John S. Smeda

The Role of Blood Pressure and Aldosterone in the Production of Hemorrhagic Stroke in Captopril-Treated Hypertensive Rats

BACKGROUND AND PURPOSE We tested the hypothesis that the lowering of plasma aldosterone levels contributed to the antistroke effects of captopril treatment in Wistar Kyoto stroke-prone spontaneously hypertensive rats (SHRSP). METHODS The suppression of plasma aldosterone by captopril treatment (50 mg.kg-1.d-1) was prevented by the subcutaneous infusion of aldosterone into captopril-treated SHRSP. We studied the effect this had on blood pressure (BP) and stroke development. RESULTS SHRSP fed a Japanese-style diet containing 4% NaCl developed hypertension and a 100% mortality associated with intracerebral hemorrhage by 14 weeks of age. Captopril treatment from 6 weeks of age did not lower the BP but increased survival past 35 weeks of age. Hydralazine treatment (40 to 80 mg/L of drinking water) lowered BP in SHRSP but was less effective than captopril in retarding stroke. Plasma aldosterone levels were elevated with age in SHRSP after 10 weeks and were higher in poststroke versus prestroke SHRSP. Captopril treatment suppressed plasma aldosterone. When we elevated plasma aldosterone in captopril-treated SHRSP to levels between those present in untreated pre- and poststroke SHRSP, the ability of captopril to retard stroke development was negated. The effects of aldosterone were mimicked by deoxycorticosterone (40 mg/kg, SC2 times/wk) but not by dexamethasone (0.1 mg.kg-1.d-1, SC). Spironolactone treatment (20 mg.kg-1.d-1, SC) of SHRSP reduced BP but had little effect on stroke development. CONCLUSION Elevations in plasma aldosterone enhance stroke development within captopril-treated SHRSP through mechanisms that do not involve stimulation of mineralocorticoid receptors or the enhancement of hypertension. The antistroke effects of captopril treatment may be partially mediated through the suppression of plasma aldosterone.

Stroke-prone spontaneously hypertensive rats lose their ability to auto-regulate cerebral blood flow prior to stroke.

OBJECTIVES We hypothesized that the loss of cerebral blood flow (CBF) auto-regulation under hypertensive conditions could promote cerebrovascular over-perfusion and haemorrhage formation. The possibility that CBF auto-regulation becomes defective prior to haemorrhagic stroke development was assessed in Wistar- Kyoto stroke-prone spontaneously hypertensive rats (SHRsp) and related to the myogenic responsiveness of the cerebrovasculature to pressure. METHODS Laser Doppler techniques were used to measure relative CBF in relation to mean arterial pressure (MAP 130-260 mmHg) within the perfusion domains of the middle (MCA) and posterior (PCA) cerebral arteries. The ability of isolated MCAs and PCAs to constrict to a 120 mmHg pressure step (pressure-dependent constriction) was measured using a pressure myograph. RESULTS Two weeks prior to stroke, 10-week-old pre-stroke SHRsp exhibited near-constant CBF regulation to a 200 mmHg MAP. Thirteen-week-old pre-stroke SHRsp and age-matched post-stroke SHRsp lost their ability to auto-regulate CBF in the MCA and PCA perfusion domains. CBF increased at a high rate and in a linear manner with MAP. A distinct upper limit to CBF auto-regulation was absent. Pressure-dependent constriction was attenuated prior to stroke, and lost after stroke in isolated MCAs, but not the PCAs, of SHRsp. CONCLUSIONS The loss of CBF auto-regulation prior to stroke in SHRsp could enhance cerebral perfusion and facilitate the initiation of haemorrhage. Such dysfunction after stroke could produce secondary haemorrhages. Defects in pressure-dependent constriction cannot fully account for the pattern of CBF auto-regulation loss observed in post-stroke SHRsp.

Alterations in Autoregulatory and Myogenic Function in the Cerebrovasculature of Dahl Salt-Sensitive Rats

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.

Alterations in the modulation of cerebrovascular tone and blood flow by nitric oxide synthases in SHRsp with stroke

AIMS The modulation of myogenic function and cerebral blood flow (CBF) by nitric oxide (NO) synthases (NOS) was assessed in the middle cerebral arteries (MCAs) of Kyoto Wistar stroke prone hypertensive rats (SHRsp) in relation to haemorrhagic stroke development. METHODS AND RESULTS MCAs were studied with a pressure myograph. CBF in MCA perfusion domain was measured using laser Doppler techniques. NOS isozymes were identified using immunohistochemistry. MCAs expressed endothelial, neuronal, and inducible NOS (eNOS, nNOS, and iNOS, respectively) in the endothelium, nNOS and traces of iNOS in smooth muscle and adventitial cells. Before stroke, MCA pressure-dependent constriction (PDC) was superimposed over basal non-pressure-dependent tone (BNPDT). Endothelial NO generation and non-endothelial nNOS but not iNOS reduced BNPDT and increased the lumen diameter at which PDC initiated without altering the amplitude of PDC. NOS inhibition decreased CBF and increased the upper blood pressure limit of autoregulation. PDC, CBF autoregulation, and NOS dilatory influence were lost, and BNPDT was increased in MCAs from SHRsp with stroke. The expression of NOS isozymes and MCA reactivity to NO donors was not altered. NOS activity was not recovered by in vitro l-arginine or tetrahydrobiopterin supplementation, l-arginase inhibition or superoxide scavengers. CONCLUSION The loss of PDC and CBF autoregulation during hypertension may facilitate over-perfusion and cerebral haemorrhage formation in SHRsp. NOS dysfunction in MCAs preceded stroke and involved the inactivation of eNOS and nNOS in areas not subjected to hyper-distension. The elevation in BNPDT due to NOS inactivation may oppose over-perfusion in the absence of CBF autoregulation.

Troubleshooting tissue specificity and antibody selection: Procedures in immunohistochemical studies.

Optimal antigen detection and identification is dependent on the tissue of interest, the method of fixation, processing, and antibody specificity. We evaluated specific antigens in frozen middle cerebral artery (MCA) sections from rat brains under various conditions of fixation and differing primary and secondary antibody concentrations. Fresh MCAs were frozen, cryosectioned (8 microm), and adhered to chrom-alum coated slides. The effects of different fixation and antigen retrieval/pretreatments were tested for detection of enzymes and receptors involved in MCA tone regulation. Antigen localization was determined with specific primary antibodies and detected using fluorochrome-conjugated secondary antibodies. Spatial distribution of localized antigens was imaged using confocal microscopy. Frozen sections preserved the morphology of the endothelium and/or vessel wall within the tissue in a manner comparable to formalin-fixed sections. Fixation and tissue processing methods were modified based on the primary antibody used. Optimal antigen detection was obtained using fixatives such as 4% paraformaldehyde, 100% acetone or 100% methanol. Pretreatments, such as 1% SDS, enzymatic digestion using 0.1% trypsin, or application of heat were used to optimize antigen-antibody interaction. Stringent background and control checks were performed to ensure specificity of staining in both single and multiple labeling techniques. In a research setting where epitope detection is not used for diagnostic purposes, there is more latitude in tissue fixation. Frozen samples offer a more versatile method of linking the appropriate fixation and tissue processing to the primary antibodys unique needs. At the same time, it stabilizes the tissue in a format that allows for later analysis of multiple antigens with specific detection requirements in same tissue.

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

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.

Electromechanical Alterations in the Cerebrovasculature of Stroke-Prone Rats

BACKGROUND AND PURPOSE Cerebrovascular pressure-dependent constriction (PDC) is associated with smooth muscle (SM) depolarization and Ca(2+) influx through voltage-gated channels. We studied the alterations in electromechanical contraction in the middle cerebral arteries (MCAs) of stroke-prone Wistar-Kyoto spontaneously hypertensive rats (SHRsp) in relation to the stroke-related loss of PDC. METHODS Constriction to pressure, elevated [K(+)](o) and/or [Ca(2+)](o), and SM membrane potentials (E(m)) were measured in isolated pressurized MCAs of SHRsp and stroke-resistant SHR. RESULTS MCAs of SHRsp exhibited an age-related decrease in PDC before hemorrhagic stroke and a loss of PDC after stroke. At 100 mm Hg, the MCAs of poststroke SHRsp maintained partial constriction that was not altered with pressure but was inhibited by nifedipine (1 micromol/L). The MCAs of poststroke SHRsp constricted to vasopressin (0.17 micromol/L) but not to elevated [K(+)](o). When pressure was reduced from 100 to 0 mm Hg, the MCAs from young prestroke SHRsp exhibited SM hyperpolarization (-38 to -46 mV), whereas those of poststroke SHRsp maintained a constant, depolarized E(m) (-34 mV). Alterations in E(m) with varying [K(+)](o) suggested that there was a decrease in SM K(+) conductance in the MCAs of poststroke SHRsp. CONCLUSIONS The observation that the MCAs of poststroke SHRsp depolarize but do not constrict to elevated [K(+)](o) suggests the presence of dysfunctional voltage-gated Ca(2+) channels. The inability to alter E(m) with pressure or to constrict to depolarization could partially contribute to the loss of PDC in the MCAs of poststroke SHRsp.

Effects of Poststroke Losartan Versus Captopril Treatment on Myogenic and Endothelial Function in the Cerebrovasculature of SHRsp

Background and Purpose— We assessed the ability of poststroke captopril and losartan treatment to reverse myogenic and endothelial dysfunction in the middle cerebral arteries of Kyoto-Wistar stroke-prone spontaneously hypertensive rats (SHRsp) that developed intracerebral hemorrhagic stroke. Methods— SHRsp were sampled before and after stroke development and after up to 37 days of captopril (50 mg/kg per day) or losartan (35 mg/kg per day) treatment initiated after stroke. Pressure-dependent constriction to a 100-mm Hg pressure step, constriction to nitric oxide synthase inhibition (100 &mgr;mol/L N&ohgr;-nitro-l-arginine methyl ester), and endothelium-dependent vasodilation to bradykinin (1.6 &mgr;mol/L), 2-f-LIGRLO-NH2 (1 &mgr;mol/L, a protease-activated receptor-2 agonist), and A23187 (2 &mgr;mol/L) were evaluated in middle cerebral arteries at 100 mm Hg with a pressure myograph. Results— Middle cerebral arteries from SHRsp with stroke could not constrict to pressure or nitric oxide synthase inhibition, lacked the ability to vasodilate to bradykinin, and exhibited attenuated dilation and vasomotion in response to A23187. Vasodilation to 2-f-LIGRLO-NH2 was unaltered. The aforementioned cerebrovascular alterations were reversed after 31 days of poststroke losartan but not of captopril treatment in the absence of an antihypertensive effect. Captopril treatment restored middle cerebral artery constriction to pressure, NOS inhibition, and bradykinin vasodilation temporarily after 7 to 18 days of treatment, after which function deteriorated to a level observed in SHRsp at stroke. Conclusions— Aspects of poststroke cerebrovascular dysfunction, which likely play an important role in altering and modulating cerebral blood flow autoregulation, can be reversed in SHRsp more effectively after stroke development by blocking angiotensin II type 1 receptors as opposed to lowering angiotensin II levels.

PERINDOPRIL TREATMENT IN THE PREVENTION OF STROKE IN EXPERIMENTAL ANIMALS

Objective To determine the effect of perindopril treatment and treatment withdrawal in the prevention of stroke in male stroke-prone spontaneously hypertensive rats (SPSHR). Design After weaning at 4 weeks of age, male SPSHR were given a Japanese-style rat diet which induces stroke in these animals. Beginning at 6 weeks of age, SPSHR were treated with either distilled water (control) or different daily dosages of perindopril (1 or 4 mg/kg) by gavage for 24 weeks followed by treatment withdrawl. Additional subgroups were treated with the 4 mg/kg dose for different durations (8, 12 or 24 weeks) before treatment withdrawal. Treatment effects on blood pressure, heart rate and body weight were studied during the treatment period and after the withdrawal of the treatment Myogenic and mechanical properties of the middle cerebral arteries were studied in control SPSHR that had developed stroke, in treated SPSHR at the end of the treatment period, and at certain intervals after the withdrawal of the treatment. Methods Systolic blood pressure, heart rate and body weight of control and treated SPSHR were determined at regular intervals before, during and after the treatment withdrawal periods until they died from stroke, or until 42 or 43 weeks of age when the study was terminated. Functional studies of the cerebral arteries were carried out using a pressurized artery system. At necropsy, macroscopic and microscopic examinations were made of the kidneys and brain. Results Untreated SPSHR usually died of stroke-related complications by 14 weeks of age. The middle cerebral arteries from these animals had lost their ability to contract in response to pressure increase. Chronic treatment of SPSHR with perindopril when initiated at 6 weeks of age attenuated the sharp blood pressure rise, and prevented the development of stroke during the treatment period. This was associated with the preservation of the myogenic response of the middle cerebral arteries to pressure increase, and the prevention of tissue damage in the kidneys and brain. After withdrawl of the treatment, SPSHR treated for a longer period (12 or 24 weeks) also survived longer than those treated for a shorter period (8 weeks). The subsequent loss of myogenic response in the middle cerebral arteries was associated with the development of stroke and death in these treatment withdrawl groups. Conclusion Chronic treatment with perindopril is beneficial for the prevention of stroke in SPSHR, through the preservation of the myogenic response properties of the cerebral arteries, and the attenuation of tissue damage in the brain and kidneys.

Cerebrovascular alterations in pressure and protein kinase C-mediated constriction in Dahl salt-sensitive rats

Background Dahl salt-sensitive (DSS) rats fed an 8.7% sodium chloride diet from weaning spontaneously developed hypertension and a 50% mortality rate by 5 weeks. Before death the rats exhibited behavioural signs of stroke and disruption of the blood–brain barrier. Objectives To test the hypothesis that rats exhibiting stroke had middle cerebral arteries (MCAs) that had lost the ability to constrict in response to pressure, and to assess whether this defect was associated with abnormalities in protein kinase C (PKC)-mediated constriction. Methods MCAs were sampled from DSS rats before and after stroke and from Dahl salt-resistant (DSR) rats fed 8.7% NaCl. Constrictions in response to a 100 mmHg pressure step and to PKC activation by phorbol dibutyrate (PDB) (0.1 μmol/l) in the presence of nifedipine (3 μmol/l) were measured. Results MCAs from DSS rats after stroke constricted in response to vasopressin but were unable to constrict in response to pressure or PDB in the presence of nifedipine, whereas those from DSS rats before stroke and from DSR rats constricted in response to all the stimuli. The PKC inhibitors, chelerythrine (12 μmol/l) and bisindolylmaleimide (5 μmol/l) inhibited constrictions in response to pressure and to PDB in the presence of nifedipine. Conclusions Constriction of the MCA in response to pressure is dependent on functional PKC signalling. Development of stroke in DSS rats fed a high-salt diet is associated with an inability of the MCAs to constrict in response to pressure, possibly because of the presence of an incompetent PKC system. The inability to constrict in response to pressure may cause blood flow abnormalities that contribute to disruption of the blood–brain barrier in these rats.