Lysophosphatidic Acid Reduces Microregional Oxygen Supply/Consumption Balance after Cerebral Ischemia-Reperfusion.

BACKGROUND: Lysophosphatidic acid (LPA) is a small phospholipid-signaling molecule, which can alter responses to stress in the central nervous system. OBJECTIVE: We hypothesized that exogenous LPA would increase the size of infarct and reduce microregional O2 supply/consumption balance after cerebral ischemia-reperfusion. METHODS: This was tested in isoflurane-anesthetized rats with middle cerebral artery blockade for 1 h and reperfusion for 2 h with or without LPA (1 mg/kg, at 30, 60, and 90 min after reperfusion). Regional cerebral blood flow was determined using a C14-iodoantipyrine autoradiographic technique. Regional small-vessel (20-60 µm in diameter) arterial and venous oxygen saturations were determined microspectrophotometrically. RESULTS: There were no significant hemodynamic or arterial blood gas differences between groups. The control ischemic-reperfused cortex had a similar O2 consumption to the contralateral cortex. However, microregional O2 supply/consumption balance was significantly reduced in the ischemic-reperfused cortex with many areas of low O2 saturation (43 of 80 veins with O2 saturation below 50%). LPA did not significantly alter cerebral blood flow, but it did significantly increase O2 extraction and consumption of the ischemic-reperfused region. It also significantly increased the number of small veins with low O2 saturations in the reperfused region (76 of 80 veins with O2 saturation below 50%). This was associated with a significantly increased cortical infarct size after LPA administration (11.4 ± 0.5% control vs. 16.4 ± 0.6% LPA). CONCLUSION: This suggests that LPA reduces cell survival and that it is associated with an increase in the number of small microregions with reduced local oxygen balance after cerebral ischemia-reperfusion.

Lysophosphatidic acid increased infarct size in the early stage of cerebral ischemia-reperfusion with increased BBB permeability.

BACKGROUND: We investigated whether exogenous lysophosphatidic acid (LPA), a phospholipid extracellular signaling molecule, would increase infarct size and blood-brain barrier (BBB) disruption during the early stage of cerebral ischemia-reperfusion, and whether it works through Akt-mTOR-S6K1 intracellular signaling. MATERIAL AND METHODS: Rats were given either vehicle or LPA 1 mg/kg iv three times during reperfusion after one hour of middle cerebral artery (MCA) occlusion. In another group, prior to administration of LPA, 30 mg/kg of PF-4708671, an S6K1 inhibitor, was injected. After one hour of MCA occlusion and two hours of reperfusion the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid and the volume of 3H-dextran distribution were determined to measure the degree of BBB disruption. At the same time, the size of infarct was determined and western blot analysis was performed to determine the levels of phosphorylated Akt (p-Akt) and phosphorylated S6 (pS6). RESULTS: LPA increased the Ki in the ischemic-reperfused cortex (+43%) when compared with Control rats and PF-4708671 pretreatment prevented the increase of Ki by LPA. LPA increased the percentage of cortical infarct out of total cortical area (+36%) and PF-4708671 pretreatment prevented the increase of the infarct size. Exogenous LPA did not significantly change the levels of p-Akt as well as pS6 in the ischemic-reperfused cortex. CONCLUSION: Our data demonstrate that the increase in BBB disruption could be one of the reasons of the increased infarct size by LPA. S6K1 may not be the major target of LPA. A decrease of LPA during early cerebral ischemia-reperfusion might be beneficial for neuronal survival.

Improvement in Microregional Oxygen Supply/Consumption Balance and Infarct Size After Cerebral Ischemia-Reperfusion With Inhibition of p70 Ribosomal S6 Kinase (S6K1).

BACKGROUND: We tested the hypothesis that inhibition of p70 ribosomal S6 kinase (S6K1) would decrease infarct size and improve microregional O2 supply/consumption balance after cerebral ischemia-reperfusion. METHODS: This was tested in isoflurane-anesthetized rats with middle cerebral artery blockade for 1 hour and reperfusion for 2 hours with or without PF-4708671 (S6K1 inhibitor, 75 mg/kg, 15 minutes after blockade). Regional cerebral blood flow was determined using a C14-iodoantipyrine autoradiographic technique. Regional small vessel (20-60 µm diameter) arterial and venous oxygen saturations were determined microspectrophotometrically. RESULTS: There were no significant hemodynamic or arterial blood gas differences between groups. The control ischemic-reperfused cortex had a similar O2 consumption to the contralateral cortex. However, microregional O2 supply/consumption balance was significantly reduced in the ischemic-reperfused cortex with many areas of low O2 saturation (23 of 80 veins with O2 saturation below 45%). PF-4708671 did not significantly alter cerebral blood flow or O2 consumption. However, it significantly reduced the number of small veins with low O2 saturations in the reperfused region (6 of 80 veins with O2 saturation below 45%). This was associated with a significantly reduced cortical infarct size after S6K1 inhibition (12.9 ± .8% control versus 6.6 ± .3% PF-4708671). CONCLUSION: This suggests that S6K1 inhibition is important for cell survival and that it reduces the number of small microregions with reduced local oxygen balance after cerebral ischemia-reperfusion.

Local O2 Balance in Cerebral Ischemia-Reperfusion Improved during Pentobarbital Compared with Isoflurane Anesthesia.

BACKGROUND: Most anesthetics affect cerebral blood flow and metabolism. We compared microregional O2 balance in cerebral ischemia-reperfusion during pentobarbital and isoflurane anesthesia. METHODS: After 1 hour of middle cerebral artery occlusion and a 2-hour reperfusion under isoflurane (1.4%, n = 14) or pentobarbital (50 mg/kg, n = 14) anesthesia in rats, regional cerebral blood flow using (14)C-iodoantipyrine autoradiography, microregional arterial and venous O2 saturation (20-60 µm in diameter) using cryomicrospectrophotometry, and the size of cortical infarct were determined. RESULTS: Ischemia-reperfusion decreased the average cortical venous O2 saturation in both pentobarbital and isoflurane groups (P < .0001), which was higher (P < .05) with pentobarbital despite a similar average regional cerebral blood flow and O2 consumption. The heterogeneity of venous O2 saturation reported as a coefficient of variation (100 × standard deviation/mean) was smaller (P < .005) with pentobarbital than that with isoflurane (7.5 versus 16.1). The number of veins with low venous O2 saturation (<50%) was smaller (P < .005) with pentobarbital (5 of 80 versus 24 of 80). The percentage of cortical infarct in total cortex was smaller with pentobarbital (5.2 ± 2.5% versus 12.3 ± 2.6%, P < .001). CONCLUSIONS: In the cerebral ischemic-reperfused cortex, the average venous O2 saturation was higher, and its heterogeneity and the number of veins with low O2 saturation were smaller under pentobarbital than isoflurane anesthesia. This improvement in microregional O2 balance with pentobarbital was accompanied by the reduced cortical infarct. Our data suggest that the neurologic outcome could vary during cerebral ischemia-reperfusion depending on the anesthetics used.

Effects of dexmedetomidine on microregional O2 balance during reperfusion after focal cerebral ischemia.

BACKGROUND: This study was performed to determine whether there is an association between microregional O2 balance and neuronal survival in cerebral ischemia-reperfusion using dexmedetomidine, an α2-adrenoreceptor agonist and a sedative. METHODS: Rats were subjected to 1 hour middle cerebral artery occlusion and a 2-hour reperfusion. During reperfusion, normal saline (n = 14) or dexmedetomidine 1 µg/kg/minute (n = 14) was infused intravenously. At 2 hours of reperfusion, regional cerebral blood flow using (14)C-iodoantipyrine autoradiography, microregional arterial and venous (20-60 µm in diameter) O2 saturation (SvO2) using cryomicrospectrophotometry, and the size of cortical infarction were determined. RESULTS: Ischemia-reperfusion decreased microregional SvO2 (52.9 ± 3.7% vs. 61.1 ± .6%, P < .005) with increased variation or heterogeneity (P < .0001) with similar regional cerebral blood flow and O2 consumption. Dexmedetomidine during reperfusion decreased the heterogeneity of SvO2 that was analyzed with an analysis of variance (P < .01) and reported as coefficient of variation (100 × standard deviation/Mean) (11.8 vs. 16.4). The number of veins with O2 saturation less than 50% decreased with dexmedetomidine (13/80 vs. 27/81, P < .01). The percentage of cortical infarct in total cortex was smaller with dexmedetomidine (8.3 ± 2.2% vs. 12.6 ± 1.5%, P < .005). CONCLUSIONS: In the cerebral ischemic reperfused cortex, dexmedetomidine decreased the heterogeneity of SvO2 and the number of small veins with low O2 saturation suggesting improved microregional O2 supply/consumption balance. The improvement was accompanied by the reduced size of cortical infarction.

Inhibition of p70 Ribosomal S6 Kinase (S6K1) Reduces Cortical Blood Flow in a Rat Model of Autism-Tuberous Sclerosis.

The manifestations of tuberous sclerosis complex (TSC) in humans include epilepsy, autism spectrum disorders (ASD) and intellectual disability. Previous studies suggested the linkage of TSC to altered cerebral blood flow and metabolic dysfunction. We previously reported a significant elevation in cerebral blood flow in an animal model of TSC and autism of young Eker rats. Inhibition of the mammalian target of rapamycin (mTOR) by rapamycin could restore normal oxygen consumption and cerebral blood flow. In this study, we investigated whether inhibiting a component of the mTOR signaling pathway, p70 ribosomal S6 kinase (S6K1), would yield comparable effects. Control Long Evans and Eker rats were divided into vehicle and PF-4708671 (S6K1 inhibitor, 75 mg/kg for 1 h) treated groups. Cerebral regional blood flow (14C-iodoantipyrine) was determined in isoflurane anesthetized rats. We found significantly increased basal cortical (+ 32%) and hippocampal (+ 15%) blood flow in the Eker rats. PF-4708671 significantly lowered regional blood flow in the cortex and hippocampus of the Eker rats. PF-4708671 did not significantly lower blood flow in these regions in the control Long Evans rats. Phosphorylation of S6-Ser240/244 and Akt-Ser473 was moderately decreased in Eker rats but only the latter reached statistical significance upon PF-4708671 treatment. Our findings suggest that moderate inhibition of S6K1 with PF-4708671 helps to restore normal cortical blood flow in Eker rats and that this information might have therapeutic potential in tuberous sclerosis complex and autism.

Cerebral ischemia and reperfusion increases the heterogeneity of local oxygen supply/consumption balance.

BACKGROUND AND PURPOSE: After cerebral vessel blockage, local blood flow and O2 consumption becomes lower and oxygen extraction increases. With reperfusion, blood flow is partially restored. We examined the effects of ischemia-reperfusion on the heterogeneity of local venous oxygen saturation in rats in order to determine the pattern of microregional O2 supply/consumption balance in reperfusion. METHODS: The middle cerebral artery was blocked for 1 hour using the internal carotid approach in 1 group (n=9) and was then reperfused for 2 hours in another group (n=9) of isoflurane-anesthetized rats. Regional cerebral blood flow was determined using a C(14)-iodoantipyrine autoradiographic technique. Regional small vessel arterial and venous oxygen saturations were determined microspectrophotometrically. RESULTS: After 1 hour of ischemia, local cerebral blood flow (92±10 versus 50±10 mL/min per 100 g) and O2 consumption (4.5±0.6 versus 2.7±0.5 mL O2/min per 100 g) decreased compared with the contralateral cortex. Oxygen extraction increased (4.7±0.2 versus 5.4±0.3 mL O2/100 mL) and the variation in small vein (20-60 µm) O2 saturation as determined by its coefficient of variation (=100×SD/mean) increased (5.5 versus 10.5). With 2 hours of reperfusion, the blood flow decrement was reduced and O2 consumption returned to the value in the contralateral cortex. Oxygen extraction remained elevated in the ischemic-reperfused area and the coefficient of variation of small vein O2 saturation increased further (17.3). CONCLUSIONS: These data indicated continued reduction of O2 supply/consumption balance with reperfusion. They also demonstrated many small regions of low oxygenation within the reperfused cortical region.

Effects of the thioredoxin-1 inhibitor PX-12 on blood-brain barrier permeability in the early stage of focal cerebral ischemia.

BACKGROUND/AIMS: Since a thioredoxin-1 (Trx-1) inhibitor, 1-methylpropyl-2-imidazolyl disulfide (PX-12) which is an antitumor agent, significantly decreased vascular permeability in tumor xenografts within a few hours of treatment, we tested whether PX-12 would attenuate blood-brain barrier (BBB) disruption in the early stage of focal cerebral ischemia and whether its action could be affected by vascular endothelial growth factor (VEGF) which interacts with the Trx-1 system. METHODS: In rats, 40 min after intravenous infusion of either 25 mg/kg of PX-12 (PX-12 group) or normal saline (control group), a middle cerebral artery (MCA) was occluded. In half of each group, VEGF (10(-10) mol/l) was applied topically in the ischemic cortex (IC). Ninety minutes after MCA occlusion, the transfer coefficient (Ki) of (14)C-α-aminoisobutyric acid and the volume of (3)H-dextran distribution were determined to measure the degree of BBB disruption. VEGF protein levels were determined using Western blot analysis. RESULTS: MCA occlusion increased the Ki in the control (+196%) as well as in the PX-12-treated rats (+90%), but the Ki of the IC of the PX-12 group was lower (-42%) than that of the control rats. VEGF protein levels were decreased in both the IC (-9.5%) and the contralateral cortex (CC; -10.2%) with PX-12 treatment. In the VEGF-treated rats, PX-12 also attenuated (-41%) the Ki of the IC. The difference in the volume of dextran distribution between the IC and the CC became insignificant with PX-12 treatment with or without VEGF application. CONCLUSION: Our data demonstrated that PX-12 was effective in decreasing BBB disruption in the early stage of focal cerebral ischemia and that VEGF is not an important factor involved in the action of PX-12 on BBB permeability.

Leucine Reduced Blood-Brain Barrier Disruption and Infarct Size in Early Cerebral Ischemia-Reperfusion.

A disruption of the blood-brain barrier (BBB) is a crucial pathophysiological change that can impact the outcome of a stroke. Ribosomal protein S6 (S6) and protein kinase B (Akt) play significant roles in early cerebral ischemia-reperfusion injury. Studies have suggested that branched-chain amino acids (BCAAs) may have neuroprotective properties for spinal cord or brain injuries. Therefore, we conducted research to investigate if leucine, one of the BCAAs, could offer neuroprotection and alter BBB disruption, along with its effects on the phosphorylation of S6 and Akt during the early phase of cerebral ischemia-reperfusion, specifically within the thrombolytic therapy time window. In rats, ten min after left middle cerebral artery occlusion (MCAO), 5 µL of 20 mM L-leucine or normal saline was injected into the left lateral ventricle. After two hours of reperfusion following one hour of MCAO, we determined the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid to assess the BBB disruption, infarct size, and phosphorylation of S6 and Akt. Ischemia-reperfusion increased the Ki (+143%, p < 0.001) and the intra-cerebroventricular injection of leucine lowered the Ki in the ischemic-reperfused cortex (-34%, p < 0.001). Leucine reduced the percentage of cortical infarct (-42%, p < 0.0001) out of the total cortical area. Ischemia-reperfusion alone significantly increased the phosphorylation of both S6 and Akt (p < 0.05). However, the administration of leucine had no further effect on the phosphorylation of S6 or Akt in the ischemic-reperfused cortex. This study suggests that an acute increase in leucine levels in the brain during early ischemia-reperfusion within a few hours of stroke may offer neuroprotection, possibly due to reduced BBB disruption being one of the major contributing factors. Leucine did not further increase the already elevated phosphorylation of S6 or Akt by ischemia-reperfusion under the current experimental conditions. Our data warrant further studies on the effects of leucine on neuronal survival and its mechanisms in the later stages of cerebral ischemia-reperfusion.

Effects of cannabinoid receptor agonist WIN 55,212-2 on blood-brain barrier disruption in focal cerebral ischemia in rats.

This study was performed to investigate whether WIN 55,212-2 (WIN), a cannabinoid receptor agonist, could attenuate blood-brain barrier (BBB) disruption in focal cerebral ischemia in rats and whether the CB 1 receptor antagonist rimonabant could prevent this attenuation. A total of 0.3 or 1 mg/kg of WIN was injected intravenously before and after permanent middle cerebral artery (MCA) occlusion. Some animals were pretreated with rimonabant 2 mg/kg i.p. before receiving 0.3 mg/kg of WIN. At 1 h after MCA occlusion, BBB permeability was determined by measuring the transfer coefficient (K(i)) of (14)C-α-aminoisobutyric acid and the volume of dextran distribution. With MCA occlusion, K(i) increased in the ischemic cortex (IC) in all of the experimental groups. However, the K(i) of the IC of the WIN 0.3 and 1 mg/kg groups was lower (­46 and ­42%, respectively, p < 0.05) than that of the control group. With rimonabant pretreatment, the K(i) of the IC became higher ((+)88%, p < 0.05) than with WIN 0.3 mg/kg alone and similar to that of the control rats. The difference in the volume of dextran distribution between the IC and the contralateral cortex was significant in the control but not in the WIN-treated rats. With rimonabant pretreatment, however, the difference became significant. Our data demonstrated that WIN could attenuate BBB disruption in focal cerebral ischemia and this attenuation could be prevented with rimonabant. Our data suggest an involvement of CB(1) receptors in the regulation of BBB disruption in the early stage of stroke.

The effects of dexmedetomidine on regional cerebral blood flow and oxygen consumption during severe hemorrhagic hypotension in rats.

BACKGROUND: We performed this study to determine how dexmedetomidine would affect regional cerebral blood flow (rCBF) and microregional O(2) consumption during nonhemorrhagic normovolemia and during severe hemorrhagic hypotension in rats. METHODS: Forty-eight male rats were anesthetized with isoflurane and their lungs were mechanically ventilated. Half of the rats were bled to reach a mean arterial blood pressure of 40 to 45 mm Hg and were maintained at this level for at least 30 minutes before rCBF or microregional arterial oxygen saturation (Sao(2)) and venous oxygen saturation (Svo(2)) were determined. The other half were not bled and served as nonhemorrhagic controls. Half of each group was given dexmedetomidine 1 µg/kg/min IV for 45 minutes and the other half was given the same amount of normal saline infusion. The infusion started 10 minutes before blood withdrawal for the hemorrhagic groups. The rCBF was determined using (14)C-iodoantipyrine, and the microregional Sao(2) and Svo(2) were determined using cryomicrospectrophotometry at 45 minutes of infusion. RESULTS: Dexmedetomidine decreased heart rate by 25%, but the decrease of mean arterial blood pressure was not significant. The total amount of blood withdrawn and hemoglobin were similar between the normal saline-treated and the dexmedetomidine-treated groups. In normovolemia, dexmedetomidine significantly decreased rCBF (-58%) in the lateral cortex with a similar percentage decrease (-57%) of calculated O(2) consumption. Microregional Svo(2) was similar between the normal saline-treated group (62.8% ± 1.3% [mean ± SD]) and the dexmedetomidine-treated group (60.7% ± 1.8%) despite a large difference in rCBF. Hemorrhage significantly decreased rCBF (-44%) in the lateral cortex in the normal saline-treated rats with no significant change in regional cerebrovascular resistance. In contrast, in the lateral cortex of the dexmedetomidine-treated rats, the decrease of rCBF was not significant but there was a significant decrease in regional cerebrovascular resistance. A decrease (-25%) in the O(2) consumption was observed in the lateral cortex of the normal saline-treated rats with hemorrhage, but hemorrhage did not decrease O(2) consumption in the dexmedetomidine-treated rats. Despite significantly lower rCBF (-34%) in the dexmedetomidine-treated rats, the Svo(2) was similar between the normal saline-treated (42.8% ± 2.5%) and the dexmedetomidine-treated rats (43.2% ± 2.7%). CONCLUSIONS: Our data showed that in normovolemia, dexmedetomidine produced a proportionate decrease of rCBF and O(2) consumption. Hemorrhage decreased rCBF more than O(2) consumption. Dexmedetomidine prevented rCBF and O(2) consumption from decreasing after hemorrhage. Our data suggest that dexmedetomidine may help provide optimal O(2) supply and consumption balance during hemorrhage.

Rapalink-1 Increased Infarct Size in Early Cerebral Ischemia-Reperfusion With Increased Blood-Brain Barrier Disruption.

It has been reported that the mechanistic target of rapamycin (mTOR) pathway is involved in cerebral ischemia-reperfusion injury. One of the important pathological changes during reperfusion after cerebral ischemia is disruption of blood-brain barrier (BBB). Rapamycin, a first-generation mTOR inhibitor, produces divergent effects on neuronal survival and alteration in BBB disruption. In this study, we investigated how Rapalink-1, a third-generation mTOR inhibitor, would affect neuronal survival and BBB disruption in the very early stage of cerebral ischemia-reperfusion that is within the time window of thrombolysis therapy. The middle cerebral artery occlusion (MCAO) was performed in rats under isoflurane anesthesia with controlled ventilation. Of note, 2 mg/kg of Rapalink-1 or vehicle was administered intraperitoneally 10 min after MCAO. After 1 h of MCAO and 2 h of reperfusion, the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid (104 Da) and the volume of 3H-dextran (70,000 Da) distribution were determined to assess the degree of BBB disruption. At the same time points, phosphorylated S6 (Ser240/244) and Akt (Ser473) as well as matrix metalloproteinase-2 (MMP2) protein level were determined by Western blot along with the infarct size using tetrazolium stain. Rapalink-1 increased the Ki in the ischemic-reperfused cortex (IR-C, +23%, p < 0.05) without a significant change in the volume of dextran distribution. Rapalink-1 increased the percentage of cortical infarct out of the total cortical area (+41%, p < 0.005). Rapalink-1 significantly decreased phosphorylated S6 and Akt to half the level of the control rats in the IR-C, which suggests that both of the mechanistic target of rapamycin complex 1 and 2 (mTORC1 and mTORC2) were inhibited. The MMP2 level was increased suggesting that BBB disruption could be aggravated by Rapalink-1. Taken together, our data suggest that inhibiting both mTORC1 and mTORC2 by Rapalink-1 could worsen the neuronal damage in the early stage of cerebral ischemia-reperfusion and that the aggravation of BBB disruption could be one of the contributing factors.

Inhibition of serum and glucocorticoid regulated kinases by GSK650394 reduced infarct size in early cerebral ischemia-reperfusion with decreased BBB disruption.

Blood-brain barrier (BBB) disruption is one of the most important pathological changes following cerebral ischemia-reperfusion. We tested whether inhibition of the serum and glucocorticoid regulated kinase 1 (SGK1) would decrease BBB disruption and contribute to decreasing infarct size in the first few hours of cerebral ischemia-reperfusion within the thrombolysis therapy time window. After transient middle cerebral artery occlusion (MCAO), an SGK1 inhibitor GSK650394, or vehicle was administered into the lateral ventricle of rats. After one hour of MCAO and two hours of reperfusion, we determined BBB disruption using the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid, and also determined infarct size, phosphorylation of NDRG1, and MMP2 protein level. Ischemia-reperfusion increased (+34%, p < 0.05) and GSK650394 decreased (-25%, p < 0.05) the Ki in the ischemic-reperfused cortex. GSK650394 decreased the percentage of cortical infarct (-31%, p < 0.001). At the same time GSK650394 reduced NDRG1 phosphorylation and MMP2 protein level in the ischemic-reperfused cortex suggesting that SGK1 was inhibited by GSK650394 and that lower MMP2 could be one of the mechanisms of decreased BBB disruption. Collectively our data suggest that GSK650394 could be neuroprotective and one of the mechanisms of the neuroprotection could be decreased BBB disruption. SGK1 inhibition within the thrombolysis therapy time window might reduce cerebral ischemia-reperfusion injury.

Adhesion molecule L1 inhibition increases infarct size in cerebral ischemia-reperfusion without change in blood-brain barrier disruption.

OBJECTIVE: Neural cell adhesion molecule L1CAM (L1) is involved in neuroprotection. To investigate a possible neuroprotective effect of L1 during ischemia, we determined whether blocking L1 with an antagonistic antibody would worsen the outcome of focal cerebral ischemia-reperfusion and increase blood-brain barrier (BBB) disruption. METHODS: Transient middle cerebral artery occlusion (MCAO) was performed in anesthetized rats. Five µg of antagonistic mouse IgG monoclonal L1 antibody 324 or non-immune control mouse IgG was applied on the ischemic-reperfused cortex during one hour of MCAO and two hours of reperfusion. At two hours of reperfusion, BBB permeability, size of infarct using tetrazolium staining, number of TUNEL-labeled apoptotic cells, and immunohistochemistry for expression of PTEN and p53 were studied. RESULTS: The antagonistic L1 antibody 324 increased the percentage of cortical infarct area (+36%), but did not affect BBB permeability in the ischemic-reperfused cortex. The antagonistic L1 antibody increased number of apoptotic neurons and p53 expression, but decreased PTEN expression. CONCLUSION: Functional antagonism of L1 increases infarct size by increasing numbers of apoptotic neurons without affecting BBB permeability during the early stage of cerebral ischemia-reperfusion. Our data suggest that L1 affects primarily the brain parenchyma rather than BBB during early stages of cerebral ischemia-reperfusion and that endogenous brain L1 may be neuroprotective.

The effects of isoflurane pretreatment on cerebral blood flow, capillary permeability, and oxygen consumption in focal cerebral ischemia in rats.

BACKGROUND: We performed experiments to test whether isoflurane pretreatment produces vascular effects, especially at the levels of arterioles and capillaries affecting regional cerebral blood flow (rCBF), O(2) supply and consumption, or capillary permeability in focal cerebral ischemia. Because inducible nitric oxide synthase (iNOS) was implicated as one of the mechanisms of isoflurane preconditioning, the effect of iNOS inhibition on rCBF was also studied. METHODS: Twenty-four hours before middle cerebral artery (MCA) occlusion, rats were pretreated with 2% isoflurane for 30 minutes using an endotracheal tube and mechanical ventilation for the isoflurane preconditioned (IsoPC) group. For the group of iNOS inhibition, aminoguanidine 200 mg/kg was injected IP 30 minutes before isoflurane pretreatment. One hour after MCA occlusion, rCBF was measured using (14)C-iodoantipyrine autoradiography. Alternate slices of the tissue were used to determine arteriolar and venular O(2) saturation using cryo microspectrophotometry. Capillary permeability was determined by measuring the transfer coefficient (Ki) of (14)C-alpha-aminoisobutyric acid. Additional measurements of rCBF were performed at 3 hours after MCA occlusion. RESULTS: MCA occlusion decreased rCBF and O(2) consumption and increased Ki in both the control and the IsoPC groups at 1 hour after MCA occlusion. In the ischemic cortex (IC), the rCBF and O(2) consumption were significantly greater in the IsoPC group than in the control group (+40% and +41%, respectively), but they were similar in the contralateral cortex between the 2 groups. There was no difference in Ki between the groups in the IC or in the contralateral cortex. The increase of rCBF in the IC (+50%) was sustained in the IsoPC group at 3 hours after MCA occlusion. With iNOS inhibition, the increase of rCBF in the IC with isoflurane pretreatment became insignificant. CONCLUSIONS: Our data demonstrate that isoflurane pretreatment improved rCBF and increased the regional O(2) supply and consumption in the focal ischemic area but did not affect capillary permeability during the early stage of focal cerebral ischemia. The isoflurane-induced increase in rCBF in the ischemic area became insignificant with inhibition of iNOS.

Effects of blockade of ionotropic glutamate receptors on blood-brain barrier disruption in focal cerebral ischemia.

To determine whether blockade of ionotropic glutamate receptors such as NMDA or AMPA receptors would attenuate blood-brain barrier (BBB) disruption in focal cerebral ischemia, 15 min before middle cerebral artery (MCA) occlusion, CGS-19755 or NBQX was injected intraperitoneally in rats. At 1 h after MCA occlusion, BBB permeability was determined by measuring the transfer coefficient (K(i)) of (14)C-α-aminoisobutyric acid and the volume of dextran distribution. With MCA occlusion, K(i) was increased in the ischemic cortex (IC) (316%). CGS-19755 attenuated the increase in K(i) in the IC (-46%), but NBQX did not significantly decrease it. The difference in the volume of dextran distribution between the IC and the contralateral cortex became insignificant with the blockade of NMDA or AMPA receptors. Our data demonstrated that blockade of NMDA or AMPA receptors could attenuate the BBB disruption in focal cerebral ischemia and suggest that ionotropic glutamate receptors are involved in part in BBB disruption.

Effects of fentanyl pretreatment on regional cerebral blood flow in focal cerebral ischemia in rats.

BACKGROUND: There are reports that opioid preconditioning induces opioid-receptor-dependent neuroprotection against cerebral ischemia. This experiment was performed to test whether pretreatment with fentanyl, a synthetic primary mu-opioid receptor agonist, would affect the regional cerebral blood flow (rCBF) in focal cerebral ischemia in rats. METHODS: Twenty-four hours before permanent and unilateral middle cerebral artery (MCA) occlusion, rats were pretreated with normal saline, 200 microg/kg of fentanyl i.p. or 500 microg/kg of fentanyl i.p. The rats were anesthetized with isoflurane and were mechanically ventilated to cannulate the vessels and to occlude MCA. One hour after MCA occlusion, the rCBF was measured using (14)C-iodoantipyrine. RESULTS: The cortical rCBF decreased 1 h after MCA occlusion in all the experimental groups. In the ischemic cortex, the rCBF of the rats treated with 500 microg/kg of fentanyl was significantly greater (+80%, p < 0.05) than that of the control animals. The rCBF of the ischemic cortex of the rats treated with fentanyl 200 microg/kg seemed higher than in the control animals, but the difference was not statistically significant. The rCBF was similar in the nonischemic brain regions such as the contralateral cortex or pons among the experimental groups. CONCLUSION: Our data demonstrated that pretreatment with fentanyl improved the rCBF in the focal ischemic area without change in rCBF in the nonischemic cortex. Our data suggest that fentanyl could be effective in improving the rCBF in the focal ischemic area when used as a preconditioning agent and that improvement of rCBF could be one of the contributing factors of neuroprotection by opioid preconditioning.

Effects of exogenous excitatory amino acid neurotransmitters on blood-brain barrier disruption in focal cerebral ischemia.

This study was performed to determine whether exogenous N-methyl-D: -aspartate (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) would aggravate blood-brain barrier (BBB) disruption in focal cerebral ischemia in rats. Forty-five minutes after middle cerebral artery (MCA) occlusion, one of the following patches was applied to the exposed ischemic cerebral cortex of each rat: normal saline (control), 10(-5) M AMPA, 10(-4) M AMPA, 10(-5) M NMDA, or 10(-4) M NMDA. At 1 h after MCA occlusion, BBB permeability was determined by measuring the transfer coefficient (Ki) of (14)C-alpha-aminoisobutyric acid ((14)C-AIB). In all experimental groups, the Ki of the ischemic cortex (IC) was higher than that of the corresponding contralateral cortex (CC). The Ki of the IC of the animals treated with 10(-4) M AMPA or 10(-4) M NMDA was higher (+41%: P < 0.05 and +33%: P < 0.05, respectively) than that of the control animals. Our data demonstrated that exogenous NMDA or AMPA could further aggravate the BBB disruption in focal cerebral ischemia. Any insult increasing the release of excitatory neurotransmitters could further aggravate BBB disruption and brain edema during the ischemic period.

Inhibition of p70 ribosomal S6 kinase 1 (S6K1) by PF-4708671 decreased infarct size in early cerebral ischemia-reperfusion with decreased BBB permeability.

It is not clear whether inhibition of p70 ribosomal S6 kinase 1 (S6K1) is neuroprotective in cerebral ischemia-reperfusion. Decreasing blood-brain barrier (BBB) disruption has been associated with a better neuronal outcome in cerebral ischemia. We hypothesized that inhibition of S6K1 would decrease BBB disruption and infarct size in the early stage of cerebral ischemia-reperfusion. Middle cerebral artery occlusion (MCAO) was performed in rats under isoflurane anesthesia with controlled ventilation. 75 mg/kg of PF-4708671, an S6K1 inhibitor, was administered intraperitoneally 15 min after MCAO. After 1 h of MCAO and 2 h of reperfusion, the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid and the volume of 3H-dextran distribution were determined to assess the degree of BBB disruption. At the same time point, phosphorylated Rictor (pT1135) and the infarct size were measured to evaluate S6K1 activity. In the PF-4708671 treated rats, the Ki of the ischemic-reperfused cortex was lower than the untreated rats (-22%, P < 0.05) and the volume of dextran distribution was significantly lower in most brain regions. With PF-4708671, a significant decrease in pT1135 Rictor was observed and the percentage of cortical infarct out of total cortical area was decreased (11.6 ±â€¯2.0% vs 7.2 ±â€¯1.1%, P < 0.0001). Our data demonstrate that PF-4708671 decreased the size of the cortical infarct in the ischemic-reperfused cortex with a decrease in BBB disruption suggesting that inhibition of S6K1 may induce neuronal survival in early cerebral ischemia-reperfusion and that a decrease of BBB disruption could be one of the contributing factors.

Cerebral O(2) consumption in young Eker rats, effects of GABA blockade: implications for autism.

Since there is a strong correlation between tuberous sclerosis and autism, we used a tuberous sclerosis model (Eker rat) to test the hypothesis that the increased regional cerebral O(2) consumption in the Eker rat might be associated with autism. We also examined whether this increased cerebral O(2) consumption was related to changes in the activity of the gamma-aminobutyric acid (GABA) inhibitory system. Young (4 weeks) male control Long Evans (n=14) and Eker (n=14) rats (70-100g) were divided into control and bicuculline (1mg/kg/min for 2 min then 0.1mg/kg/min for 13 min, GABA(A) receptor antagonist) treated animals. Cerebral regional blood flow ((14)C-iodoantipyrine) and O(2) consumption (cryomicrospectrophotometry) were determined in isoflurane anesthetized rats. We found significantly increased basal O(2) consumption in the cortex (6.3+/-0.7 ml O(2)/min/100g Eker vs. 5.1+/-0.2 ml O(2)/min/100g control), hippocampus and cerebellum, but not the pons. Regional cerebral blood flow was also elevated in the cortex and hippocampus in Eker rats at baseline, but cerebral O(2) extractions were similar. Bicuculline significantly increased O(2) consumption in the cortex (6.5+/-0.3) and all other regions of the control rats, but had no effect on cortex (5.9+/-1.5) or other regions of the Eker rats. Cerebral blood flow followed a similar pattern. In conclusion, Eker rats had significantly elevated cerebral O(2) consumption and blood flow, but this was not affected by GABA receptor blockade. This suggested a reduced activity of the GABA(A) receptor in the brains of Eker rats. This may have important implications in the treatment of autism.