Alterations in Autoregulatory and Myogenic Function in the Cerebrovasculature of Dahl Salt-Sensitive Rats John S.Smeda.PhD.Geoffrey W.Payne.MSc Bmekgroumd mud Purpose-D)shl salt-sensitrve rats fed an 8.7%Nal diet exhibied hypertensive eneephnlopathy and developed seizures associated with areas of'hlood-hmin harrier (RBR]disnuption without brin ischemin.The inciderce of hemorrhagic stroke was low (7/47).We tested the hypochesis that a defect in cerebral blood flow (CBF) autregulation under hypertensive conditions preceded hypertensive encephalpathy. Meno-Brain ischemia and BDB disruption were assessed with the use of tetrazulium red staining and Evans blue dye extmvnsntion,respectively Myogenic constriction to pressure wns mensured in isolated middle cerebral ntenes (MCAs) with a pressure myograph.CBF aregulation wus assessed with the use of laser-Doppler techniques. Resmlp-Asymptomati rals fod 8.7%NaCI had MCAs that developed an agrelaed atteration in their abdlity to coostrict to pressure.which was amplified in rats exhibiting hypertensive encephalopathy.The MCAs of rats with bemorrhagic stroke lost this fimction and developed large degrees of basal tone.The msority (4/6)of asymptomatic rats fed high salt for longer than 3 weeks exchibued a linear relationship between CBF and blood pressure.The characteristics of CBF regulation were cunsistent with the possable absence of autoregulation coupled with cerebruvascular vasoconstricbon. Comclursions-Both MCA pressure-dependert constriction and CBF autoregulstion in the MCA perfusion domain were lost before the development of lypertensive enoephslopmthy or hemorrhege stroke.These detects could contnbute to the development of BRB disruption during hypertension.Cerehrovaseular vasoconstriction in the shsence of CBF autoregulation may protect the brain from excessive overperfusion during hypertension and could account for the kw ncidence of cerebral hemorhage in this model.(Sroke.2003;34:1484-1490.) Key Words:blood-brain barrier cerebral blood flow hypertensive encephalopathy hypertersion ■niddle cerebral8rley■nuscle,smooth.vascular■stroke,morg1e Typertensive animals and humans have cerehrowascula- expected to occur Thus CBF remains constant despite the esthat are capae of aoregularing blood flow w买mBP higher blood presres(BPs)than those of their nomocensive Dahl sal-sensitive [Dahl-SS)rats fol a high-Nacl diet counterpartsElevating the upper BP limit of cerebral blood develop hypertenesion,bchanvioral signes of stroke,and distup- flow (CBF)aulegulation under hypericrsne condtiuns is a tion uf'the BBB.75 The present study teslo the hypothesis beneficinl feature in th it prevents wverperfusion af'the leain that stmoke developeent in Dshl-55 rats was precelod ty a during hypertensson This protective effect can be demon defect in the ability of the cerebrovasculature to autoregulate strated in normal animals.Elevations in sympathetic nerve blood fluw and that this defect was associatd with an stimula1 o the ccrebruvasculture1 e upper lim美 inblily of the ecrdbral aleries to consriel mn tespunse lo of CRF aoregulation 1.Such manipulmions redhce the peessure (mycgenk responsel. disnuption of the blnod-brin bamrier (BBB)in response to experimenal clevations in BP.1.M while the inhibition of Materials and Methods CBF4lins(y the inlinson吧 The experiments were consisteat with the gudelines set by the Cachannel hlockers)uder conditions when BP is elevated Cinadin Courcil on Arimal Care.Mae Duhl-SS tals used in the study were beed it our irstitue und Sed a fommal or hghol diet enhances日3B disruptioe' fmum weinng (5 werks of age)The nemmal-salt diet tumiskd ofa Pressure-dependent constriction helps to promoee CBP Polal RMH 3200 formula (PMI Feeda Inel the.oomained 0.7% aulorcpulatin Elvalions in RP pomote crehnoasor N(026%Na',044%C)wherers the high-silt dict onerined 3.7%NaCl (5.11%Na'3.59%C Rat were monilcced daily foe constriction,which elevines vascular resistance in fow and sigrs ot hperensive excephalputhy or stroke (seteures head and counterncts the potential increase in CBF that mighe be frelimb repetilive twnching belwvice,forelirab ard hindimb parl Roocivod Maich 4,2002,finl tevison teerived Jumary 16,2003.uoxxpood Juury 21,2003. Roptit ietsts w D J.5.Sarsle,Divbiou uf Beaie Meditl Seioe Rim E4354,Muutial Unieraiy.Heall Seirte Cerke,SI Jutn's, 2003 Amican Hart Axociiot Ie. Srote b aaible al bllpYwwsirakeshs.erz D0L:10116101STR00735421224AA 14g4
Alterations in Autoregulatory and Myogenic Function in the Cerebrovasculature of Dahl Salt-Sensitive Rats John S. Smeda, PhD; Geoffrey W. Payne, MSc Background and Purpose—Dahl salt-sensitive rats fed an 8.7% NaCl diet exhibited hypertensive encephalopathy and developed seizures associated with areas of blood-brain barrier (BBB) disruption without brain ischemia. The incidence of hemorrhagic stroke was low (7/47). We tested the hypothesis that a defect in cerebral blood flow (CBF) autoregulation under hypertensive conditions preceded hypertensive encephalopathy. Methods—Brain ischemia and BBB disruption were assessed with the use of tetrazolium red staining and Evans blue dye extravasation, respectively. Myogenic constriction to pressure was measured in isolated middle cerebral arteries (MCAs) with a pressure myograph. CBF autoregulation was assessed with the use of laser-Doppler techniques. Results—Asymptomatic rats fed 8.7% NaCl had MCAs that developed an age-related attenuation in their ability to constrict to pressure, which was amplified in rats exhibiting hypertensive encephalopathy. The MCAs of rats with hemorrhagic stroke lost this function and developed large degrees of basal tone. The majority (4/6) of asymptomatic rats fed high salt for longer than 3 weeks exhibited a linear relationship between CBF and blood pressure. The characteristics of CBF regulation were consistent with the possible absence of autoregulation coupled with cerebrovascular vasoconstriction. Conclusions—Both MCA pressure-dependent constriction and CBF autoregulation in the MCA perfusion domain were lost before the development of hypertensive encephalopathy or hemorrhagic stroke. These defects could contribute to the development of BBB disruption during hypertension. Cerebrovascular vasoconstriction in the absence of CBF autoregulation may protect the brain from excessive overperfusion during hypertension and could account for the low incidence of cerebral hemorrhage in this model. (Stroke. 2003;34:1484-1490.) Key Words: blood-brain barrier cerebral blood flow hypertensive encephalopathy hypertension middle cerebral artery muscle, smooth, vascular stroke, hemorrhagic Hypertensive animals and humans have cerebrovasculatures that are capable of autoregulating blood flow at higher blood pressures (BPs) than those of their normotensive counterparts.1,2 Elevating the upper BP limit of cerebral blood flow (CBF) autoregulation under hypertensive conditions is a beneficial feature in that it prevents overperfusion of the brain during hypertension. This protective effect can be demonstrated in normal animals. Elevations in sympathetic nerve stimulation to the cerebrovasculature increase the upper limit of CBF autoregulation.1,3 Such manipulations reduce the disruption of the blood-brain barrier (BBB) in response to experimental elevations in BP,1,3,4 while the inhibition of CBF autoregulation in rats (by the infusion of high levels of Ca2 channel blockers) under conditions when BP is elevated enhances BBB disruption.5 Pressure-dependent constriction helps to promote CBF autoregulation.1,6 Elevations in BP promote cerebrovascular constriction, which elevates vascular resistance to flow and counteracts the potential increase in CBF that might be expected to occur. Thus, CBF remains constant despite the change in BP. Dahl salt-sensitive (Dahl-SS) rats fed a high-NaCl diet develop hypertension, behavioral signs of stroke, and disruption of the BBB.7,8 The present study tested the hypothesis that stroke development in Dahl-SS rats was preceded by a defect in the ability of the cerebrovasculature to autoregulate blood flow and that this defect was associated with an inability of the cerebral arteries to constrict in response to pressure (myogenic response). Materials and Methods The experiments were consistent with the guidelines set by the Canadian Council on Animal Care. Male Dahl-SS rats used in the study were bred at our institute and fed a normal or high-salt diet from weaning (5 weeks of age). The normal-salt diet consisted of a Prolab RMH 3000 formula (PMI Feeds Inc) that contained 0.7% NaCl (0.26% Na, 0.44% Cl), whereas the high-salt diet contained 8.7% NaCl (5.11% Na, 3.59% Cl). Rats were monitored daily for signs of hypertensive encephalopathy or stroke (seizures, head and forelimb repetitive twitching behavior, forelimb and hindlimb paralReceived March 4, 2002; final revision received January 16, 2003; accepted January 21, 2003. From the Division of Basic Medical Sciences, Memorial University, St John’s, Newfoundland, Canada. Reprint requests to Dr J.S. Smeda, Division of Basic Medical Sciences, Room H4354, Memorial University, Health Science Center, St John’s, Newfoundland, Canada A1B 3V6. E-mail jsmeda@mun.ca © 2003 American Heart Association, Inc. Stroke is available at http://www.strokeaha.org DOI: 10.1161/01.STR.0000073842.18224.AA 1484
Smeda and Payne Cerebrevascular Function in DahlSS Rats JS ysi线severe lehir款：igns are desenbed in grealer detail前Revelts The sysdlie BP wis measued by a tail cufl'compression method (model 59.IITCX Examination of Brains The brairs of 47 Dahl-SS rats exhibting behavioeal abnommalities were fised in 10%PO-huffered foemlin Forty heains were sec- tioned in 1-mm-Thick crermal sectinns with the use of a jig (RFM 4000C,ASI Instruments)and examnined by microocope bor cerebral hemocrhages.Seven brairs were inbedded in puraffin and histolog. cally sectuned The 5-um-ick seclioes were sarnal with cresv 42 vinlel hle or hemnatnxylin and eosin ee assessed for the presrnce af glial fibnllary acidic prottin (which accumulates in astrocytes afer Bchemic damaze")and examined for ischemi alteratioes and the presence of hemoen巾hge. ylo世were sliced with the use of a ji电REM40C)and ad ischemic lesiors with tiued tochniq letrazolium red is taken up by the brain cells,and dehy dropereses Wccks cn Diet convert ths compound的m白teed4 ye tht remirs within the cells.Echemie damige ot'the hnen inetives cellulr Figur 1.Mortalty profie of Dan-5S rats fd an 8.7%Nacl diat dehydropen现，Icadingo a faiain fom waaning (5 wenks of agel.Dal-8S fed roral NaCl 1.7%) (n=10 remained healthy for the duration of the experiment [14 Measurements of Plasma Extravasation Acrosx wek籽Cfag the BBB Rats were anesthetiped with sodium penoberbital 165 m/kg IP)and CBF value [e,ICBF a:a given MAP=(fux at MAPMx t MAP Evies hlue dye (10 mgml.of 0.9%ie)wies infirsed (30 mg dye cdI间mme划CF was then pltted时MAP per kikgren)ino the frmreal vrin.Ader 12 mirules,the ches caviy was opened,and a polyethylene catheter (PE-50)wus inserted Statistical Analysis into the borta and tied.The right and lett ventncles were cut to allow A1-wZy ANOVA and a Fiher port hoe test were iged to determie free oflow.and xdie (09%)wies perfirsed irto the acet aa presure of 200 rtm Hg This cleared the intncular dye fmme the aroup ditereaces A generl lineur mod of MANOVA was ued o ahne the CBF verais HP curve drta.Chanpes in CHF with HP and cereeral visculalure,lening ony exinn ascular dye te had craked the dffrrentol imerxctive effect of ClF with BP were sssrssed the BBH.Evans blue dye likely corjugaes to plasrta albumin,and Rerression anthus (usin吧the least squsres对dand devintine合 udes have sho4 n that a direct尾lationship oocurs betwcer血e enterion)ond the assessnent of the Pearson cocrelation coellcien. extravasaloe of dye and albumi during canditions of voue]were used to deoemmine whether sigrilcan.relatonships evinl between pinmrters Resuits wrre comsilered diffirrm Measurement of Pressure-Dependent Constriction Pc005.D台presertod as the mean+S乐mGmn us【cpiesent山e rursber of【ats used in the expenmem. in Middle Cerebral Arteries The technique used to mesare pressure-dependent oonstnction and Results pressure-independert tone in the middle cerebral arteres (MCAs) has bemn described in ather publictions A segment of the Hypertension and Mortality in Dahl-SS Rats MCA spirming the thinlis fissire of the hrain wars excised and The onset of deatch started when Dahl-SS rats were fed 8.7% mounied on a pipetie in a pressure inyogragh.The lumen and NaCl for periods >2.5 weeks,and 50%mortality was exterioe of te artery were tiled and suffased with oxygenated (95% ohserved alter 4.5 wecks of'fioding (Figure 1)Refore death O%CC)Krehs'solution,With the use of videomicropythe lumen diameler wars meaoured at x 322 magre fication Chrpes in of the animals exhibited scrcures.The symplms ob- lumen damneter I seoond to 4 mimtes afler the ipplicaliun of a scrved were the thythmic,abrupt movements of the head in pressure step of 100 mm Hz,subsequert to a 6-minute equilibeation an up-and-down mocion often associated with a laceral de- 国0 mm Ha pe5se,wcee)same5正eofp国e5 sure-depen3cet flection or repetitive forearm flexion on the left or right side constriction (myogrnie respone)Press-re-independken lone (hasgl The balance (30%)of the animals exhibited severe lethargy. seoond amer MCA pressunzation to 100 mm Hg (betore the signi Such animals were poorly groomed and remained huddled icant ergagemert of pressuredeperdent constnctionl versus the with urine-soaked bedding srrounding the posterior of the animal (indiciting that the rat had not moved for a consider- ilidipine国Cr“-fiee2 5 mmolL EGTA Krebs'3lik) able time).Lethargy was not due to puralysis since the animals could he influenced to move The rats often devel- Measurement of CBF Autoregulation oped a persisten penile erection (only male rats were stud- The laser-Doppler tochniques used to measure CBF are described elsewhere.Kelative CBF (rCHF)was measured in the pertusion iod).suggesting aulonomic dysfunction.Forclimb or hind dnman of the MCA.Blood Pacch.pH.HCO.ind O.salumton were limb paralysis was rare (1/20 rats fed 8.%%Nacl for >4 meirared ficmn a lemonl ireral bloed simple.Norepinephnne weeks)Dahl-SS mts fed normal (0%)Natl for 10 weeks (which cumnot constrict the cercbrovascuat.re')was efuased into the remained asympcomatic and healthy. rats (viz the temoral vein)to slowhy raise the BP (measured through In rats.a systolic BP of 150 mm Hg is considered the a tiertond catheter)The CBF sigral from the lser-Doppler probe aed the syechronied femon BP seadings were ored as dgilal threshold foc hypertension.Maximal systolic BPs were dala The CBF present al a given mean arteral BP [MAP]wis established al 00 mm Hg afler 3 wecks of'highsalt normalized to the CHF present at MAP of 100 mm Hg to gie the feeding (Figure 2)Dahl-SS rats fed 0.7%NaC]for 9
ysis, severe lethargy; signs are described in greater detail in Results). The systolic BP was measured by a tail cuff compression method (model 59, IITC). Examination of Brains The brains of 47 Dahl-SS rats exhibiting behavioral abnormalities were fixed in 10% PO4-buffered formalin. Forty brains were sectioned in 1-mm-thick coronal sections with the use of a jig (REM- 4000C, ASI Instruments) and examined by microscope for cerebral hemorrhages. Seven brains were imbedded in paraffin and histologically sectioned. The 5-m-thick sections were stained with cresyl violet blue or hematoxylin and eosin or assessed for the presence of glial fibrillary acidic protein (which accumulates in astrocytes after ischemic damage9) and examined for ischemic alterations and the presence of hemorrhage. The unfixed brains of 6 rats exhibiting abnormal behavioral symptoms were sliced with the use of a jig (REM-4000C) and assessed for ischemic lesions with a tetrazolium red technique.10 Tetrazolium red is taken up by the brain cells, and dehydrogenases convert this compound to an impermeable red dye that remains within the cells. Ischemic damage of the brain inactivates cellular dehydrogenase, leading to a failure in staining. Measurements of Plasma Extravasation Across the BBB Rats were anesthetized with sodium pentobarbital (65 mg/kg IP), and Evans blue dye (30 mg/mL of 0.9% saline) was infused (30 mg dye per kilogram) into the femoral vein. After 12 minutes, the chest cavity was opened, and a polyethylene catheter (PE-50) was inserted into the aorta and tied. The right and left ventricles were cut to allow free outflow, and saline (0.9%) was perfused into the aorta at a pressure of 200 mm Hg. This cleared the intravascular dye from the cerebral vasculature, leaving only extravascular dye that had crossed the BBB. Evans blue dye likely conjugates to plasma albumin, and studies have shown that a direct relationship occurs between the extravasation of dye and albumin during conditions of inflammation.11 Measurement of Pressure-Dependent Constriction in Middle Cerebral Arteries The technique used to measure pressure-dependent constriction and pressure-independent tone in the middle cerebral arteries (MCAs) has been described in other publications.7,12–14 A segment of the MCA spanning the rhinalis fissure of the brain was excised and mounted on a pipette in a pressure myograph. The lumen and exterior of the artery were filled and suffused with oxygenated (95% O2/5% CO2) Krebs’ solution. With the use of videomicroscopy, the lumen diameter was measured at 322 magnification. Changes in lumen diameter 1 second to 4 minutes after the application of a pressure step of 100 mm Hg, subsequent to a 6-minute equilibration at 0 mm Hg pressure, were used as a measure of pressure-dependent constriction (myogenic response). Pressure-independent tone (basal tone) was measured as the difference in lumen diameter present at 1 second after MCA pressurization to 100 mm Hg (before the significant engagement of pressure-dependent constriction) versus the diameter present at 100 mm Hg after maximal dilation (3 mol/L nifedipine or Ca2-free/2.5 mmol/L EGTA Krebs’ solution). Measurement of CBF Autoregulation The laser-Doppler techniques used to measure CBF are described elsewhere.12 Relative CBF (rCBF) was measured in the perfusion domain of the MCA. Blood PaCO2, pH, HCO3, and O2 saturation were measured from a femoral arterial blood sample. Norepinephrine (which cannot constrict the cerebrovasculature1) was infused into the rats (via the femoral vein) to slowly raise the BP (measured through a femoral catheter). The CBF signals from the laser-Doppler probe and the synchronized femoral BP readings were stored as digital data. The CBF present at a given mean arterial BP (MAP) was normalized to the CBF present at a MAP of 100 mm Hg to give the rCBF value [ie, rCBF at a given MAP(flux at MAP/flux at MAP of 100 mm Hg)]. rCBF was then plotted against MAP. Statistical Analysis A 1-way ANOVA and a Fisher post hoc test were used to determine group differences. A general linear model of MANOVA was used to analyze the CBF versus BP curve data. Changes in CBF with BP and the differential interactive effect of CBF with BP were assessed. Regression analysis (using the least squares standard deviation fit criterion) and the assessment of the Pearson correlation coefficient (r value) were used to determine whether significant relationships existed between parameters. Results were considered different at P0.05. Data are represented as the meanSE measurement; n values represent the number of rats used in the experiment. Results Hypertension and Mortality in Dahl-SS Rats The onset of death started when Dahl-SS rats were fed 8.7% NaCl for periods 2.5 weeks, and 50% mortality was observed after 4.5 weeks of feeding (Figure 1). Before death, 70% of the animals exhibited seizures. The symptoms observed were the rhythmic, abrupt movements of the head in an up-and-down motion often associated with a lateral deflection or repetitive forearm flexion on the left or right side. The balance (30%) of the animals exhibited severe lethargy. Such animals were poorly groomed and remained huddled with urine-soaked bedding surrounding the posterior of the animal (indicating that the rat had not moved for a considerable time). Lethargy was not due to paralysis since the animals could be influenced to move. The rats often developed a persistent penile erection (only male rats were studied), suggesting autonomic dysfunction. Forelimb or hindlimb paralysis was rare (1/20 rats fed 8.7% NaCl for 4 weeks). Dahl-SS rats fed normal (0.7%) NaCl for 10 weeks remained asymptomatic and healthy. In rats, a systolic BP of 150 mm Hg is considered the threshold for hypertension. Maximal systolic BPs were established at 200 mm Hg after 3 weeks of high-salt feeding (Figure 2). Dahl-SS rats fed 0.7% NaCl for 9 Figure 1. Mortality profile of Dahl-SS rats fed an 8.7% NaCl diet from weaning (5 weeks of age). Dahl-SS fed normal NaCl (0.7%) (n10) remained healthy for the duration of the experiment (14 weeks of age). Smeda and Payne Cerebrovascular Function in Dahl-SS Rats 1485
1486 Stroke June 2003 Dahl-55 Rats ,&%uC白Ud 。07%4CDt Weeks on Dint Figure 2.Systolie BP profie of Dahl-83 rats fod high-Nacl (d./)and norma-NsCl dets.Dahl-SS rats fed high salt after wcaning 5 wocks of agci chbitod n rapid incroase in BF, whilc those fed 0.756 Nacl for 9 wocks cxhibted borderine hyperlensan Irats fed 87%NsCl:n-10 at 1.2 weskx:n-8 at 3 weekx:n-7 al 4 weeks;n=4 at 5 weak;rats fed 0.7%NaCe n=5 weeks exhihited borderline hypertension levels (systolic BPs near 130 mm Hg) Cerebral lesions in Dahl-SS Rats The brains of 47 Dahl SS rats exhibiting stroke-like symp Frg3Er8bh时y中88 ion in the brain0时a toms were studad.Telrazolium rod slaining (n-6)and Dahl S5 rat cxhibting sctzus.Dahl SS rats with bchaworal histological examination (using cresyl violet blu or hema oH4 f Fvrts hh.n小y件Amw妇相-cnleredd ares线ar- taxylin and eosin staining technigues or the asssesssment of'the rounding the extravasated dye comespond to areas of odema. presenxce of glial fibrillay acidi:protein n-7)failed to Ceretral bochemia was absent,and hemomhage rarely oocumed detect ischemic damage.The brairs of 7 of 47 rats exhibited 7/47 rats).On the hasis af brain pathelgy,the dys- functions observed best represent the occurrence of hyperten- iniraccrebral hemonhaage in the cerebrum.I rat bad hemor- thage in the cerebrum and brain stem.The brains of 6 rats were assessed by the Evans blue dye technique.Five rats Alterations in Cerehrovascular showed multiple sites of Evans blue dye extravasation.and I Pressure-Dependent Constriction in Dahl-SS Rats brain showed a diffuse extravasation of dye.Figure 3 repre- Asymptomatic Duhl-SS rals fod 8.7%NaCl for 1 to 7 wocks sents the typica apperrance ot'the brain fiom one ot'these (aged6tol2 ecks)had MCAs th山hite时n age-relalod rats.We concluded that the stroke-like bcbaviocal dysfunc- decline in their abulity to elicit pressure-dependent constric tions (seizures,head and forelimb repetitive twitching beha tion (Figure 4A and Table 1:PDC.constriction obeerved ior,forelimb and hindlimb paralysis,and severe lethargy) fiom 1 sccund t 4 minules aller a pessure step ol obeerved in Dahl-SS rats were more consistent with the 100 mm Hgl.Because of'the attemation of'pressure- presence of hypertensive encephalopathy than true stroke dependent cocriction,the rals maintained progressively (discussed later).Hypertensive ercephuloputhy is a cocdition larger MCA lumen diameters with age at 100 mm Hg promoded by BBB disruption and cerebral edema secondary pressures (Figure 4B)This feature was particularly pro to hypertension.which can occur in the ahsence of cerehral nounced in rats with hypertensive cnecphaluputhy (scc dula ischemic or hemorrhagic lesions (true stroke Edema for 4 minutes at 100 mm Hg Table 1).As shown in Figure 5 formation can be observed in the brain shon in Figure 3 as and Table 1,Dahl-SS rats fed 0.7%NaCl for 9 weeks (aged light-colored areas surrounding the Evans blue extravasation. up to 14 weeks)had MCAs that maintained robust constric We also obeerved that Dahl SS rats exhibiting hypertersive tion to a prexsure step of 100 mm lg. encephalopathy have brains with higher tissue ater contents Dahl-SS rats with hypertensive encephalopathy (and no than those in asymptomatic,younger Dabl-Ss rats (G.W. cerebral hemorrhage)had MCAs with attenused presure- Pmyne,MSc,unpublished dma,2002). dependent constriction compared with asympomatic rats fed
weeks exhibited borderline hypertension levels (systolic BPs near 150 mm Hg). Cerebral Lesions in Dahl-SS Rats The brains of 47 Dahl-SS rats exhibiting stroke-like symptoms were studied. Tetrazolium red staining (n6) and histological examination (using cresyl violet blue or hematoxylin and eosin staining techniques or the assessment of the presence of glial fibrillary acidic protein9; n7) failed to detect ischemic damage. The brains of 7 of 47 rats exhibited intracerebral hemorrhage in the cerebrum; 1 rat had hemorrhage in the cerebrum and brain stem. The brains of 6 rats were assessed by the Evans blue dye technique.11 Five rats showed multiple sites of Evans blue dye extravasation, and 1 brain showed a diffuse extravasation of dye. Figure 3 represents the typical appearance of the brain from one of these rats. We concluded that the stroke-like behavioral dysfunctions (seizures, head and forelimb repetitive twitching behavior, forelimb and hindlimb paralysis, and severe lethargy) observed in Dahl-SS rats were more consistent with the presence of hypertensive encephalopathy than true stroke (discussed later). Hypertensive encephalopathy is a condition promoted by BBB disruption and cerebral edema secondary to hypertension, which can occur in the absence of cerebral ischemic or hemorrhagic lesions (true stroke).15,16 Edema formation can be observed in the brain shown in Figure 3 as light-colored areas surrounding the Evans blue extravasation. We also observed that Dahl-SS rats exhibiting hypertensive encephalopathy have brains with higher tissue water contents than those in asymptomatic, younger Dahl-SS rats (G.W. Payne, MSc, unpublished data, 2002). Alterations in Cerebrovascular Pressure-Dependent Constriction in Dahl-SS Rats Asymptomatic Dahl-SS rats fed 8.7% NaCl for 1 to 7 weeks (aged 6 to 12 weeks) had MCAs that exhibited an age-related decline in their ability to elicit pressure-dependent constriction (Figure 4A and Table 1; PDC, constriction observed from 1 second to 4 minutes after a pressure step of 100 mm Hg). Because of the attenuation of pressuredependent constriction, the rats maintained progressively larger MCA lumen diameters with age at 100 mm Hg pressures (Figure 4B). This feature was particularly pronounced in rats with hypertensive encephalopathy (see data for 4 minutes at 100 mm Hg, Table 1). As shown in Figure 5 and Table 1, Dahl-SS rats fed 0.7% NaCl for 9 weeks (aged up to 14 weeks) had MCAs that maintained robust constriction to a pressure step of 100 mm Hg. Dahl-SS rats with hypertensive encephalopathy (and no cerebral hemorrhage) had MCAs with attenuated pressuredependent constriction compared with asymptomatic rats fed Figure 2. Systolic BP profile of Dahl-SS rats fed high-NaCl (8.7%) and normal-NaCl (0.7%) diets. Dahl-SS rats fed high salt after weaning (5 weeks of age) exhibited a rapid increase in BP, while those fed 0.7% NaCl for 9 weeks exhibited borderline hypertension (rats fed 8.7% NaCl: n10 at 1, 2 weeks; n8 at 3 weeks; n7 at 4 weeks; n4 at 5 weeks; rats fed 0.7% NaCl: n5). Figure 3. Evans blue dye extravasation in the brain of a Dahl-SS rat exhibiting seizures. Dahl-SS rats with behavioral abnormalities exhibited BBB disruption and the extravascular movement of Evans blue dye (arrows). Light-colored areas surrounding the extravasated dye correspond to areas of edema. Cerebral ischemia was absent, and hemorrhage rarely occurred (7/47 rats). On the basis of brain pathology, the behavioral dysfunctions observed best represent the occurrence of hypertensive encephalopathy. 1486 Stroke June 2003
Smeda and Payne Cerebrovascular Functiun in Dahl-SS Rats 1467 A Figure 4.Age-related attenuation of 10 11 12 10 11 12 e4的in MCA&al helthy asyrp- tomstic Dshl-SS rats fed 8.7%NaCl trom 鳞eaning weeks of ap脉A,Lumen diam- 2on1 ictian个nes印onse to a pres- sure step of 100 mm Hg.As shewn in B.a decrease in the of the MCAs to constrict to a prossure stop of 100 mm Hg resubed in the maimonance of a larger MCA luen dismele ist 100 mm Hal wih g An age-elsted decine in presssun件 dependent constriction was not obecrved in MCA xmpled fnm Dahl-S8 rata fad 0.7%NaCl ke 9 wanks (14 weeks af ag see Figure bl.In A,peroert MCA pressure dependemt constrction=b.boxage waka-7.3:n=20,f-0.761,P-c0.001. in B,MCA lmen demeter um=68/×35tw6w+阳念-20. -0702,P0.01 high salt fi 1 k 3 wecks or rals fod low salL Pressure- in rals with ccrebtal hemuuluge.These prediclod values were dependenl constriction was abolished in the MCAs of grer(P<005)than the actual pressure-deperent constric- Dahl-SS mts with cerehral hemorrhage (Figure s and Table tion responses measured in rats with hyperensive encepha- 1).The average age of mts exhihiting hypertensive enceph- lopnhy (-113+38%)or cerebral hemorrhage (-22 alopauthy and cerehral bemocrhage was 9.30.I and 10.30.I 2.1%).We also selected age-matched asymplomatic Dal SS ees,respectively(fed high salt for4.3±0.】and53±0.l rats fed high salt and compared them with the rats with wecks,respedively)We allempled to determin whether the rypertensive enccphalo减ly里d crebeal hemoufupe.he attenualed pressure-dependent constriction in the MCAs chosen asymplomatic rals had group ages of 9.30.]wecks could be accounted for by the nge-related decline in this (n=7)nnd 10.30.3 weeks (n=5]and exhihited function In Figure 4A.asymtomatic Dahl-SS mts fed high -266±01%and-1S8士0%MCA pressure-dependen salt exhibited the following relationship between pressure constriction respanses,respectively,whuich were greater dependent consstriction and age:percent pressure-dependent (P0.05)than those in rats with hypertensive encephalopathy GIk0-5.86×e(wek)-T9.3r-0.761,Pc0.001. (-11.3上3.8%)ot cercbral hemotti(-2.2±2.1% n-20).On the basis of age,the latler equation prodiclod a Despie the abeence of pressure-dependent cunstriction -24%pressure-dependent constriction response in the rats with cerehml hemorrhage maincained substantinl MCA MCAs of raes with hypertensive encephalopathy and -19% pressure-independent tone This is demonstrated hy the high TABLE 1.AHerations in MCA Lumen Diameter in Response to a 100 mm Hg Pressure Step L山Ten dameler of MCAs.an PrESS Sxp Ampltude of Detary g 1 Scand al 4NU8减 为10n物+ POC 险D% gn Diot Swhs 100 mn Ho 100 mn Hg Maximol Vasociaton T m A 8.7 103 Aoymplenate 10+49 11a+58 3m+57 713+59 79+28 47 3u5 feymplertalc 161±91 13±45 1周±101 501±97 185±79 37 507 6 Acymptonalc 72±B1 151±55 20笛±29 22.1±7.6 340±84 D 8.7 43+01 Hypertersiv 12+51 181+4.1 190+41 205+71 103+55 2p12o E 87 5.3±0.I Irocercbral 149二165 145113 199二45 40±40 3.4上122 nNt为 0.7 W也c 10上145 127±9.1 213±86 6闪8±98 2习6±8了 <D.C5 P<0.05 物nitcart P:005 05 E第AB.0F A w C D.E riffn A4我G,QE BnC E Datwren groups U%0 UV C U.E C野AU FsC D F vsC,D.E EBA且.D CC pregsare-dpertrmtw1n4wel1T,ar Independot tore,dmoree In luren daneer bebuoon mdnal vocodlktion (wth 3 un af nfod pin?)and the dlamoter af I socond arter prssutzdot to 1001n均 S业水gO%A图post hoc test
high salt for 1 to 3 weeks or rats fed low salt. Pressuredependent constriction was abolished in the MCAs of Dahl-SS rats with cerebral hemorrhage (Figure 5 and Table 1). The average age of rats exhibiting hypertensive encephalopathy and cerebral hemorrhage was 9.30.1 and 10.30.1 weeks, respectively (fed high salt for 4.30.1 and 5.30.1 weeks, respectively). We attempted to determine whether the attenuated pressure-dependent constriction in the MCAs could be accounted for by the age-related decline in this function. In Figure 4A, asymptomatic Dahl-SS rats fed high salt exhibited the following relationship between pressuredependent constriction and age: percent pressure-dependent constriction5.86age (weeks)79.3 (r0.761, P0.001; n20). On the basis of age, the latter equation predicted a 24.8% pressure-dependent constriction response in the MCAs of rats with hypertensive encephalopathy and 19% in rats with cerebral hemorrhage. These predicted values were greater (P0.05) than the actual pressure-dependent constriction responses measured in rats with hypertensive encephalopathy (11.33.8%) or cerebral hemorrhage (2.2 2.1%). We also selected age-matched asymptomatic Dahl-SS rats fed high salt and compared them with the rats with hypertensive encephalopathy and cerebral hemorrhage. The chosen asymptomatic rats had group ages of 9.30.1 weeks (n7) and 10.30.3 weeks (n5) and exhibited 26.60.1% and 15.80.3% MCA pressure-dependent constriction responses, respectively, which were greater (P0.05) than those in rats with hypertensive encephalopathy (11.33.8%) or cerebral hemorrhage (2.22.1%). Despite the absence of pressure-dependent constriction, rats with cerebral hemorrhage maintained substantial MCA pressure-independent tone. This is demonstrated by the high Figure 4. Age-related attenuation of pressure-dependent constriction (myogenic response) in MCAs of healthy asymptomatic Dahl-SS rats fed 8.7% NaCl from weaning (5 weeks of age). A, Lumen diameter constriction (%) in response to a pressure step of 100 mm Hg. As shown in B, a decrease in the ability of the MCAs to constrict to a pressure step of 100 mm Hg resulted in the maintenance of a larger MCA lumen diameter (at 100 mm Hg) with age. An age-related decline in pressuredependent constriction was not observed in MCAs sampled from Dahl-SS rats fed 0.7% NaCl for 9 weeks (14 weeks of age; see Figure 5). In A, percent MCA pressuredependent constriction5.86age (weeks)79.3; n20, r0.761, P0.001. In B, MCA lumen diameter (m)6.97age (weeks)69.3; n20, r0.702, P0.01. TABLE 1. Alterations in MCA Lumen Diameter in Response to a 100 mm Hg Pressure Step Lumen diameter of MCAs, m Pressure Step Amplitude of Dietary NaCl, % Weeks on Diet n Status 1 Second at 100 mm Hg 4 Minutes at 100 mm Hg At 100 mm Hg Maximal Vasodilation PDC, m PIT, m A 8.7 1 to 3 7 Asymptomatic 1924.9 1185.8 2005.7 73.35.9 7.92.8 B 8.7 3 to 5 7 Asymptomatic 1819.1 1314.5 19810.1 50.19.7 16.57.9 C 8.7 5 to 7 6 Asymptomatic 1728.1 1505.5 2062.9 22.17.6 34.08.4 D 8.7 4.30.1 6 Hypertensive encephalopathic 1825.1 1619.1 1924.1 20.57.1 10.35.5 E 8.7 5.30.1 3 Intracerebral hemorrhage 14916.5 14513 1994.5 4.04.0 49.412.2 F 0.7 9 5 Asymptomatic 19014.5 1279.1 2138.6 63.89.8 23.68.7 P0.05 P0.05 No significant differences between groups P0.05 P0.05 E vs A, B, D, F A vs C, D, E A vs B, C, D, E B vs C, E B vs D B vs C, D, E C vs A, D F vs C, D F vs C, D, E E vs A, B, D PDC indicates pressure-dependent constriction, difference in lumen diameter between 1 second and 4 minutes after pressurization to 100 mm Hg; PIT, pressure independent tone, difference in lumen diameter between maximal vasodilation (with 3 m of nifedipine) and the diameter at 1 second after pressurization to 100 mm Hg. Statistics: ANOVA plus Fisher post hoc test. Smeda and Payne Cerebrovascular Function in Dahl-SS Rats 1487
148器 Stroke 1une2003 时E.T%4白 5mpmn南 40 HeC 「的n0t,15745g 15 Aymplomaic Dat-SS Fan s B.T%MCI D et kor 'vt. 30 。.%C口女hn 26 20 16 10 05 6 5101251481间10200225702面 0h-55R3落 Mean Arterial Preessura (mmHg) A■sTEm G.☐TT Figun 6.Atarations in CAF aiorg.lion in asymplomatis Dah-SS rats fed 5.7%NaCi for varying durations.Belore the G☐+0tawm9te mad3oan92n J年a对Pndh8a网snc8 of a distinct inllection poin请U方 Figure 6.Pressure-dependent constriction (myogenic responsel a8ht5 arved in同s fed high明or1Cr2wwek,ep- sonting an upper EP lmt to CBF rogulation.Such an altoration is consisoent with a lss in CBF a.toregulation.Dahl SS rats fad hyperensive aneephalopetry or hamarhagic strac.The per- cent decrease in lumen dameter after the applcstion of a pres- 07%N米o9wn+ks mantain4dGBf将orecutstion (Ta sure stap cr100mmHg图shoan.Asmptcm的cDhl8srs 2.The CBF sutoregulatory ourve represenang this group was ed 8.756 NaCl for 1 to 3 wocks (n-7:agod 6.9=0.2 wocls)3 identical to that of rats fed high sat tor 2 wecks.These cata o5wwe*8们”行8yw时83士0.1ek.nd51e7w88门-6： were removed fom the fgura to cacrease cengestion.Fach g时112+03ws8ng5ww8of3的hd curve is signiticandy Pe0.0b)dferert from all other curves in MCAs that progreasiely devdopod an attenuated abiity to dicit 止s3 ute levels ower common BP5 nd in temm形ct宁 imerac- prextuire-depandlent.Presire-dlependert- Tve efect of CBF with BP P<0.001 in al cmea (MANOVA an 6nh线hrtr Afeniied in ralswih Pr/nM8神iphA- n-6 rats in eoch group. lopatny [+Hyp.Enoeph.;n=6;aged 9.3-0.1 wocks]and was abolshed in rats with cerebral hemomhage l+Hem.:n=3;aped Alterations in CBF Autoregulation in 10.3=0.01 weeks).Dol-85 rets fed 0.7%NaCl diat for 9 waeks Dahl-SS Rats after weanng In=ai had MCAs that maintained pressure- C月F焦repulation was assessad in asvmptomatie门l-SS nstnction.P20.05.A vs B.C.D,E:U vs C.D.E: FvC.D.ELANOVA and Fisher poat hoe testl. rats fed 8.7%NaCl for I to 3 weeks or 0.7%NaCl for 9 weeks afler weuning.Through the control of tespiralory rles and level of constriction present at I second after equilihration to the administraticn of oxygen,blood Pa (402 mm Hg) 100 mm Hg in compurison to maximal dilation (see pressure- pH(7.0±0.01，andH0O1aels25.3±08 mmol/L}were independent tone data in Table 1).This suggested that nomal,and oxygen sanumtion was alwmys996%(n=25 cerebral hemorhage is associated with the development of ruts). intrinsie tone in the isolated MCAs Dahl-SS rats fed 7%NaCl for I week or 0.T%NaCl for The MCAs of asympomatic rats and rats with hyperten 9 weeks autoregulated blood flow to an upper mean BP limit sive crepislopall or ccrctrral Icmnonlge led comgarsblc af168上63204上I2mmH北.tsn少(Fgue6J lumen diameters under conditions of maximum dilation (see Table 2)One of 6 rats fed 8.7%NaCl for 2 weeks and 4 of Maximal Vasodilation at 100 mm Hg pressure duta in Tabl 6 rats fod the dict for 3 wocks exhibited linear increases in 1)The ability of 3 jmol/I.nifedipine to produce maximal CHF with HP and the ahsence of an upper BP limit to CBF vasodilstion was validated.After administration of nifed regulaticn (Figure 6 and Table 2).These characteristics are pine,no further vasodilation oeeurred in Ca-free consistent with the loss of'CBF auroregulation Dahl-S5 rats 25 mmolL EGTA Krebs'solution (mean percent difference fed 0.7%NaCl for 9 weeks maintained CBF sutoregulation in lumen diamda.miliedipine versus ETA Kiels'sulution. (Table 2).The CBF autoregullory eve tepresenling this asymptomatic rats,0.441.17%,n=5;rats with hypertensive group was identical to that of rats fed high salt for 2 weeks enoephaloputhy.0.01-0.93%.n-5)We aisu obescrvod no and was removod from Figure 6 to docrcase congestion differences in dilatory sensitivry to nifedipine (ie compar- The relative change in CBF hetween BPs of %0 to bl ED values for dose-response curves from 10mol/L 120 mm Hg was comnparable between the groups of rats [subchreshold]to 3X10*mol/l.[maximal vasodilarion ] (Tahle 2.discussed later).The upper BP limit of regulation when MCAs sampled fiom aymptomatic Duhl SS rats shified to higher pressures with the duration af salt feeding in (ye信-5.6，-S)rats with lypertensive cnecpha Dahl-SS rats fied 8.7%NaCl and was remskubly high lopathy (n=5)or cerehral hemorrhage in=3)were compared 20412 mm lg)in rats fed low salt that exhihited border- (data not shown). line bypertension (appruximately 150 mm Hg)
level of constriction present at 1 second after equilibration to 100 mm Hg in comparison to maximal dilation (see pressureindependent tone data in Table 1). This suggested that cerebral hemorrhage is associated with the development of intrinsic tone in the isolated MCAs. The MCAs of asymptomatic rats and rats with hypertensive encephalopathy or cerebral hemorrhage had comparable lumen diameters under conditions of maximum dilation (see Maximal Vasodilation at 100 mm Hg pressure data in Table 1). The ability of 3 mol/L nifedipine to produce maximal vasodilation was validated. After administration of nifedipine, no further vasodilation occurred in Ca2-free/ 2.5 mmol/L EGTA Krebs’ solution (mean percent difference in lumen diameter, nifedipine versus EGTA Krebs’ solution: asymptomatic rats, 0.441.17%, n5; rats with hypertensive encephalopathy, 0.010.93%, n5). We also observed no differences in dilatory sensitivity to nifedipine (ie, comparable ED50 values for dose-response curves from 109 mol/L [subthreshold] to 3106 mol/L [maximal vasodilation]) when MCAs sampled from asymptomatic Dahl-SS rats (young, n5; old, n8) or rats with hypertensive encephalopathy (n5) or cerebral hemorrhage (n3) were compared (data not shown). Alterations in CBF Autoregulation in Dahl-SS Rats CBF autoregulation was assessed in asymptomatic Dahl-SS rats fed 8.7% NaCl for 1 to 3 weeks or 0.7% NaCl for 9 weeks after weaning. Through the control of respiratory rates and the administration of oxygen, blood PaCO2 (402 mm Hg), pH (7.400.01), and HCO3 levels (25.30.8 mmol/L) were normal, and oxygen saturation was always 99.6% (n25 rats). Dahl-SS rats fed 8.7% NaCl for 1 week or 0.7% NaCl for 9 weeks autoregulated blood flow to an upper mean BP limit of 1686 and 20412 mm Hg, respectively (Figure 6 and Table 2). One of 6 rats fed 8.7% NaCl for 2 weeks and 4 of 6 rats fed the diet for 3 weeks exhibited linear increases in CBF with BP and the absence of an upper BP limit to CBF regulation (Figure 6 and Table 2). These characteristics are consistent with the loss of CBF autoregulation. Dahl-SS rats fed 0.7% NaCl for 9 weeks maintained CBF autoregulation (Table 2). The CBF autoregulatory curve representing this group was identical to that of rats fed high salt for 2 weeks and was removed from Figure 6 to decrease congestion. The relative change in CBF between BPs of 90 to 120 mm Hg was comparable between the groups of rats (Table 2; discussed later). The upper BP limit of regulation shifted to higher pressures with the duration of salt feeding in Dahl-SS rats fed 8.7% NaCl and was remarkably high (20412 mm Hg) in rats fed low salt that exhibited borderline hypertension (approximately 150 mm Hg). Figure 5. Pressure-dependent constriction (myogenic response) in MCAs of Dahl-SS rats before and after the development of hypertensive encephalopathy or hemorrhagic stroke. The percent decrease in lumen diameter after the application of a pressure step of 100 mm Hg is shown. Asymptomatic Dahl-SS rats fed 8.7% NaCl for 1 to 3 weeks (n7; aged 6.90.2 weeks), 3 to 5 weeks (n7; aged 9.30.1 weeks), and 5 to 7 weeks (n6; aged 11.20.3 weeks) after weaning (5 weeks of age) had MCAs that progressively developed an attenuated ability to elicit pressure-dependent constriction. Pressure-dependent constriction was further attenuated in rats with hypertensive encephalopathy (Hyp. Enceph.; n6; aged 9.30.1 weeks) and was abolished in rats with cerebral hemorrhage (Hem.; n3; aged 10.30.01 weeks). Dahl-SS rats fed 0.7% NaCl diet for 9 weeks after weaning (n5) had MCAs that maintained pressuredependent constriction. P0.05, A vs B, C, D, E; B vs C, D, E; F vs C, D, E (ANOVA and Fisher post hoc test). Figure 6. Alterations in CBF autoregulation in asymptomatic Dahl-SS rats fed 8.7% NaCl for varying durations. Before the development of hypertensive encephalopathy, most rats (4/6) fed high salt for 3 weeks exhibited a linear relationship between CBF and BP and the absence of a distinct inflection point (such as that observed in rats fed high salt for 1 or 2 weeks), representing an upper BP limit to CBF regulation. Such an alteration is consistent with a loss in CBF autoregulation. Dahl-SS rats fed 0.7% NaCl for 9 weeks maintained CBF autoregulation (Table 2). The CBF autoregulatory curve representing this group was identical to that of rats fed high salt for 2 weeks. These data were removed from the figure to decrease congestion. Each curve is significantly (P0.05) different from all other curves in absolute levels over common BPs and in terms of the interactive effect of rCBF with BP (P0.001 in all cases) (MANOVA on curves); n6 rats in each group. 1488 Stroke June 2003
Smeda and Payne Cerebrovascular Function in Dahl-SS Rats I459 TABLE 2.Characteristics of CBF Automegulation in Asymptomatic Dahl-SS Rats Dietary Rats Ehitting 0wE即mtd a,% CBF Regulaton 特7女pa1 △/m &7 66 165上时 473三L17 67 2 5号 11±9 92±113片 &7 26 4 7.12±094 D.7 9 70 214±12 541±085 △83nm归n特中W打肉甲圈b解n作切力1■o×1了 The 2 of 6 rats edhitiing CEF aunoroguation tad upper BP In ts af 227 and 155 nm Hg. 930体tc源5形0%0式c0防形%a0标1水0.7% Nh0G性 We altempted to mesure CBF autoregulution in D:h-SS Thare was a lnear inerease in ICBF with BP and the ruls exhibing opertersive encephakpathy.Unfortustely. bxa动车per lim房u CBF uuloregulal知inmu时 these rats were highly suxceptibk to death within I hour of Dahl-SS fed high salt for 3 weeks This is consistent with anesthesia.This prevented us frum completing the experi- the ahsence of CHF autoregulation;however,the chanpes in ments.However,since mot asympomatic Duhl-S rats fed CBF with BP (ie,ArCBF/Amm Hg BP.Table 2)were 8.7%NaCl for 3 weeks kot ther ability to muoregulate CBF comparable to those presem in rats cupable of oregulating before hypertensive cnephlally o hemunhagic stroke CBF.We believe that higher ACBF/Amm Hg BP ratios (Figure 6 and Table 2).this function must likely remained wouldl huve occunod if CBF autoregultion wus loet under defective after hypereresive encephalopathy conditions of cerehrovascular vasodilation.However,if the loss of CBF autoregulation in Dahl-SS mats fed high salt was Discussion asociated with cerebrovascular vasococstriction,an increase Dahl-SS rats fed &Nacl exchihited srroke-like behavior in the slope of ArCBF/Amm Ig BP coud have been be.med (seizures,head and tarelimh repetitive twitching behavior lo a poin whae il win cumrgurabie to tiest olsavod in ials forelimb and hindlimb paralysis.severe lethargy)The brains capubk of uuloreulating CBF,despite the loes of autoregu- lation.An altemative possibelity also exists.Our experiments of these rats indicated plasma extravasation and edema in the cerehrumm.Ischemia was absert,ard the incidence of cerehral were limited hy the fact that the macimal mean BPs that could hemorrhage wus low (7/47).The rats best represerted a be achieved by the intravenous infision of norepinephrine modal of hyperensive encephalopathy s oppased to true into Dahl-SS mts were approximately 260 mm Hg If the uppa limit of CBF autoregulalion was 250 mn Hg.the strokke.In humins,hypertersive encephalopathy is produced inability to surpass this point wodld have produced a linear by BBB disruption The developenert of beain edema CHF versus BP curve with the absence of an upper HP limit secondary to hypertensicn applies pressure to the neural to autoregulation.This could hive resultod in the mistaken tissue and promotes neurological dysfunction.Hypertensive impression that no upper BP lim existed and that CBF enoephaleputhy is associated with confusion.stupor.and utoregulation was lost.In our view.this would require convulsions,symptoms similar to those observed in our ruts. unpreocdenod large shill in the upper BP limit of autoregu- and is distinguished from stroke in that it can occur in the lation far above that ohserved in any cther study involving ahsence of cerebral ischemia or hemorrhage. hypertensive rats.Although our experiments cannot disprove Myogerc dysfunctioes preceded the onset of hypertensive the latter scenario,we believe that it is the less plausible of enocphaloputhy.Pressure-dependemt constriction was alenu- the 2 hypotheses presemed ated in the MCAs of asymptomatic Dahl-SS rats in relation to The eerebrovascular alermions occumng in the Dal-55 the duration of high salt feeding.As a result of the loss of tats cthibit similtios and cuntransts lo the types of chanes pressure-dependent constriction,the MCAs mantain peogres present in stroke-peone spontaneously hypertensive rats sively larger en diameers at 100mm Ig pressure.In the (SHRSP)Unlike the SHRSP we have studied, presence ofhypertension,this alteration could increase down- Duhl-SS rats developed a lower incidence of cerebral hem stream flow and peessure to the microvasculature and facili orrhage.We believe that seizures observed in Dahl SS rats tate the formation of brain edema and hypertensive enceph were assoctkd with lperensive eneepulopalhy.In this alopathy.The MCAs of rats with hypertensive regind.the Dahl-S5 model and the presem sludy ae uque encephalopathy or cerebral hemorrhage exhibited a greater because tew studies have assessed cerebrovascular alteraticns atteraation of pressure-dependent constriction than thal preceding and following the development of hypertensive which could be accounted for by an age-relaked decline in this encephaloputhy.Both SHRSP and Dahl-SS rats lose their function This added dysfunction could be promoed by ability to autoregulate CBF and develop defects i pressure secondary aherations after hypertensive encephalopathy or deperdert orsiridn within the MCAs befre the developmet cerclal hemmonhage.Alemnalicly.loypertensive cnepha of hemomhagic ske or hypertersive encephalopachy lopathry and ccrebral hemonhape could be farored in rats However,unlike Dabl-SS rats the loss of autcregulaticn in with cerebrovasculatures that exchibit a low capacity to elicit SHRSP is isociaed with incre in△CBF△nm Hg BP.e pressure-dependent cortriction. This type of chang would facilitale overperfusian to a grester
We attempted to measure CBF autoregulation in Dahl-SS rats exhibiting hypertensive encephalopathy. Unfortunately, these rats were highly susceptible to death within 1 hour of anesthesia. This prevented us from completing the experiments. However, since most asymptomatic Dahl-SS rats fed 8.7% NaCl for 3 weeks lost their ability to autoregulate CBF before hypertensive encephalopathy or hemorrhagic stroke (Figure 6 and Table 2), this function most likely remained defective after hypertensive encephalopathy. Discussion Dahl-SS rats fed 8.7% NaCl exhibited stroke-like behavior (seizures, head and forelimb repetitive twitching behavior, forelimb and hindlimb paralysis, severe lethargy). The brains of these rats indicated plasma extravasation and edema in the cerebrum. Ischemia was absent, and the incidence of cerebral hemorrhage was low (7/47). The rats best represented a model of hypertensive encephalopathy as opposed to true stroke. In humans, hypertensive encephalopathy is produced by BBB disruption.16 The development of brain edema secondary to hypertension applies pressure to the neural tissue and promotes neurological dysfunction. Hypertensive encephalopathy is associated with confusion, stupor, and convulsions, symptoms similar to those observed in our rats, and is distinguished from stroke in that it can occur in the absence of cerebral ischemia or hemorrhage.16 Myogenic dysfunctions preceded the onset of hypertensive encephalopathy. Pressure-dependent constriction was attenuated in the MCAs of asymptomatic Dahl-SS rats in relation to the duration of high-salt feeding. As a result of the loss of pressure-dependent constriction, the MCAs maintain progressively larger lumen diameters at 100 mm Hg pressure. In the presence of hypertension, this alteration could increase downstream flow and pressure to the microvasculature and facilitate the formation of brain edema and hypertensive encephalopathy. The MCAs of rats with hypertensive encephalopathy or cerebral hemorrhage exhibited a greater attenuation of pressure-dependent constriction than that which could be accounted for by an age-related decline in this function. This added dysfunction could be promoted by secondary alterations after hypertensive encephalopathy or cerebral hemorrhage. Alternatively, hypertensive encephalopathy and cerebral hemorrhage could be favored in rats with cerebrovasculatures that exhibit a low capacity to elicit pressure-dependent constriction. There was a linear increase in rCBF with BP and the absence of an upper limit to CBF autoregulation in most Dahl-SS fed high salt for 3 weeks. This is consistent with the absence of CBF autoregulation; however, the changes in rCBF with BP (ie, rCBF/mm Hg BP; Table 2) were comparable to those present in rats capable of autoregulating CBF. We believe that higher CBF/mm Hg BP ratios would have occurred if CBF autoregulation was lost under conditions of cerebrovascular vasodilation. However, if the loss of CBF autoregulation in Dahl-SS rats fed high salt was associated with cerebrovascular vasoconstriction, an increase in the slope of rCBF/mm Hg BP could have been blunted to a point where it was comparable to that observed in rats capable of autoregulating CBF, despite the loss of autoregulation. An alternative possibility also exists. Our experiments were limited by the fact that the maximal mean BPs that could be achieved by the intravenous infusion of norepinephrine into Dahl-SS rats were approximately 260 mm Hg. If the upper limit of CBF autoregulation was 260 mm Hg, the inability to surpass this point would have produced a linear CBF versus BP curve with the absence of an upper BP limit to autoregulation. This could have resulted in the mistaken impression that no upper BP limit existed and that CBF autoregulation was lost. In our view, this would require an unprecedented large shift in the upper BP limit of autoregulation far above that observed in any other study involving hypertensive rats. Although our experiments cannot disprove the latter scenario, we believe that it is the less plausible of the 2 hypotheses presented. The cerebrovascular alterations occurring in the Dahl-SS rats exhibit similarities and contrasts to the types of changes present in stroke-prone spontaneously hypertensive rats (SHRSP).12,13,17 Unlike the SHRSP we have studied,17 Dahl-SS rats developed a lower incidence of cerebral hemorrhage. We believe that seizures observed in Dahl-SS rats were associated with hypertensive encephalopathy. In this regard, the Dahl-SS model and the present study are unique because few studies have assessed cerebrovascular alterations preceding and following the development of hypertensive encephalopathy. Both SHRSP and Dahl-SS rats lose their ability to autoregulate CBF and develop defects in pressuredependent constriction within the MCAs before the development of hemorrhagic stroke or hypertensive encephalopathy.12,13 However, unlike Dahl-SS rats, the loss of autoregulation in SHRSP is associated with an increase in CBF/mm Hg BP.12 This type of change would facilitate overperfusion to a greater TABLE 2. Characteristics of CBF Autoregulation in Asymptomatic Dahl-SS Rats Dietary NaCl, % Weeks on Diet n Rats Exhibiting CBF Regulation Upper BP Limit of CBF Regulation rCBF/mm Hg 8.7 1 6 6/6 1686† 4.731.17 8.7 2 6 5/6 1819 9.21.13‡ 8.7 3 6 2/6 NA* 7.120.94 0.7 9 7 7/7 20412 5.410.85 rCBF/mm Hg indicates change in relative CBF between BPs of 90 to 120 mm Hg (103 ). *The 2 of 6 rats exhibiting CBF autoregulation had upper BP limits of 227 and 185 mm Hg. Statistics (ANOVA): †P0.05 vs 0.7% NaCl diet; ‡P0.05 vs 8.7% NaCl diet for 1 week and 0.7% NaCl diet. Smeda and Payne Cerebrovascular Function in Dahl-SS Rats 1489
1490 Stroke June 2003 dgrc than il'(as prodictod in Dahl-S5 ns)reulation wa loe 4.Hill MA.2 u H Porenk s人cirirger GA,Dema MI.Swu虹a urder conditions of'cerebrascular vasoconstnction.This codld modunolatalasiun:C sgrling palbwerys urdcrlying rryuemic account for the higher incidence of cerebral hemorhage ob y/为edm1:17- served in SHRSP7 versus Duhl SS rats in the present stuy. T.Payne t.Sreda Is Cerehrosascilaralteutons in preoure and protein D-55 rats exhibiting seurs ard rypertersne crecphalope- kitne C-nodited ootitnctin in Dall sat-sstalive fts fopertrs. were abso highly susoplible lo death and surivod for orly 38位1355-161 B.Von Lunerogi N.Canargo M.Canpbell Jr.Mueller FB. a fiw days.It is poeesble fhul curebral hemocrtge might hive Tmemars PB.Seaky JE,Laragh JH Arpoteesin ll receptor onep- developed afier hypertensive enoephslopethy in Dahl-SS rats if aris debryx nomd daran:ard sruoe in xalt-kuaked Lhl il-wmalnve the animals survived for a longer time in the presence of .J0ew.192:199-57 象.Butrn D.Sihend.:n上s.Hy pucic-indwmric bran irjary xtinabica dia efiedne C书F auloregatio Rp#l19:12k11-11R Acknowledgments 10.Lusdy EF,Soli BS.Fenk RS.Lacy P5,Cortbo Dl,Zclesock GB. DyAlacy L Morphemetric ealemn ne hrain inencts is rats and Ih信study was suppored by p限rcrs空p funding trom the Heart and IL.UdaK,Takeuchi Y,ovt HZ Smple inetho时e4ue的d Reseech. mnhasood vecube perebuty.Prac Soc Er Mal Mnd IvilkI5: 384-1387. References 12.Smeda Van BN.King S.Stroke-prone sporrancoushy hyper L.hdam0.snd时发Tdwirgen L Cerheal aroegalnon J公ew城.19591719-1705 2.Heisad DD.Baurtbech GL.Cenebal vescuer chatges durine chronic nlcs1 n.Car Fhsol%anacof1为2，]0s2-554 4.sm.kg1m 71371-s75 ue0山48 ore rals.S0ab.200,31.7S1-759. 3.Edvinsson 1.Hardebo JE.Cumnon C.Iflgenoe of the cerebeevascular 15.Tarrak K.Sadeshirra 5.Fumniuch (il..Ladacna C.Iris DU,Heigad 号dck日natann on regeral ion,egilatant,and biod-tan 1X1),Eviderce tal diruption of the Hood-hrain bamer prooodes eetir farclin.Cha Found Spmt.17:56cB-15 peahctic nerve in园cd里rupy,CirealaNon9gi,5小- hr国n Arch四d 10T--152 192152938-45 5.Giahh MR.llDerhge IXG,W'shter GiF.locheder M.ILyeiz 1 Brain I7.Smce J5.Hemunba起k:dk+clprierl it apurla3ay与1 with rinod pire.Af Nevral 1993:52.391-400 1217-121w
degree than if (as predicted in Dahl-SS rats) regulation was lost under conditions of cerebrovascular vasoconstriction. This could account for the higher incidence of cerebral hemorrhage observed in SHRSP17 versus Dahl-SS rats in the present study. Dahl-SS rats exhibiting seizures and hypertensive encephalopathy were also highly susceptible to death and survived for only a few days. It is possible that cerebral hemorrhage might have developed after hypertensive encephalopathy in Dahl-SS rats if the animals survived for a longer time in the presence of defective CBF autoregulation. Acknowledgments This study was supported by partnership funding from the Heart and Stroke Foundation of Canada and the Canadian Institutes of Heath Research. References 1. Paulson OB, Strandgaard S, Edwinsson L. Cerebral autoregulation. Cerebrovasc Brain Metab Rev. 1990;2:161–192. 2. Heistad DD, Baumbach GL. Cerebral vascular changes during chronic hypertension: good guys and bad guys. J Hypertens. 1992;10(suppl 7):S71–S75. 3. Edvinsson L, Hardebo JE, Owman C. Influence of the cerebrovascular sympathetic innervation on regional flow, autoregulation, and blood-brain barrier function. Ciba Found Symp. 1978;56:69–95. 4. Mayhan WG, Werber AH, Heistad DD. Protection of cerebral vessels by sympathetic nerves and vascular hypertrophy. Circulation. 1987;75:I- 107–I-112. 5. Gabb MR, Hollerhage HG, Walter GF, Hocheder M, Haubitz I. Brain edema, autoregulation, and calcium antagonism: an experimental study with nimodipine. Adv Neurol. 1990;52:391–400. 6. Hill MA, Zou H, Potoenik SJ, Meininger GA, Davis MJ. Signal transduction in smooth muscle: invited review: arteriolar smooth muscle mechanotransduction: Ca2 signaling pathways underlying myogenic reactivity. J Appl Physiol. 2001;91:973–983. 7. Payne GW, Smeda JS. Cerebrovascular alterations in pressure and protein kinase C-mediated constriction in Dahl salt-sensitive rats. J Hypertens. 2002;20:1355–1363. 8. Von Lutterotti N, Camargo MJ, Campbell WG Jr, Mueller FB, Timmermans PB, Sealey JE, Laragh JH. Angiotensin II receptor antagonist delays renal damage and stroke in salt-loaded Dahl salt-sensitive rats. J Hypertens. 1992;10:949–957. 9. Burtrum D, Silverstein FS. Hypoxic-ischemic brain injury stimulates glial fibrillary acidic protein mRNA and protein expression in neonatal rats. Exp Neurol. 1994;126:112–118. 10. Lundy EF, Solik BS, Frank RS, Lacy PS, Combs DJ, Zelenock GB, D’Alecy LG. Morphometric evaluation of brain infarcts in rats and gerbils. J Pharmacol Methods. 1986;16:201–214. 11. Udaka K, Takeuchi Y, Movat HZ. Simple method for quantitation of enhanced vascular permeability. Proc Soc Exp Biol Med. 1970;133: 1384–1387. 12. Smeda JS, Van Vliet BN, King S. Stroke-prone spontaneously hypertensive rats lose their ability to auto-regulate cerebral blood flow. J Hypertens. 1999;17:1697–1705. 13. Smeda JS. Cerebral vascular changes associated with hemorrhagic stroke in hypertension. Can J Physiol Pharmacol. 1992;70:552–564. 14. Smeda JS, King S. Electromechanical alterations in the cerebrovasculature of stroke-prone rats. Stroke. 2000;31:751–758. 15. Tamaki K, Sadoshima S, Baumbach GL, Iadecola C, Reis DJ, Heistad DD. Evidence that disruption of the blood-brain barrier precedes reduction in cerebral blood flow in hypertensive encephalopathy. Hypertension. 1984;6:175–181. 16. Phillips SJ, Whisnant JP. Hypertension and the brain. Arch Intern Med. 1992;152:938–945. 17. Smeda JS. Hemorrhagic stroke development in spontaneously hypertensive rats fed a North American, Japanese-style diet. Stroke. 1989;20: 1212–1218. 1490 Stroke June 2003