been nted previusly. 3 Based n this previus bservatin, an arbitrary anatmic distinctin was made, separating telencephalic frm nntelencephalic brain regins. N effect f plasma glucse cncentratin was apparent in the telencephalic regins (p=0.29), but such an effect was present in the nntelencephalic regins (p=0.002), where mean rcbf reductin was 17%, which is cnsistent with previus reprts. 23 The Nrm Hyper Sham Nrm Dilute Hyper FIGURE 1. Scatterplt fcerebral bld flw in individual telencephalic (left) and nntelencephalic (right) brain regins f cntrl (Nrm) and streptztcin-treated (Hyper) rats subjected t sham hemdilutin (Sham) r isvlemic hemdilutin (Dilute). Seven rats in each subgrup. Linked data pints indicate difference in bld flw in individual brain regins between subgrups. Average bld flw reductin f 12% was measured in hyperglycemic rats ( p<0.02 fr telencephalic and nntelencephalic regins cmbined); effect was mre prnunced in nntelencephalic brain regins (17% reductin, p<0.002). Hemdilutin increased mean bld flw by 14% ( p<0.02). There was n interactin between these effects.

4 Duckrw Hematcrit, Hyperglycemia, and rcbf 1075 TABLE 2. Reginal Cerebral Bld Flw in Nrmglycemic and Hyperglycemic Rats With and Withut Hemdilutin Withut hemdilutin With hemdilutin Brain regin Mtr crtex* Sensry crtex* Auditry crtex* Visual crtex* Olfactry lbe* Caudate-putamen* Thalamus Hippcampus* Hypthalamus Superir clliculus Inferir clliculus Mesencephaln Pns Pyramidal tract Medulla Subcrtical white matter* Cerebellum Nrmglycemic 132±5 154 ±7 155±6 135±5 112±7 109±5 124±7 92±5 107 ±5 131±10 159±10 109±6 108 ±7 89±4 112±8 70±7 100±6 Hyperglycemic 132±8 153±8 155±13 139±6 106±6 103±4 109±6 82±4 88±8 112±6 142±10 96±7 87±5 73± ±4 82±5 Nrmglycemic 153±6 174±6 175 ±10 158±10 129±6 125±9 143±9 105±6 124 ±8 152±8 193±9 129 ±7 123 ±7 103 ±7 130 ±9 73±6 119±6 Hyperglycemic 150±6 172±13 162± ± ±5 95±7 99± ± ± ±5 68±6 96±6 P <0.05 <0.05 Data are mean±sem ml/100 g/min f 7 rats., n significant glucse effect. *Telencephalic regins. cause f this reginal variatin in rcbf respnse remains unexplained. Hwever, rstral-caudal gradients in the rcbf respnse bserved in nenatal dgs during asphyxia 12 have been ascribed t the knwn density gradient f adrenergic innervatin f the cerebral bld vessels. 13 Recently, the attenuatin f cerebral hyperemia by indmethacin during seizures was reprted t be mre prminent in the diencephaln-mesencephaln, pns, and medulla f piglets. 14 Althugh the cause f reduced rcbf during hyperglycemia is nt knwn, the presence f reginal variatins in bld flw cntrl must be recgnized. Hemdilutin increased glbal CBF in bth nrmglycemic and hyperglycemic rats. A hematcrit decrease f 6 units (13%, frm 47% t 41%) increased rcbf by 14%. This hematcrit span includes the nrmal hematcrit fr rats, which in ur labratry is 44%. Althugh there are few studies f the effects f hematcrit n rcbf in rats, 15 the magnitude f this change in rcbf is similar t that reprted fr dgs, 16 lambs, 9 and humans. 17 It has been nted that hematcrit changes f <15% d nt prduce detectable changes in CBF in humans. 18 The rcbf increase that I measured ccurred after a 13% decrease in hematcrit, indicating that hematcrit shuld be cnsidered a relevant parameter in studies f rcbf in rats. CBF varies inversely with hematcrit because f the cntributin f erythrcytes t brain metablism and bld rhelgy. rcbf is regulated, in part, by the ability f bld t supply xygen t the tissue, as evidenced by the inverse relatin f CBF t bld xygen cntent 9 and the direct relatin f CBF t xyhemglbin affinity. 19 These factrs are defined by the structure and amunt f hemglbin in the erythrcytes and are thereby indirectly influenced by acute changes in hematcrit. Erythrcyte cncentratin directly influences whle bld viscsity. 8 Althugh the cntributins f the rhelgic and metablic influences f changing hematcrit n rcbf may vary within the range ver which hematcrit changes, 15 at near-nrmal hematcrits their cntributins appear t be rughly equal. 9 It is cnceivable that chrnic hyperglycemia alters the usual relatin between erythrcyte cncentratin and rcbf, prducing a lwer rcbf at a given hematcrit. Chrnic hyperglycemia induced by streptztcin culd increase erythrcyte rigidity 20 and adhesiveness. 21 These factrs wuld reduce rcbf by increasing whle bld viscsity. Chrnic hyperglycemia increases xyhemglbin affinity 22 and glycsylates hemglbin Increased amunts f defective hemglbin with increased xygen affinity wuld act t increase rcbf. The relative cntributins f these ppsing factrs t ttal cerebrvascular resistance are unknwn. Hwever, these influences wuld act equally n all brain regins. The presence f reginal variatins in the rcbf reductin measured during chrnic hyperglycemia cntrasted with the glbal respnse t hemdilutin is evidence that these factrs d nt explain the rcbf reductin. Chrnic hyperglycemia culd increase the cncentratins f plasma cmpnents that cntribute t bld viscsity, such as fibringen and a-2-macrglbulin, and lwer rcbf by a viscsity-mediated mechanism that is independent f the hematcrit. Althugh this pssibility cannt be excluded by my data, plasma fibringen cncentratin decreases during streptztcin-induced hyperglycemia (unpublished data), making this pssibility less likely. Again,

1076 Strke Vl 21, N 7, July 1990 the presence f reginal variatins in the reductin f rcbf during chrnic hyperglycemia argues against a viscsity-mediated mechanism fr the reductin in rcbf.

5 1076 Strke Vl 21, N 7, July 1990 the presence f reginal variatins in the reductin f rcbf during chrnic hyperglycemia argues against a viscsity-mediated mechanism fr the reductin in rcbf. My data indicate that erythrcyte cncentratin cannt explain the reductin in rcbf during chrnic hyperglycemia and that the effects f hyperglycemia and hemdilutin n CBF are independent. Acknwledgments Technical assistance was prvided by Daniel C. Beard. Statistical cnsultatin was prvided by the Center fr Bistatistics and Epidemilgy f the Miltn S. Hershey Medical Center f the Pennsylvania State University. References 1. Ginsberg MD, Welsh RA, Budd WW: Deleterius effect f glucse pretreatment n recvery frm diffuse cerebral ischemia in the cat. Strke 1980;ll: Duckrw RB, Beard DC, Brennan RW: Reginal cerebral bld flw decreases during hyperglycemia. Ann Neurl 1985; 17: Duckrw RB, Beard DC, Brennan RW: Reginal cerebral bld flw decreases during chrnic and acute hyperglycemia. Strke 1987;18: Harik SI, LaManna JC: Vascular perfusin and bld-brain glucse transprt in acute and chrnic hyperglycemia. / Neurchem 1988;51: Nedergaard M, Jakbsen J, Diemer NH: Autradigraphic determinatin f cerebral glucse cntent, bld flw, and glucse utilizatin in fcal ischemia f the rat brain: Influence f the plasma glucse cncentratin. / Cereb Bld Flw Metab 1988;8: Duckrw RB, Bryan RM: Reginal cerebral glucse utilizatin during hyperglycemia. / Neurchem 1987;48: Kikan GE, LaManna JC, Harik SI: Brain perfusin in acute and chrnic hyperglycemia in rats. Strke 1989;20: Stne HO, Thmpsn HK, Schmidt-Nielsn K: Influence f erythrcytes n bld viscsity./lm// > /y<.sf/1968;214: Hudak ML, Kehler RC, Rsenberg AA, Traystman RJ, Jnes MD Jr: Effect f hematcrit n cerebral bld flw. Am JPhysil 1986;251:H63-H Sakurada O, Kennedy C, Jehle J, Brwn JD, Carbin GL, Sklff L: Measurement f lcal cerebral bld flw with id[ 14 C]antipyrine.^m JPhysil 1978;234:H59-H Winer BJ: Statistical Principles in Experimental Design. New Yrk, McGraw-Hill Bk C, 1971, pp Hernandez MJ, Brennan RW, Hawkins RA: Reginal cerebral bld flw during nenatal asphyxia, in Passnneau JV, Hawkins RA, Lust WD, Welsh FA (eds): Cerebral Metablism and Neural Functin. Baltimre, Williams & Wilkins C, 1980, pp Nielsen KC, Owman C: Adrenergic innervatin f pial arteries related t the circle f Willis in the cat. Brain Res 1967; 6: Busija DW, Leffler CW: Rle f prstanids in cerebrvascular respnses during seizures in piglets. Am J Physil 1989; 256:H120-H Brgstrm L, Jhannssn H, Siesj BK: The influence f acute nrmvlemic anemia n cerebral bld flw and xygen cnsumptin f anesthetized rats. Ada Physil Scand 1975; 93: Fan F-C, Chen RYZ, Schuessler GB, Chien S: Effects f hematcrit variatins n reginal hemdynamics and xygen transprt in the dg. Am J Physil 1980;238:H545-H Gaehtgens P, Marx P: Hemrhelgical aspects f the pathphysilgy f cerebral ischemia. / Cereb Bld Flw Metab 1987;7: Wd JH, Kee DB Jr: Hemrhelgy f the cerebral circulatin in strke. Strke 1985;16: Kehler RC, Traystman RJ, Rsenberg AA, Hudak ML, Jnes MD Jr: Rle f O 2 -hemglbin affinity n cerebrvascular respnse t carbn mnxide hypxia. Am J Physil 1982;245:H1019-H McMillan DE, Utterback NG, LaPuma J: Reduced erythrcyte defrmability in diabetes. Diabetes 1978;27: Wautier J-L, Patn RC, Wautier M-P: Increased adhesin f erythrcytes t endthelial cells in diabetes mellitus and its relatin t vascular cmplicatins. N Engl J Med 1981; 305: Ditzel J: Oxygen transprt impairment in diabetes. Diabetes 1976;25(suppl 2): Rubin MJ, Bhlen HG: Cerebral vascular autregulatin f bld flw and tissue P 2 in diabetic rats. Am J Physil 1985;249:H540-H546 KEY WORDS flw rats hematcrit hyperglycemia cerebral bld

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