Post-stroke losartan and captopril treatments arrest hemorrhagic expansion in SHRsp without lowering blood pressure.

We assessed the ability of poststroke losartan and captopril treatment to attenuate hematoma expansion and plasma extravasation after intracerebral hemorrhagic stroke in Kyoto-Wistar stroke-prone hypertensive rats (SHRsp). Cerebrum volume, herniation and surface areas exhibiting new and old hemorrhages and albumin extravasation were measured prior to and after stroke and following 30 and 60 days of post-stroke losartan or captopril treatment in Evans Blue dye perfused brains. Lesion morphology was studied in serial sections. Losartan or captopril treatment initiated at stroke prevented death for 60 days without lowering BP. Stroke onset was associated with the development and subsequent expansion of cerebrum volume, herniation, hematoma and plasma albumin extravasation. Losartan arrested cerebral volume expansion and herniation, and produced the virtual disappearance of hematoma and plasma albumin extravasation after 60 days. Captopril treatment equally attenuated cerebral herniation and hematoma expansion but was less effective in stopping albumin extravasation and allowed cerebrum volume to increase to post-stroke levels after 60 days of treatment. Both treatments resolved hematomas into cortical fluid filled spaces and prevented new hemorrhage formation. We believe that the treatments attenuated death after stroke by inhibiting hemorrhagic expansion through non-pressure related physiological changes mediated by the inhibition of the renin-angiotensin system.

Cerebrovascular recovery after stroke with individual and combined losartan and captopril treatment of SHRsp.

We assessed whether the superior restoration of cerebrovascular function after hemorrhagic stroke by losartan versus captopril treatment was due to better BP, uremia, uricaemia, or aldosterone control in Kyoto Wistar stroke-prone-hypertensive rats and evaluated whether elevated angiotensin II (A2) levels enhanced the effectiveness of losartan treatment. Constriction was studied in the middle cerebral arteries (MCAs) using a pressure myograph. Post-stroke survival increased from 21 to 310 and 189days respectively with losartan and captopril treatment. Neither treatment reduced BP, both reversed uremia and hyperaldosteronism equally after 7days. Plasma uric acid remained low. At stroke, MCA constriction to pressure (PDC), protein kinase C (PKC) activation, depolarization, and sarcoplasmic Ca2+ were attenuated. Endothelial-dependent-vasodilation by bradykinin and endogenous NO release were lost. Both treatments recovered these functions within 7days. These functions deteriorated after 116days of captopril but not losartan treatment. Inhibiting A2 formation during losartan treatment didn't alter BP or vascular recovery. The superior recovery of PDC by losartan over captopril was not produced by better BP, uremia or aldosterone control or elevated A2. PDC recovery was associated with improved PKC function and enhanced basal NO release. The re-establishment of PDC could reduce cerebrovascular over-perfusion and hematoma expansion after stroke.

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.

Captopril treatment temporarily restores cerebral blood flow autoregulation in spontaneously hypertensive rats after hemorrhagic stroke.

Hemorrhagic stroke development in stroke-prone spontaneously hypertensive Kyoto Wistar rats (SHRsp) is associated with a loss of cerebral blood flow (CBF) autoregulation and death. We assessed the ability of poststroke captopril treatment to retard death and restore CBF autoregulation in SHRsp. Laser Doppler techniques were used to measure alterations in CBF with varying mean arterial pressure (MAP) in anesthetized SHRsp. Three weeks before stroke, all SHRsp autoregulated near constant CBF to an upper MAP limit of 155 +/- 4 mm Hg. CBF autoregulation was absent in half of the SHRsp at 0.5-2 weeks before stroke and nonexistent in SHRsp with stroke. Captopril treatment (50 mg kg(-1) d(-1)) initiated at the first signs of stroke (seizures) increased the lifespan of SHRsp from 10 +/- 3 to 124 +/- 18 days without lowering blood pressure and restored CBF autoregulation within 10 days. CBF autoregulation subsequently deteriorated where after 25 days of treatment, only 2 of 5 SHRsp maintained the ability to autoregulate CBF. We concluded that captopril treatment retarded death and new hemorrhage formation after stroke. The early restoration of autoregulation could prevent sudden death after stroke, but other mechanisms associated with poststroke captopril treatment act to prolong life in the presence of hypertension and absence of CBF autoregulation.

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 antibody's 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.

Protease-activated receptor 2 and bradykinin-mediated vasodilation in the cerebral arteries of stroke-prone rats.

Protease-activated receptor 2 (PAR(2)) expression is up-regulated during vascular injury associated with edema. PAR(2) and bradykinin subtype 2 receptor (B(2)) expression and function were assessed in relation to hypertensive encephalopathy (HE) and cerebral hemorrhage (CH) in middle cerebral arteries (MCA) of Kyoto Wistar stroke-prone spontaneously hypertensive rats (SHRsp). Before stroke, bradykinin and PAR(2) activation by 2-furoyl-leucine-isoleucine-glycine-arginine-leucine-ornithine-amide (2Fly) produced endothelium-dependent vasodilation that was inhibited by K(+) depolarization, carbenoxolone, and the blockade of intermediate (IK(Ca)) plus small (SK(Ca)) and (in the case of bradykinin) smooth muscle (SM) large conductance (BK(Ca)) calcium-activated K(+) channels. Responses were not altered by N omega-nitro-L-arginine methyl ester, indomethacin, 17-octadecynoic acid or Ba(2+)+ouabain. We concluded that vasodilation to 2Fly or bradykinin was not mediated by NO, cyclooxygenases, arachidonic acid-metabolizing cytochrome P450s or SM K(ir) channels+Na(+)/K(+) ATPase activation. Vasodilation likely involved the spread of endothelial hyperpolarization (generated by IK(Ca)+SK(Ca)) through myoendothelial junctions and in some cases SM BK(Ca) activation. SHRsp with HE or CH had MCA that could not constrict to pressure and did not vasodilate to bradykinin. Their responses to 2Fly remained unaltered. The patterns and densities of PAR(2) and B(2) immunoreactivity in frozen MCA sections were not altered with stroke. MCA function remained normal in SHRsp subjected to dietary manipulations that prevented stroke without altering hypertension. Despite the presence of vascular injury, edema, inflammation and the loss of endothelium-dependent bradykinin vasodilation we found no evidence that PAR(2) expression or vascular function was altered in MCA after stroke.

Increased homocysteine-induced release of excitatory amino acids in the striatum of spontaneously hypertensive stroke-prone rats.

BACKGROUND AND PURPOSE: Increased plasma [homocysteine] is associated with stroke but its direct effects on the brain during a stroke are unknown. Since excitatory amino acids are important in inducing brain damage, we examined the effect of homocysteine on the release of various amino acids in the striatum of spontaneously hypertensive stroke-prone (SHSP) rats before and after a stroke. METHODS: In vivo microdialysis was carried out in the striatum of anesthetized SHSP rats before and after signs of stroke. Animals were exposed to 20 and 200 muM homocysteine in the microdialysis solution and then the microdialysates were analyzed 30 min later for amino acid content. Brain cryosections were silver-stained to quantify infarcts in the non-ischemic and the damaged tissues in pre-stroke and post-stroke rats. RESULTS: Both pre-stroke and post-stroke animals had similar levels of all amino acids in the striatum. Homocysteine did not alter amino acid release in rats prior to stroke but induced a significant increase in the release of all amino acids tested in the post-stroke rats. However, the increase was significantly greater with the excitatory amino acids glutamate and aspartate, and with tyrosine in post-stroke animals as compared to those in pre-stroke, normal animals. The mean pixel density of the gray matter of post-stroke animals was significantly decreased following homocysteine treatment indicating the presence of neurological damage. CONCLUSIONS: Homocysteine-induced neurological damage in post-infarct SHSP rats was associated with a hypersecretion of excitatory amino acids. Patients with hyperhomocysteinemia may be at risk for augmented brain damage from an ischemic infarct due to a selective activation of neuronal excitatory amino acids.

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 micromol/L N(omega)-nitro-l-arginine methyl ester), and endothelium-dependent vasodilation to bradykinin (1.6 micromol/L), 2-f-LIGRLO-NH(2) (1 micromol/L, a protease-activated receptor-2 agonist), and A23187 (2 micromol/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-NH(2) 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.

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.

Stroke development in stroke-prone spontaneously hypertensive rats alters the ability of cerebrovascular muscle to utilize internal Ca2+ to elicit constriction.

BACKGROUND AND PURPOSE: The ability of middle cerebral arteries (MCAs) to utilize intracellular smooth muscle (SM) Ca2+ to produce constriction in response to pressure and agonists was assessed in relation to hemorrhagic stroke development in Wistar-Kyoto stroke-prone (SHRSP) and stroke-resistant (srSHR) spontaneously hypertensive rats. METHODS: MCAs were studied with the use of a pressure myograph at 100 mm Hg. RESULTS: MCAs from srSHR and prestroke SHRSP exhibited pressure-dependent constriction and constricted in response to vasopressin or serotonin in the presence of nifedipine or the absence of [Ca2+]o. MCAs from poststroke SHRSP lost the latter functions and could only constrict in response to vasopressin/serotonin in Krebs' solution containing Ca2+ in the absence of nifedipine. This indicated that the SM could not utilize internal Ca2+ for constriction and maintained constriction by Ca2+ entry through L-type channels. The MCAs of poststroke SHRSP could not constrict to [K+]o-induced depolarization, suggesting that the agonist-induced opening of the L-type channels occurred by mechanisms other than SM depolarization. Depletion of the sarcoplasmic SM Ca2+ stores of MCAs from srSHR with cyclopiazonic acid did not prevent pressure-dependent constriction. CONCLUSIONS: Stroke in SHRSP produced a defect in the ability of MCAs to constrict in response to vasopressin or serotonin via the use of an intracellular source of Ca2+. This could be promoted by an inability of the SM to release intracellular Ca2+, by the depletion of internal Ca2+ stores, or by a decrease in the contractile sensitivity to Ca2+ released from the internal stores.

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 micromol/l) in the presence of nifedipine (3 micromol/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 micromol/l) and bisindolylmaleimide (5 micromol/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.