Subarachnoid hemorrhage model in the rat: modification of the endovascular filament model.

The present study describes modifications to the endovascular filament model of subarachnoid hemorrhage (SAH) in rats. Specifically, we sought to improve the percentage yield of SAH, reduce mortality rates and better simulate human cerebral aneurysmal rupture. Instead of using a 4-0 prolene suture to induce SAH in the existing endovascular filament model, a hollow and flexible polyetrafluoroethylene (PTFE) tube was maneuvered into the proximal anterior cerebral artery (ACA) to ensure that advancement occurred without producing trauma to the vessels. SAH was induced by advancing a tungsten wire through this tube, perforating the ACA at the desired location. These modifications produced significant improvements over the endovascular filament model. Mortality rate declined from 46 to 19%, and SAH was produced more frequently. With the prolene suture, only 48% of our attempts produced a SAH, and unsuccessful attempts typically resulted in an acute subdural hematoma (ASDH). In contrast, the wire/tubing technique was 90% successful at inducing SAH, and led to a significant reduction of ASDH incidence from 44 to 6%. Additionally, the modified technique produced vasospasm in basilar and middle cerebral arteries post-SAH as well as pseudoaneurysms in the proximal ACA which indicated the location of vessel perforation.

Correlation of intrinsic optical signal, cerebral blood flow, and evoked potentials during activation of rat somatosensory cortex.

OBJECT: This study was undertaken to test the hypothesis that cerebral blood flow (CBF) and the intrinsic optical signal could be dissociated by altering adenosine receptor activity and to uncover the origin of the optic signal using a cranial window in the anesthetized rat. METHODS: In anesthetized, ventilated, and temperature-controlled rats with closed cranial windows, the authors evaluated simultaneously the alterations in pial arteriolar diameter, intrinsic optical signals (690 nm), and somatosensory evoked potentials during cortical activation evoked by contralateral sciatic nerve stimulation (SNS). To dissociate the vascular and intrinsic signal, they topically applied the adenosine receptors antagonists theophylline (5 microM), which affects A1 and A2A receptors, and 8-cyclopentyl-1,3-dipropylxanthine (CPX, 1 microM), which blocks the A(1) receptor. The former interacts primarily with the vasculature whereas the latter influences the parenchyma exclusively. RESULTS: During 20 seconds of contralateral SNS, pial arterioles in the hindlimb somatosensory cortex dilated in a characteristic peak and shoulder pattern. As compared with mock cerebrospinal fluid alone, theophylline significantly (p<0.05) attenuated SNS-induced vasodilation (mean+/-standard deviation 8.1+/-2.5% vs 21.7+/-1.9%; 4 rats in each group). In contrast, CPX potentiated vasodilation significantly (p<0.05) during SNS (54.7+/-15.8% for the CPX group vs 20.1+/-1.9% for the controls; 5 rats in each group). The change in optical signal persisted after cessation of SNS in all the animals. Thus, the pattern of change of the optical signal was distinctly different from the pattern of changes in arteriolar diameter (which returned rapidly to baseline). Moreover, the optical signal during SNS was increased by 50% by theophylline and by almost 5-fold by CPX (p<0.05). The area of change of the intrinsic signal was also increased by the topical application of theophylline and CPX. The somatosensory evoked potential recordings revealed no significant changes after theophylline application, but CPX caused a small diminution of the N1 wave (p<0.01). CONCLUSIONS: The noncongruent temporal profiles of the changes in pial arteriolar diameter and optical signal, imaged at 690 nm, indicate that the optical signal at 690 nm is not related to CBF. Alteration of adenosine receptor activity independently changed cortical activity, as measured by the optical signal, and CBF, as determined by pial arteriolar diameter. Manipulation of the adenosine receptor activity during increased cortical activity confirmed the temporal dissociation of optical signal and CBF and provided further evidence for the role of adenosine in regulating CBF.

Postischemic augmentation of conducted dilation in cerebral arterioles.

BACKGROUND AND PURPOSE: Conducted vasomotor responses likely play an important role in cerebrovascular regulation, but it is unclear how these responses may be affected by ischemia. The purpose of this study was to evaluate the hypothesis that cerebral ischemia and reperfusion (I/R) alters vascular conduction in cerebral arterioles. METHODS: Middle cerebral artery occlusion (MCAO) was induced by an intraluminal filament technique in 4 groups of rats: (A) 2-hour MCAO/24-hour reperfusion (n=14); (B) 2-hour MCAO/1-hour reperfusion (n=7); (C) 1-hour MCAO/24-hour reperfusion (n=6); and (D) 1-hour MCAO/1-hour reperfusion (n=5). Neurological status and infarction (2,3,5-triphenyltetrazolium chloride staining) were evaluated after I/R. Conducted vasomotor responses were assessed in intracerebral branches of the MCA, by following the longitudinal spread of vasodilation or vasoconstriction to localized microapplication of ATP or adenosine. RESULTS: Local microapplication of ATP evoked a biphasic constriction (17+/-3%) and dilation (7+/-2%) response, whereas adenosine elicited only dilation (11+/-2%). These local responses spread longitudinally along sham-control arterioles (1 mm conduction distance) with rapid spatial decay. Ischemia followed by 24-hour reperfusion (groups A and C) led to a marked potentiation of conducted dilation responses: dilation to ATP conducted with virtually no decay in I/R arterioles. Augmentation of conductivity was not observed in the 1-hour reperfusion groups (B and D). Moreover, I/R did not alter conducted constriction. CONCLUSIONS: Ischemia-reperfusion led to a specific augmentation of conducted vasodilation in cerebral arterioles. Presumably, enhanced conductivity may improve cerebral perfusion after ischemia.

Time-dependent alterations in functional and pharmacological arteriolar reactivity after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Disturbances in cerebral arteriolar function, in addition to large vessel vasospasm, may be responsible for ischemia after subarachnoid hemorrhage. The purpose of this study was to test the hypothesis that subarachnoid hemorrhage alters cerebral microvascular reactivity. METHODS: An endovascular filament model was used to induce subarachnoid hemorrhage in halothane-anesthetized male Sprague-Dawley rats. We evaluated pial arteriolar responses to sciatic nerve stimulation, topically applied vasoactive agents (adenosine or sodium nitroprusside), and CO(2) inhalation in rats subjected to subarachnoid hemorrhage at 1 to 5 days after insult. RESULTS: In sham-operated rats, sciatic nerve stimulation evoked a 23.5+/-1.8% increase in arteriolar diameter, which was significantly attenuated to 13.7+/-0.9%, 12.8+/-2.5%, and 18.8+/-2.9% at 24, 48, and 72 hours after subarachnoid hemorrhage, respectively (P<0.05; n> or =7). At 96 and 120 hours after subarachnoid hemorrhage, sciatic nerve stimulation-induced dilation recovered to sham levels. Somatosensory-evoked potentials were unaltered by subarachnoid hemorrhage. Pial vasodilatation to adenosine (10 micromol/L) and sodium nitroprusside (1 micromol/L) were significantly impaired, by 47% and 41%, respectively, at 48 hours after subarachnoid hemorrhage (P<0.05; n=7). In contrast, CO(2) reactivity was unaffected by subarachnoid hemorrhage. CONCLUSIONS: Pial arteriolar responses to cortical activation may be decreased in the initial 2 to 3 days after experimental subarachnoid hemorrhage.

Effect of caffeine on cerebral blood flow response to somatosensory stimulation.

Despite caffeine's wide consumption and well-documented psychoactive effects, little is known regarding the effects of caffeine on neurovascular coupling. In the present study, we evaluated the effects of caffeine, an adenosine receptor antagonist, on intracerebral arterioles in vitro and subsequently, on the pial circulation in vivo during cortical activation induced by contralateral sciatic nerve stimulation (SNS). In our in vitro studies, we utilized isolated intracerebral arterioles to determine the effects of caffeine (10 or 50 micromol/L) on adenosine-induced vasodilatation. At the lower concentration, caffeine was without effect, but at the higher concentration, caffeine produced significant attenuation. In our in vivo studies, we determined the cerebrospinal fluid (CSF) caffeine concentrations at 15, 30, and 60 mins after intravenous administration of 5, 10 and 40 mg/kg. At the latter two concentrations, CSF levels exceeded 10 micromol/L. We then evaluated the pial arteriolar response during cortical activation caused by contralateral SNS after administering caffeine intravenously (0, 5, 10, 20 30, and 40 mg/kg). The pial circulation was observed through a closed cranial window in chloralose-anesthetized Sprague-Dawley rats. The contralateral sciatic nerve was isolated, positioned on silver electrodes and stimulated for 20 secs (0.20 V, 0.5 ms, and 5 Hz). Arteriolar diameter was quantified using an automated video dimension analyzer. Contralateral SNS resulted in a 23.8% +/-3.9% increase in pial arteriolar diameter in the hindlimb sensory cortex under control conditions. Intravenous administration of caffeine at the lowest dose studied (5 mg/kg) had no effect on either resting arteriolar diameter or SNS-induced vasodilatation. However, at higher doses (10, 20, 30, and 40 mg/kg, intravenously), caffeine significantly (P < 0.05; n = 6) attenuated both resting diameter and cerebral blood flow (CBF) responses to somatosensory stimulation. Intravenous administration of theophylline (10, 20, and 40 mg/kg), another adenosine receptor antagonist, also significantly reduced SNS-induced vasodilatation in a dose-dependent manner. Hypercarbic vasodilatation was unaffected by either caffeine or theophylline. The results of the present study show that caffeine significantly reduces cerebrovascular responses to both adenosine and to somatosensory stimulation and supports a role of adenosine in the regulation of CBF during functional neuronal activity.

Methods for isolation and characterization of intracerebral arterioles in the C57/BL6 wild-type mouse.

Vascular control mechanisms have been studied extensively in mice. However, an in vitro characterization of penetrating intracerebral arterioles has not been reported. We describe methods for isolation and cannulation for mouse intracerebral arterioles. This technique allows analysis of mouse cerebral arteriolar physiology and pharmacology without the confounding influences of the surrounding brain elements. Penetrating intracerebral arterioles from adult C57/BL6 wild-type (WT) mice were isolated at 4 degrees C, transferred to an inverted microscope and cannulated at both ends using a dual glass micropipette system, wherein intraluminal flow (0.2 microl/min) and pressure (60 mmHg) were maintained. The arterioles developed spontaneous tone when the chamber was warmed to 37 degrees C, with the resulting diameter reaching 68.4+/-0.9% of passive diameter (29.8+/-1.1 microm). After the development of spontaneous tone, incremental changes in luminal pressure from 20 to 140 mmHg induced myogenic responses. Acidosis (pH 6.8) and alkalosis (pH 7.6) caused dilation (20.0+/-1.4%) and constriction (17.2+/-1.4%), respectively. Extraluminal adenosine (ADO (10 microM); 24.3+/-3.6%) and sodium nitroprusside (SNP (10 microM); 28.6+/-4.1%) and intraluminal adenosine 5'-triphosphate (ATP (10 microM); 20.0+/-3.9%) resulted in vasodilation similar in magnitude to that observed in rat arterioles. This information provides a foundation for elucidating cerebral vascular control mechanisms in genetically engineered mice.

Systemic theophylline augments the blood oxygen level-dependent response to forepaw stimulation in rats.

BACKGROUND AND PURPOSE: Functional MR imaging with blood oxygen level-dependent (BOLD) contrast enhancement is believed to rely on changes in cerebral blood flow and deoxyhemoglobin level to estimate the location and degree of neural activation. We studied the relationship between neural activation and the observed BOLD response by using theophylline, an antagonist of the inhibitory neurotransmitter adenosine and a potent inhibitor of the vasodilatory response to neural activation. METHODS: Using a rat model with electrical forepaw stimulation, we performed fMRI measurements before and after the systemic injection of either theophylline (0.1 mmol/kg) or an equivalent volume of saline. Changes in the BOLD response were quantified by determining the number of activated voxels and the amplitude of the BOLD response for each animal in the theophylline and saline groups. RESULTS: The theophylline group had a significantly Tincreased BOLD response (70-150% increased activated voxel count and 60-65% increased BOLD response amplitude) at 45 and 60 minutes after systemic injection compared with baseline. The response of the saline-injected control group did not change significantly. CONCLUSION: The administration of systemic theophylline significantly augmented the BOLD response due to either an elevation of resting deoxyhemoglobin levels or the neuroexcitatory effect of theophylline. This effect potentially could be used in human fMRI studies to increase the sensitivity of the BOLD response.

Hippocampal injury and neurobehavioral deficits following hyperglycemic cerebral ischemia: effect of theophylline and ZM 241385.

OBJECT: The effects of the adenosine receptor antagonists theophylline (for A1 and A2) and ZM 241385 (for A2A) on hippocampal injury and Morris water maze (MWM) performance in rats were investigated following normoglycemic and hyperglycemic cerebral ischemia (induced by four vessel occlusion for 10 minutes). METHODS: Theophylline (36 mg/kg), ZM 241385 (1 mg/kg), or an equivalent volume of saline was administered to rats intraperitoneally 30 minutes before ischemia was induced. Moderate hyperglycemia was achieved by intraperitoneal administration of D-glucose (3 g/kg, 15 minutes before induction of ischemia). Morris water maze trials were performed on the 6th. 7th, and 8th days after ischemic insult. After the conclusion of the performance tests, the rat brains were cut into 8-microm sections, stained with cresyl violet and acid fuchsin, and evaluated in a blinded fashion to determine the extent of injury. Theophylline worsened injury in the hippocampus following normoglycemic and hyperglycemic ischemia. Moreover, theophylline significantly (p < 0.05, six animals) worsened latency and learning index (LI) scores during the MWM trials in both normoglycemic and hyperglycemic animals. On the other hand, ZM 241385 had no effect on either ischemic injury or MWM performance in normoglycemic animals. In the animals in the hyperglycemic ischemia group, however, ZM 241385 significantly (p < 0.05, five animals) reduced injury in the CA1 (94.6 +/- 1.7% compared with 79.2 +/- 10.9%), CA3 (26 +/- 12.5% compared with 11.2 +/- 4.3%), and hilum (22.4 +/- 8.1% compared with 11 +/- 5.5%) regions. In addition, ZM 241385 significantly improved latency (52 +/- 29.7 seconds compared with 24.8 +/- 11.2 seconds, p < 0.05) and LI scores (203.2 +/- 33.3 compared with 152.1 +/- 31.8, p < 0.05) in the MWM trials. A statistically significant correlation was also found between hippocampal injury (CA1, CA3, and hilum) and MWM performance. CONCLUSIONS: The results of this study provide further evidence for a neuromodulatory role of adenosine during normoglycemic and hyperglycemic ischemia.

Adenosine-induced modulation of excitatory amino acid transport across isolated brain arterioles.

OBJECT: Excitatory amino acid (EAA) uptake by neurons and glia acts synergistically with stereoselective transport across the blood-brain barrier (BBB) to maintain EAA homeostasis in the brain. The endogenous neuroprotectant adenosine counteracts many aspects of excitotoxicity by increasing cerebral blood flow and by producing pre- and postsynaptic actions on neurons. In the present study, the authors explored the effect of adenosine on EAA transport across the BBB. METHODS: The effects of adenosine on the permeability of the BBB and transport of aspartate and glutamate across the BBB were studied in a well-characterized isolated penetrating cerebral arteriole preparation suitable for simultaneous investigations of changes in diameter and permeability. At concentrations within the physiological to low pathophysiological range (10(-7)-10(-6) M), the net vectorial transport of [3H]L-glutamate or [3H]L-aspartate from blood to brain was significantly attenuated, whereas there was no effect of adenosine on paracellular BBB permeability to [14C]sucrose or [3H]D-aspartate. With higher concentrations of adenosine (10(-4) M and 10(-3) M) the net vectorial transport of [3H]L-glutamate and [3H]Laspartate returned toward baseline. At 10(-3) M, the permeability to [14C]sucrose was significantly altered, indicating a breakdown in the BBB. The effect of adenosine (10(-6) M) was blocked by theophylline, a blocker of the A1 and A2 receptors of adenosine. CONCLUSIONS: Adenosine-mediated modulation of glutamate and aspartate transport across the BBB is a novel physiological finding.

Impairment of intracerebral arteriole dilation responses after subarachnoid hemorrhage. Laboratory investigation.

OBJECT: Cerebrovascular dysfunction after subarachnoid hemorrhage (SAH) may contribute to ischemia, but little is known about the contribution of intracerebral arterioles. In this study, the authors tested the hypothesis that SAH inhibits the vascular reactivity of intracerebral arterioles and documented the time course of this dysfunction. METHODS: Subarachnoid hemorrhage was induced using an endovascular filament model in halothane-anesthetized male Sprague-Dawley rats. Penetrating intracerebral arterioles were harvested 2, 4, 7, or 14 days postinsult, cannulated using a micropipette system that allowed luminal perfusion and control of luminal pressure, and evaluated for reactivity to vasodilator agents. RESULTS: Spontaneous tone developed in all pressurized (60 mm Hg) intracerebral arterioles harvested in this study (from 66 rats), with similar results in the sham and SAH groups. Subarachnoid hemorrhage did not affect dilation responses to acidic pH (6.8) but led to a persistent impairment of endothelium-dependent dilation responses to adenosine triphosphate (p < 0.01), as well as a transient attenuation (p < 0.05) of vascular smooth muscle-dependent dilation responses to adenosine, sodium nitroprusside, and 8-Br-cyclic guanosine monophosphate (cGMP). Impairment of NO-mediated dilation was more sustained than adenosine- and 8-Br-cGMP-induced responses (up to 7 days postinsult compared with 2 days). All smooth muscle-dependent responses returned to sham levels by 14 days after SAH. CONCLUSIONS: Subarachnoid hemorrhage led to a persistent impairment of endothelium-dependent dilation and a transient attenuation of vascular smooth muscle-dependent dilation responses to adenosine. Impairment of NO-mediated dilation occurred when the response to cGMP was intact, suggesting a change in cGMP levels rather than an alteration in intracellular mechanisms downstream from cGMP.

Hippocampal injury and neurobehavioral deficits are improved by PD 81,723 following hyperglycemic cerebral ischemia.

We investigated the effects of PD 81,723, an allosteric enhancer for the adenosine A(1) receptor subtype, on hippocampal injury and Morris water maze (MWM) performance following hyperglycemic cerebral ischemia and reperfusion (4-VO, 10 min) in the rat. PD 81,723 (3 or 10 mg/kg) or the equivalent volume of saline was administered intraperitoneally 30 min prior to ischemia. Moderate hyperglycemia was achieved by administration of D-glucose (3g/kg, i.p.) 15 min prior to induction of ischemia. Morris water maze trials were performed on the 6th, 7th, and 8th days after the ischemic insult. The rat brains were sectioned (8 microm), stained with cresyl violet/acid fuchsin, and evaluated for hippocampal ischemic injury by an experimenter blinded to the treatment conditions. At the higher dose, PD 81,723 (10 mg/kg) had no effect on hippocampal injury or MWM performance following hyperglycemic ischemia compared to corresponding saline-treated animals. In contrast, a lower dose of PD 81,723 (3 mg/kg) resulted in significant (P < 0.05, n = 8) reduction of hippocampal injury following hyperglycemic ischemia. Furthermore, corresponding Morris water maze performance (latency, learning index, and cumulative distance swum) was significantly improved by PD 81,723 (P < 0.05, n = 8). The results of the present study suggest that, in the presence of PD 81,723, an A(1) allosteric enhancer, endogenously produced adenosine is sufficient to exert significant neuroprotection during hyperglycemic ischemia. Moreover, the present study provides further evidence for a neuromodulatory role of adenosine during hyperglycemic ischemia.

cGMP-dependent and not cAMP-dependent kinase is required for adenosine-induced dilation of intracerebral arterioles.

Adenosine (ADO) is a potent cerebral vasodilator and has been proposed as a metabolic regulator of cerebral blood flow. However, the signal transduction pathway by which ADO causes vasodilation in cerebral microvessels is currently unknown. The current study was designed to investigate the role of cyclic nucleotides and cyclic nucleotide-dependent protein kinases in ADO-induced dilation of resistance-sized rat cerebral arterioles that develop spontaneous tone. Arterioles were cannulated and perfused intraluminally at constant flow (2 microl/min) and pressure (60 mm Hg). ADO (29.7 +/- 2.0%; 1 microM), CGS-21680 (16 +/- 4%, 1 microM), 8-bromo-cyclic guanosine monophosphate (8 Br-cGMP; 29.9 +/- 3.9%; 100 microM), sodium nitroprusside (SNP; 30.6 +/- 3.3%, 1 microM), cyclic guanine monophosphate-dependent protein kinase activator (Sp-8-pCPT-cGMPS, 25.9 +/- 4.2%; 10 microM), forskolin (30.5 +/- 5.9%; 0.1 microM), and pH 6.8 all produced large dilations. The selective cGMP-dependent protein kinase inhibitor, Rp-8-pCPT-cGMPS (10 microM), had no effect on resting diameter or reactivity to acidic pH, but significantly ( < 0.05) attenuated arteriolar dilations to ADO (59%, n = 8), CGS-21680 (60%, n = 4), SNP (62%, n = 3), 8 Br-cGMP (88%, n = 3), and Sp-8-pCPT-cGMPS (98%, n = 3). H8, the less-selective cyclic nucleotide-dependent protein kinase inhibitor, had similar effects as Rp-8-pCPT-cGMPS. Additionally, the inhibitor of the soluble guanylate cyclase, 1H-[1,24]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), blocked the response to SNP (70% inhibition) and significantly inhibited the ADO response (43% inhibition). In contrast, inhibition of the cyclic ADO monophosphate (cAMP)-dependent protein kinase Rp-8-CPT-cAMPS had no effect on the ADO, SNP, or pH responses, but significantly blocked forskolin-induced vasodilation (53%). It is concluded that ADO-induced vasodilation in cerebral microvessels, at least in part, involves cGMP and cGMP-dependent protein kinase, but not cAMP or cAMP-dependent kinase. Our data therefore provides a new insight into mechanisms by which ADO invokes vasodilation in cerebral microvascular arterioles.