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Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2010 Jul 21;31(2):476–485. doi: 10.1038/jcbfm.2010.110

The effects of poststroke captopril and losartan treatment on cerebral blood flow autoregulation in SHRsp with hemorrhagic stroke

John S Smeda 1,*, Noriko Daneshtalab 1
PMCID: PMC3049503  PMID: 20648036

Abstract

The ability of captopril and losartan treatment to restore cerebral blood flow (CBF) autoregulation after intracerebral hemorrhagic stroke (HS) was assessed in Kyoto–Wistar stroke-prone hypertensive rats (SHRsp). Laser Doppler techniques assessed CBF autoregulation in the middle cerebral artery (MCA) perfusion domain and a pressure myograph was used to measure pressure-dependent constriction (PDC) in isolated MCAs before and after stroke and after 13, 33, and 63 days of poststroke captopril or losartan treatment. The treatments did not lower blood pressure (BP) and equally suppressed plasma aldosterone after HS. The HS development was associated with the loss of CBF autoregulation, high CBF, increased CBF conductance to elevations in BP, and the loss of PDC in the MCAs. Both treatments restored these functions to prestroke levels within 13 days. The PDC and CBF autoregulation subsequently deteriorated after 63 days of captopril treatment while being maintained at prestroke levels over all durations of losartan treatment. The SHRsp subjected to 35 days of poststroke losartan treatment exhibited less blood–brain barrier (BBB) disruption and brain herniation than captopril-treated SHRsp. The superior ability of losartan to restore CBF autoregulation and myogenic function may have contributed to the more effective attenuation of cerebral damage after HS.

Keywords: aldosterone, angiotensin, blood–brain barrier, hypertension, middle cerebral artery, myogenic response

Introduction

Intracerebral hemorrhagic stroke (HS) development in stroke-prone hypertensive rats (SHRsp) is associated with the activation of the renin-angiotensin system (Camargo et al, 1991) and the development of cerebrovascular dysfunctions (Sironi et al, 2003, 2004). The loss of pressure-dependent constriction (PDC) within the MCAs of SHRsp precedes cerebral hemorrhage development in SHRsp (Smeda and King, 2000) and coincides with the loss of cerebral blood flow (CBF) autoregulation in the MCA perfusion domain (Daneshtalab and Smeda, 2010). In the absence of such regulation, elevations in blood pressure (BP) could produce cerebral over-perfusion and high microvascular pressures. This could enhance the probability of blood–brain barrier (BBB) disruption and cerebral hemorrhage formation. The continued presence of this dysfunction after HS would facilitate secondary hemorrhage formation and increase the risk of disability and death after HS (Neunhoeffer et al, 2010), whereas the restoration of these functions after HS could limit the progression of brain damage.

We observed that poststroke treatment of SHRsp with captopril (an angiotensin-converting enzyme inhibitor (ACEI) that blocks the formation of angiotensin II (AII)) retarded the onset of death after stroke in SHRsp (Smeda et al, 1999). Death (which occurred on average within 12 days) was attenuated, and all rats survived for >10 weeks following poststroke captopril treatment. Similar poststroke treatment with losartan (an AII type-1 receptor (AT-1R) blocker (ARB)), prevented death in all SHRsp for >14 weeks (the maximum duration of the study), suggesting that losartan may be even more effective than captopril in retarding poststroke mortality in our SHRsp (Smeda and McGuire, 2007). Neither captopril nor losartan treatment reduced the BP of poststroke SHRsp. Both treatments proved to be superior in preventing death after stroke when compared with more effective antihypertensive treatments (hydralazine) (Smeda et al, 1999). These studies suggested that the presence of an active renin-angiotensin system may facilitate the onset of death after HS in SHRsp.

The current study assessed the comparative ability of captopril and losartan treatment to restore CBF autoregulation after HS. Laser Doppler techniques were used to measure alterations in CBF in response to elevations in BP within SHRsp before and after stroke and after 13, 33, and 63 days of poststroke captopril and losartan treatment. The capacity to restore CBF autoregulation within the MCA perfusion domain in vivo was related to the ability of isolated MCAs to elicit PDC over the same time points. The BBB disruption was assessed by examining the extravasation of albumin-conjugated Evans-blue dye within the brain before and after stroke and after 35 days of poststroke captopril or losartan treatment. The study was undertaken with the premise that an overactive renin-angiotensin system may be involved in promoting cerebrovascular autoregulatory dysfunction in SHRsp after stroke. If this occurred, the suppression of the system after HS with captopril or losartan treatment could retard the progression of brain damage and disability by restoring CBF autoregulation and reducing CBF over-perfusion under conditions of hypertension.

Materials and methods

The experiments were in compliance with the guidelines of the Canadian Council on Animal Care (The Guide to the Care and Use of Laboratory Animals, Vol. 1, 2nd ed. ISBN:0-919087-18-3). The SHRsp were fed a Japanese-style stroke-prone diet containing 4% NaCl (Zeigler Bros, Gardners, PA, USA) from 5 weeks of age. The systolic BP (sBP) was measured before sampling using a tail-cuff compression method (Model 59, IITC Inc., Woodland Hills, CA, USA). Different SHRsp were used in the CBF, pressure myograph, and BBB permeability studies.

The characteristics of stroke development in our SHRsp has been described earlier (Smeda, 1989). It consists of an abrupt development of seizures, followed by severe lethargy and immobility, ending in death (on average) 2 weeks after the onset of stroke. The age-related development of stroke in the SHRsp used within the CBF and myogenic studies is shown in Supplementary Figure S1. The SHRsp were sampled at 10 weeks of age before stroke development. Other SHRsp were either sampled at stroke (mean age of stroke onset, 15.3±2.4 weeks, n=84) or after being orally treated with captopril or losartan (at, respectively, 50 or 35 mg/kg per day, delivered through the drinking water) from the first signs of stroke for 13, 33, or 63 days.

Cerebral Blood Flow Measurements

The techniques used to measure CBF in the SHR have been described earlier (Daneshtalab and Smeda, 2010). The SHRsp were anesthetized with Na-pentobarbitol (65 mg/kg, intraperitoneally) and maintained on a ventilator. The inhaled air contained 40% O2 and the ventilatory rate was adjusted to maintain normal blood pCO2. Blood gases were measured (Ciba Corning 278 Blood Gas Analyzer, Medfield, MA, USA) before the experiments (average blood values, O2 saturation= 99.9%±0.01%, pCO2=40.3±0.7 mm Hg, pH=7.43±0.01, n=54).

The laser Doppler probe (PF403, Periflux 4001 Master Amplifier, Perimed, Jarfalla, Sweden) was calibrated (to the Brownian motion of a standardized concentration of latex spheres (PF100, Perimed)) and positioned in a 1-mm hole drilled through the skull above the intact brain dura within the MCA perfusion domain at previously described coordinates (Daneshtalab and Smeda, 2010). The BP was measured at the femoral artery (Statham P23D transducer, Gould 8188 recorder/universal amplifier, Gould Instruments, Valley View, OH, USA). After anesthesia SHRsp typically maintained a mean BP (mBP) of 180 mm Hg. To obtain CBF measurements over a wide range of mBPs, abdominal compression (which reduces venous return to the heart) was used to initially lower BP to 60 mm Hg. The BP was then allowed to slowly return to resting levels (near 180 mm Hg) by releasing abdominal compression and was further elevated to maximal possible levels by infusing phenylephrine (350 μmol/L in lactate Ringers solution) into the femoral vein. Neither phenylephrine nor norepinephrine produced constriction within the rat cerebral vasculature. Dose–response curves demonstrating the absence of constriction in the MCAs of SHRsp in response to phenylephrine and norepinephrine in comparison to the massive constriction of mesenteric arteries from the same SHRsp is shown in Supplementary Figure S2. This allowed the BP of SHRsp to be increased without the risk of phenylephrine-induced cerebrovascular constriction.

The CBF flux values obtained from the probe were synchronized to the mBP present (C10-AD, 16LRAT, Acquire, Acquisition Program, Computer Boards Inc., Mansfield, MA, USA). The flux at a given mBP was normalized to maximum CBF values obtained during phenylephrine infusion and expressed as a percentage of maximal CBF.

Measurement of Pressure-Dependent Constriction in Middle Cerebral Arteries from Stroke-Prone Hypertensive Rats

The SHRsp were anesthetized (65 mg/kg Na-pentobarbitol, intraperitoneally) and a blood sample was taken through cardiac puncture for plasma aldosterone analysis (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA, USA). The rats were exsanguinated by removing the heart. The MCA segments were excised from the brain at a point distal to where the artery crosses the rhinalis fissure. The location of the sample site is shown in Supplementary Figure S3. The segments were mounted onto a hollow pipette within a pressure myograph. A description of the apparatus and the techniques used to measure PDC in the MCAs has been outlined earlier (Smeda and King, 2000). A pressure reservoir containing oxygenated (95%O2/5% CO2) Krebs saline solution was connected the lumen of the artery. The end of the MCA was tied forming a Krebs saline solution-filled artery that could be pressurized without luminal flow. The exterior of the artery was suffused with the same Krebs saline solution at 37°C. The changes in MCA lumen diameter (LD) were viewed with a microscope, video recorded, and measured at × 322 magnification.

The MCAs were initially equilibrated to 100 mm Hg for 30 minutes, allowing PDC to develop. Servo-null measurements of the pial arteriolar pressure in SHRsp have indicated that an mBP of 96±3 mm Hg exists at systemic mBPs>200 mm Hg (Baumbach et al, 1994). Hence, the 100 mm Hg pressure used to study the MCAs represents a realistic mBP likely experienced in vivo. After equilibration to 100 mm Hg, the pressure was reduced to near 0 mm Hg (at an actual pressure of 0.38 mm Hg to prevent arterial collapse) for 6 minutes, thereby removing the stimulus for PDC. Pressure was then reelevated to 100 mm Hg. The reapplication of pressure after the inactivation of PDC caused the MCA lumen to expand to a diameter that would be present in the absence of pressure-induced constriction. The MCAs subsequently reconstricted in response to the reapplied pressure, achieving a steady-state reduction in LD within 3 minutes. The experiment was ended by maximally vasodilating the MCAs at 100 mm Hg pressure with 3 μmol/L nifedipine. This concentration of nifedipine has been shown to be as effective as Ca+2-free Krebs saline solution containing 1 mmol/L EGTA in promoting dilation in pressurized MCAs (Smeda and King, 2000). The MCA constriction occurring between 1 second (before the significant engagement of PDC) and 4 minutes after the reapplication of 100 mm Hg pressure (the time required to produce maximal PDC) was used as a measure of PDC. The reduction in LD present at 1 second after the reapplication of 100 mm Hg pressure (after the inactivation of PDC) over that present at maximal vasodilation at 100 mm Hg represented the degree of basal nonpressure-dependent tone present within the MCAs. Using the above technique we have observed that stroke development in SHRsp is typically associated with an inability to constrict in response to a 100-mm Hg pressure step and the development of large levels of basal nonpressure-dependent tone. The rationale for using this technique has been explained in detail within a recent paper (see Online supplement to Daneshtalab and Smeda, 2010).

Assessment of Blood–Brain Barrier Disruption in Stroke-Prone Hypertensive Rats

The SHRsp were anesthetized (65 mg/kg, Na-pentobarbitol). A 30-mg/kg bolus of Evans-blue dye (30 mg/mL 0.9% saline) was infused into the femoral vein. After 20 minutes, the rats were exsanguinated, the brains were removed and photographed. Evans-blue dye conjugates with plasma albumin (Udaka et al, 1970). The extravasation of dye represents BBB disruption sufficient to permit the transvascular movement of albumin. Examples of the lesions observed in the brains of poststroke SHRsp are presented in Supplementary Figure S3. These include brain distortion and herniation produced by edema and dye extravasation in the absence of cerebral hemorrhage (BBB disruption sufficient to permit plasma and albumin but not red blood cell extravasation), as well as cerebral hematoma and the presence of fluid-filled lesions.

Data Analysis and Statistical Procedures

N-values represent SHRsp sampled. An analysis of variance was used to determine ‘between group' differences and a Fishers post hoc test assessed subgroups differences (analysis of variance+post hoc test). A general linear model of multivariate analysis determined differences in CBF with mBP between groups. Values are expressed as the mean±1 standard error. Results were considered significant at P<0.05.

Results

Physical Characteristics of Stroke-Prone Hypertensive Rats Before and After Treatment

Stroke in SHRsp was characterized by the development of seizures consisting of repetitive involuntary flexion of the left or right front paws and/or the head. Untreated SHRsp within our colony never survive stroke and follow a pattern where seizure development subsides after 1 to 3 days and is followed by severe lethargy and death (mean lifespan 1.7±0.2 weeks (n=19); Smeda, 1989). In this study, stroke was observed after 11.5 weeks of age and 90% of the SHRsp developed stroke before 18.5 weeks (n=84) (Supplementary Figure S1).

Treatment with captopril or losartan initiated after the symptoms of stroke prevented death even in SHRsp that exhibited severe disability. The treatments attenuated seizures within 1 day. The SHRsp reestablished and maintained near normal activity, motor function, and grooming after 3 to 4 days for the duration of the study (63 days of treatment).

Poststroke SHRsp exhibited elevated plasma aldosterone levels when compared with prestroke asymptomatic SHRsp sampled at 10 weeks of age (Supplementary Figure S4A). Both treatments equally suppressed plasma aldosterone in poststroke SHRsp to prestroke levels. Within the rat, aldosterone is released into the circulation in response to AII stimulation of AT-1R within the adrenal cortex. Elevated plasma aldosterone levels in poststroke SHRsp are consistent with the presence of an overactive renin-angiotensin system. The substantial and equal suppression of plasma aldosterone by captopril and losartan after 13 to 63 days of poststroke treatment (Supplementary Figure S4A) indicated that both treatments produced an equivalent suppression of the actions of the renin-angiotensin system without aldosterone escape.

Systolic BP was elevated in poststroke SHRsp over prestroke rats (Supplementary Figure S4B). These differences in sBP are consistent with the levels of BP predicted by the younger average age of the prestroke (10.4±0.1 weeks) versus poststroke (16.6±0.7 weeks). The average sBP increased slightly during captopril treatment. Losartan treatment produced a nonsignificant small suppression in sBP after 13 days followed by a reelevation of sBP to poststroke levels with longer treatment. The sBP was lower in losartan versus captopril-treated SHRsp at all treatment durations. However, neither captopril nor losartan treatment significantly lowered sBP below the levels present in nontreated poststroke SHRsp (Supplementary Figure S4B).

Cerebral Blood Flow Autoregulation in Stroke-Prone Hypertensive Rats Before and After Treatment

The changes in CBF in response to elevated mBP within SHRsp are outlined in Figure 1. Figure 2 summarizes the key CBF functional parameters.

Figure 1.

Figure 1

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Alterations in cerebral blood flow (CBF) in the middle cerebral artery perfusion domain in response to elevations in blood pressure (BP). (A) The stroke-prone hypertensive rats (SHRsp) with stroke lost the ability to autoregulate CBF and exhibited hyperperfusion when mean BP (mBP) was elevated. The CBF autoregulation was equally restored after (B) 13 days and (C) 33 days of poststroke captopril or losartan treatment. (D) The CBF autoregulation deteriorated in SHRsp subjected to 63 days of poststroke captopril treatment while being maintained after similar losartan treatment. Statistics—multivariate analysis over common ranges of mBP. (A) Before versus after stroke—P<0.001. (B, C) After stroke versus captopril or losartan treatment—P<0.001. (D) After stroke versus captopril treatment—P<0.05; after stroke versus losartan treatment—P<0.001; captopril versus losartan treatment—P<0.001. N (SHRsp) (A) before/after stroke—5/6; nontreated poststroke/captopril/losartan treated (B) 6/5/5; (C) 6/5/6; (D) 6/5/6.

Figure 2.

Figure 2

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The characteristics of cerebral blood flow (CBF) regulation in the middle cerebral artery perfusion domain of stroke-prone hypertensive rats (SHRsp). Stroke development was associated with (A) a loss of autoregulation (no upper blood pressure (BP) limit), (B) increases in relative CBF at a mean BP of 200 mm Hg, and (C) high CBF conductance in response to increases in BP. Poststroke treatment of SHRsp for 13 and 33 days with captopril or losartan restored CBF autoregulation, reduced relative CBF and conductance to prestroke levels. The CBF autoregulation deteriorated and was lost after 63 days of poststroke captopril treatment while being maintained at prestroke levels with similar losartan treatment. Statistics−analysis of variance+Fisher's post hoc, P<0.05—* versus nontreated poststroke; δ versus prestroke; φ versus captopril treatment. N (SHRsp) for (A–C) before/after stroke—5/6; captopril/losartan treated—13 days—5/5; 33 days—5/6; 63 days—5/6.

Prestroke SHRsp exhibited a moderate increase in CBF from 60 mm Hg up to an upper autoregulatory point of about 210 mm Hg mBP. Further elevations in BP produced cerebrovascular forced dilation, which was associated with a sharp increase in CBF with mBP (Figure 1A). Poststroke SHRsp could not autoregulate CBF. The CBF increased with mBP in a hyperbolic manner (Figure 1A). No upper autoregulatory mBP limit was observed when mBP was increased from 60 to 280 mm Hg (Figure 2A). The relative CBF (percentage of maximal CBF) at an mBP of 200 mm Hg (Figure 2B) and CBF conductance between mBPs of 60 to 200 mm Hg (Figure 2C) was increased twofold in poststroke versus prestroke SHRsp. Thirteen days of poststroke captopril or losartan treatment restored CBF autoregulation in SHRsp (Figure 1B), reestablished an upper mBP limit to CBF autoregulation (Figure 2A), and normalized relative CBF at an mBP of 200 mm Hg (Figure 2B) as well as CBF conductance between mBPs of 60 to 200 mm Hg (Figure 2C) to prestroke levels.

Poststroke SHRsp treated with captopril or losartan for 33 days continued to maintain their ability to autoregulate CBF. The CBF autoregulatory curves (Figure 1C), the upper mBP limit of autoregulation (Figure 2A), relative CBF at an mBP of 200 mm Hg (Figure 2B), and CBF conductance (Figure 2C) were comparable between treatments and similar to the parameters obtained after 13 days of poststroke captopril or losartan treatment. After 63 days of poststroke captopril treatment, SHRsp lost the ability to autoregulate CBF. Relative CBF increased with mBP in a manner that was on average greater than that observed in nontreated SHRsp at stroke (Figures 1D and 2B). No distinct upper mBP limit of CBF autoregulation was observed after 63 days of poststroke captopril treatment (Figures 1D and 2A) and the relative CBF at a mBP of 200 mm Hg (Figure 2B) and CBF conductance between mBPs of 60 to 200 mm Hg (Figure 2C) was comparable to that present in nontreated SHRsp at stroke. In contrast, poststroke SHRsp treated with losartan for 63 days exhibited a distinct upper mBP limit of autoregulation (Figure 2A) and maintained an ability to autoregulate CBF (Figure 1D) that was comparable to prestroke SHRsp and poststroke SHRsp treated with losartan for 13 and 33 days. After 63 days of losartan treatment, SHRsp exhibited a lower relative CBF at 200 mm Hg (Figure 1B) and reduced CBF conductance (Figure 2C) when compared with similar captopril-treated SHRsp.

Middle Cerebral Artery Myogenic Function in Stroke-Prone Hypertensive Rats Before and After Treatment

The MCAs sampled from SHRsp were initially equilibrated to 100 mm Hg pressure. Transmural pressure was reduced to 0 mm Hg for 6 minutes to deactivate PDC. The ability of the arteries to constrict to pressure was measured by reapplying 100 mm Hg pressure and measuring the change in MCA LD between 1 second to 4 minutes after the reapplication of pressure. Figure 3 summarizes the percentage decrease in LD observed in response to the latter pressure step in MCAs sampled from the various SHRsp groups. Alterations in PDC within the MCAs mirrored the changes in CBF autoregulation within the MCA perfusion domain previously described in Figures 1 and 2. The ability to constrict to pressure was lost in the MCAs at stroke and recovered to prestroke levels after 13 days of poststroke captopril or losartan treatment. This function was maintained at prestroke levels over all durations of poststroke losartan treatment but progressively deteriorated with increasing durations of captopril treatment (Figure 3).

Figure 3.

Figure 3

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Pressure-dependent constriction (PDC) in isolated middle cerebral arteries (MCAs) from stroke-prone hypertensive rats (SHRsp). The PDC was lost in the MCAs at stroke and recovered after 13 days of poststroke captopril or losartan treatment. This function progressively deteriorated with increasing durations of captopril but not losartan treatment and mimicked the pattern of cerebral blood flow (CBF) autoregulation recovery and loss following poststroke treatment (Figures 1 and 2). Statistics−analysis of variance+Fisher's post hoc, P<0.05—* versus nontreated poststroke; δ versus prestroke; φ versus captopril treatment. N (SHRsp) before/after stroke—7/7; captopril/losartan treated 13 days—7/7; 33 days—6/7; 63 days—6/6.

The LD dimensional alterations in the MCAs pertaining to the above experiment and the LDs present at 100 mm Hg under conditions of maximal dilation (at 100 mm Hg) have been presented in Supplementary Figures S5A–5D. When compared with poststroke SHRsp, MCAs from prestroke SHRsp maintained larger LDs and greater dilation 1 second after the reapplication of 100 mm Hg pressure, after a 6-minute equilibration to 0 mm Hg pressure (Supplementary Figure S5A). This indicated that in addition to producing greater PDC, MCAs from prestroke SHRsp also exhibited enhanced dilation in response to decreases in pressure. Thirteen days of poststroke captopril or losartan treatment nearly equally normalized this function in the MCAs of poststroke SHRsp (i.e., reduced the level of tone present in the MCAs 1 second after application of the 100 mm Hg pressure step; Supplementary Figure S5B). Dilation in response to decreased pressure progressively deteriorated in the MCAs after 33 and 66 days of poststroke captopril treatment while being maintained over similar durations of losartan treatment (Supplementary Figures S5C and 5D).

Blood–Brain Barrier Permeability in Stroke-Prone Hypertensive Rats Before and After Treatment

The SHRsp were infused with Evans-blue dye (30 mg/kg, intravenously). The presence of BBB disruption was indicated by the extravascular movement of Evans-blue dye within the brain, the presence of hemorrhagic lesions, and brain distortion (herniation) resulting from brain edema (for a high-resolution photo see Supplementary Figure S3). Photographs of the brains from each of the groups are shown in Figure 4. Prestroke SHRsp sampled at 10 weeks of age exhibited no visible brain lesions or evidence of Evans-blue dye extravasation. The presence of cerebral hemorrhages, fluid-filled lesions, brain herniation produced by edema, and Evans-blue dye was evident in nontreated poststroke SHRsp (Figure 4). The above lesions and cerebral abnormalities were exclusively observed in the cerebrum. The degree of brain herniation and Evans-blue dye extravasation within the cerebrum appeared to be reduced to a greater degree in the brains of SHRsp subjected to 35 days of poststroke losartan treatment over SHRsp receiving similar captopril treatment (Figure 4).

Figure 4.

Figure 4

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Cerebral lesions in stroke-prone hypertensive rats (SHRsp). The SHRsp sampled at stroke exhibited extravasation of Evans-blue dye (blue lesions), cerebral hemorrhage (red hematomas), fluid-filled lesions, and brain distortion because of edema (herniation) consistent with the presence of blood–brain barrier disruption (discussed in Supplementary Figure S3). Cerebral lesions were absent in 10-week-old prestroke SHRsp and reduced to a greater degree in SHRsp subjected to 35 days of poststroke losartan versus captopril treatment. This suggested that poststroke losartan treatment may facilitate blood–brain barrier repair more effectively than captopril treatment.

Discussion

The Chronology of Intracerebral Hemorrhagic Stroke Development

Stroke development in SHRsp coincided with the disruption of the BBB, the development of brain edema, and intracerebral hemorrhage. Magnetic resonance imaging studies of SHRsp fed a high-salt Japanese-style diet indicate a continuum of events, which starts at 11 weeks of age with the spontaneous rupture of vessels producing microhemorrhages and/or the development of pockets of edema followed by an enlargement of the hemorrhagic lesions and death within 18 weeks of age (Lee et al, 2007). This resembles HS development in hypertensive humans where initial cerebral edema and microhemorrhage formation is followed by a rapid hematoma expansion in the initial hours after stroke (Ferro, 2006). Death is also prevalent and related to cerebral hemorrhage volume (hematomas≥60 mL produce 91% mortality within 30 days) (Broderick et al, 1993). Survival is further complicated by rebleeding (18% to 36% probability within 6 hours) and secondary hemorrhage formation (Ferro, 2006). We believe that in SHRsp, as in humans, the progressive expansion of hematomas and new lesion development after initial stroke produces a rapid deterioration of health ending in death.

The Loss of Cerebral Blood Flow Autoregulation and Hemorrhagic Stroke Development

The loss of cerebrovascular PDC and CBF autoregulation in the MCA perfusion domain precedes stroke in SHRsp (Smeda and King, 2000; Daneshtalab and Smeda, 2010). These defects would favor the development of high cerebrovascular pressures and vascular over-perfusion during hypertension, which could facilitate BBB disruption and cerebral hemorrhage formation under conditions where cerebrovascular integrity is weakened by hypertensive disease. There is precedence for believing that the loss of CBF autoregulation can promote and amplify cerebral hemorrhage. BBB disruption occurs in normal rats when BP is elevated under conditions where CBF autoregulation is experimentally abolished (Gaab et al, 1990). In humans, hypertensive encephalopathy, a neurologic dysfunction associated with brain edema formation (a prelude to HS) also develops when CBF autoregulation is overwhelmed during hypertension (Port and Beauchamp, 1998). Intraventricular cerebral hemorrhage development in preterm infants is thought to be produced by over-perfusion in response to elevations in BP under conditions where CBF autoregulation is absent (Bassan, 2009). In patients with HS, the relative degree of CBF autoregulatory dysfunction present predicts clinical status and poor outcome after stroke (Neunhoeffer et al, 2010). We observed a hyperbolic relationship between CBF and mBP in poststroke SHRsp. The CBF conductance and CBF at mBPs of 200 mm Hg was twice that observed before stroke. Such conditions could facilitate the expansion of existing hematomas, new lesion formation, and the transformation of edema into hemorrhage.

High Renin-Angiotensin Activity, Cerebrovascular Dysfunction, and Hemorrhagic Stroke Development

SHRsp best model HS development in a subpopulation of human hypertensive patients exhibiting renal end organ damage associated with reduced glomerular filtration and high systemic renin and AII levels. The model is not applicable to all forms of HS development in humans. It is however useful in demonstrating the detrimental influence of an overactive renin-angiotensin system on HS development. HS is rare in animal models of hypertension but does occur within SHRsp (Smeda, 1989), two-kidney, two-clip renal hypertensive rats (Del Bigio et al, 1999) and genetically manipulated high renin-angiotensinogen mice (Lida et al, 2005), which exhibit high AII forms of hypertension. During chronic hypertension in mice (produced by high AII infusion plus nitric oxide synthase inhibition), HS development is more readily produced by acute injections of AII over norepinephrine. This occurs despite the fact that norepinephrine injections produce greater elevations in BP (Wakisake et al, 2010). As shown in the current and earlier studies (Smeda et al, 1999; Blezer et al, 2001; Sironi et al, 2004), death after HS is attenuated in SHRsp by poststroke ACEI or ARB treatments in the absence of an antihypertensive effect. The above evidence suggests that a hyperactive renin-angiotensin system during hypertension can represent a separate risk factor for HS development and attenuate survival after HS.

Magnetic resonance imaging studies of SHRsp have shown that treatment with an ACEI (enalapril) or ARBs (imidapril, losartan) at the time of stroke reverses cerebral edema and reduces hemorrhage size (Takahashi et al, 1994; Blezer et al, 2001). The unique observations made within the current study were that CBF autoregulation and cerebrovascular PDC was reestablished within poststroke SHRsp after 13 days of either captopril or losartan treatment in the absence of an antihypertensive effect. The rapid reestablishment of these functions suggest that AII is involved in perpetuating autoregulatory dysfunctions after HS. The reestablishment of CBF autoregulation after captopril or losartan treatment may have reduced cerebrovascular over-perfusion and microvascular pressures thus decreasing the probability of further BBB disruption. This would facilitate a reduction in edema and hematoma expansion. Pretreatment of SHRsp with valsartan (ARB) before stroke retards stroke development, prevents the increase in nicotinamide adenine dinucleotide phosphate oxidase activity (Yamamoto et al, 2008), and the onset of inflammation (Sironi et al, 2004). A reduction in poststroke inflammation and oxidative stress in response to ACEI and ARB treatment could have further decreased the risk of BBB disruption and produced an environment conducive to repair.

The Differential Restoration of Cerebral Blood Flow Autoregulation by Captopril and Losartan

The in vivo pattern of CBF autoregulation loss and recovery within the MCA perfusion domain coincided with a similar in vitro pattern of deterioration and recovery of PDC in the MCAs of SHRsp. PDC facilitates the maintenance of CBF autoregulation. Elevations in BP promote cerebrovascular constriction, which raises the vascular resistance to blood flow, enabling CBF to remain constant.

Poststroke losartan treatment of SHRsp produced a more permanent restoration of CBF autoregulation and PDC in the MCA perfusion domain when compared with captopril. Both treatments equally reduced plasma aldosterone in poststroke SHRsp to levels comparable to those present in prestroke SHRsp, suggesting an equivalent suppression of AII action occurred. The sBPs were slightly higher in captopril versus losartan-treated SHRsp at comparable treatment durations largely because of a slight increase in BP during captopril treatment. However, BP was not reduced below that present in nontreated SHRsp at stroke. The ability of ARB and/or ACEI treatments to increase survival in high-salt fed SHRsp in the absence of an antihypertensive effect has been observed in earlier studies (Takahashi et al, 1994; Smeda et al, 1999; Blezer et al, 2001; Sironi et al, 2004; Smeda and McGuire, 2007). A slightly greater suppression of BP with losartan verses enalapril (ACEI) treatment of SHRsp after the onset of cerebral edema (assessed through magnetic resonance imaging) was also noted by Blezer et al (2001). Both treatments promoted survival exceeding 260 days in the absence of an antihypertensive effect.

In humans, losartan inhibits the reabsorption of uric acid from the urine and decreases plasma uric acid levels (Alderman and Aiyer, 2004). Hyperuricemia has been implicated in cardiovascular disease development and the superior ability of losartan to reduce the risk of myocardial infarction and stroke over other treatments, including ACEIs, has been attributed to a reduction in plasma uric acid (Alderman and Aiyer, 2004). However, plasma uric acid is not elevated above normal in SHRsp with stroke, and we have observed that poststroke losartan or captopril treatment up to 56 days produced a small equal (in most cases nonsignificant) decrease in plasma uric acid (J Smeda, unpublished data). Therefore, it is unlikely that differences in uric acid excretion contributed to the differential effects of captopril and losartan in the current study.

Some of the benefits of ABR over ACEI treatment may be mediated through AII type-2 receptors (AT-2R). ACEIs reduce plasma AII, whereas ARB treatments increase AII levels by enhancing renin release from the afferent renal arterioles (through AT-1R blockade). The high AII levels present during AT-1R blockade could stimulate AT-2R (Chrysant, 2005). This response could be amplified by the increased expression of AT-2R observed after stroke in animals (Culman et al, 2005) and ARB treatment within the brain (Lu et al, 2005) and cerebrovasculature (Zhou et al, 2006) of SHR. In SHR (Lu et al, 2005) and normotensive rats (Culman et al, 2005), the ability of ARBs (respectively, candesartan and irbesartan) to reduce infarct volume and facilitate neurologic recovery after ischemic stroke are abolished by PD123319, an AT-2R antagonist. Valsartan (ARB) treatment has been shown to be more effective in reducing ischemia, nicotinamide adenine dinucleotide phosphate oxidase activity, superoxide production, and neurologic deficit in normal versus AT-2R knockout mice with ischemic stroke (Iwai et al, 2004). AT-2R stimulation can produce beneficial effects that may improve survival after stroke. These include the reduction of oxidative stress (Iwai et al, 2004; Okumura et al, 2005), inflammation (Okumura et al, 2005), and improved neural recovery (Iwai et al, 2004; Culman et al, 2005; Lu et al, 2005) after stroke. The ability of AT-2R stimulation to reduce renal damage in SHRsp (Gelosa et al, 2009) and promote natriuresis (Carey and Padia, 2008) may be particularly relevant in SHRsp, where the onset of stroke is accelerated by salt loading and associated with renal dysfunction. Treatment of SHRsp with Compound 21, an AT-2R agonist, retards the onset of stroke, reduces inflammation, and prolongs life (Gelosa et al, 2009). In view of the pathology attributed to AT-1R stimulation, stimulation of AT-2R under conditions of AT-1R blockade during ARB treatment of SHRsp could provide even greater benefits, possibly accounting for the superior effects losartan over captopril treatment observed in the current study.

The prospect that alternate forms of AII production, not involving ACEs produced the differing effects of losartan versus captopril was considered. Studies have shown that rat vascular chymases produce a nonactive form of AII (Miyazaki and Takai, 2001). However, a proteolytic fragment of angiotensinogen (proangiotensin 12) can be converted to AII by rat heart chymase isozymes (Prosser et al, 2009) and novel rat vascular chymases capable of creating active AII have been proposed (Guo et al, 2001). If the latter forms of AII generation contributed to the pathology of stroke in SHRsp, ARBs (but not ACEIs) would still be effective in blocking these effects.

Concluding Remarks

A prior history of hypertension and further elevations in BP at stroke are commonly observed in HS patients. The probability of death and disability are enhanced in HS patients exhibiting high BPs at admission (Asdaghi et al, 2007). The aggressive lowering of BP in hypertensive patients at the onset of HS reduces hematoma expansion (Anderson et al, 2010). However, such treatment remains controversial and it is unclear as to whether lowering BP in a HS patient produces long-term benefits. There is some consensus that BP can be safely lowered to a target sBP<140 mm Hg after HS and such treatment has been advocated (Asdaghi et al, 2007; Anderson, 2009). We believe that if a decision to lower BP is made, consideration should also be given to the type of antihypertensive therapy used. The observation of enhanced survival, the reversal of cerebral edema (Takahashi et al, 1994; Smeda et al, 1999; Blezer et al, 2001) and the restoration of cerebral vascular myogenic and endothelial function (Smeda and McGuire, 2007), as well as CBF autoregulation (current study) within animal studies of HS, particularly in response to poststroke ARB treatment, provides a premise for the investigation of the use of ARBs over other antihypertensives in the treatment of hypertension after the onset of HS in humans.

The authors declare no conflict of interest.

Footnotes

Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website (http://www.nature.com/jcbfm)

This study was supported by a grant from the Canadian Institutes of Health Research to John Smeda.

Supplementary Material

Supplementary Information
Click here for additional data file. (357.5KB, doc)

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