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.

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.

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.

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.

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.

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.

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.

Activation of Akt by SC79 decreased cerebral infarct in early cerebral ischemia-reperfusion despite increased BBB disruption.

Activation of Akt has been suggested to produce neuronal protection in cerebral ischemia. Decreasing blood-brain barrier (BBB) disruption has been associated with a better neuronal outcome in cerebral ischemia. We hypothesized that activation of Akt would decrease BBB disruption and contribute to decreasing the size of infarct in the early stage of cerebral ischemia-reperfusion within the therapeutic window. Transient middle cerebral artery occlusion (MCAO) was performed in rats under isoflurane anesthesia with controlled ventilation. Rats were treated with SC79 (a selective Akt activator which is cell and BBB permeable) 0.05 mg/kg × 3 i.p. or vehicle i.p. perioperatively. After one hour of MCAO and two hours of reperfusion, the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid (14C-AIB, molecular weight 104 Da) and the volume of 3H-dextran (molecular weight 70,000 Da) distribution were determined to measure the degree of BBB disruption. At the same time point, the size of infarction was determined using tetrazolium staining. In an additional group of rats, a higher dose of SC79 (0.5 mg/kg × 3) was administered to determine the size of infarct. Administration of SC79 increased the Ki in the ischemic-reperfused cortex (IR-C, +32%, p < 0.05) as well as in the contralateral cortex (CC, +35%, p < 0.05) when compared with the untreated animals with MCAO/reperfusion. The volume of dextran distribution was not significantly changed by SC79. SC79 treatment significantly produced a decrease in the percentage of cortical infarct out of total cortical area (12.7 ±â€¯1.7% vs 6.9 ±â€¯0.9%, p < 0.001). Increasing the dose of SC79 by ten times did not significantly affect the size of cortical infarct. Contrary to our hypothesis, our data demonstrated that SC79 decreased the size of the infarct in the ischemic-reperfused cortex despite an increase in BBB disruption. Our data suggest the importance of activation of Akt for neuronal survival in the early stage of cerebral ischemia-reperfusion within the therapeutic window and that the mechanism of neuroprotection may not be related to the BBB effects of SC79.

Akt activation improves microregional oxygen supply/consumption balance after cerebral ischemia-reperfusion.

There have been reports that activation of Akt may provide neuroprotection after cerebral ischemia-reperfusion. We tested the hypothesis that activation of Akt would decrease infarct size and improve microregional O2 supply/consumption balance after cerebral ischemia-reperfusion. This hypothesis was tested in isoflurane-anesthetized rats with middle cerebral artery blockade for 1 h and reperfusion for 2 h with or without SC-79 (Akt activator, 0.05 mg/kg, three doses). 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. Akt phosphorylation was determined by Western blot. There were no significant hemodynamic or blood gas differences between groups. The control ischemic-reperfused cortex had a similar O2 consumption, but lower blood flow and higher O2 extraction compared 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 (42 of 80 veins with O2 saturation below 50%). SC-79 did not significantly affect cerebral O2 consumption, but significantly improved O2 supply/consumption balance in the reperfused area (18 of 80 veins with O2 saturation below 50%). This was associated with a reduced cortical infarct size (13.3 ±â€¯0.5% control vs 6.7 ±â€¯0.3% SC-79). In control, Akt phosphorylation was elevated at 2 h after ischemia. With SC-79, Akt was activated at 15 min but not at 2 h in the ischemic reperfused area. These results suggest that early Akt activation is important for not only cell survival, but also for the control of local oxygen balance after cerebral ischemia-reperfusion.

Rapamycin decreased blood-brain barrier permeability in control but not in diabetic rats in early cerebral ischemia.

Diabetes causes functional and structural changes in blood-brain barrier (BBB). The mammalian target of rapamycin (mTOR) has been associated with glucose metabolism, diabetes, and altering BBB permeability. Since there is only a narrow therapeutic window (3h) for stroke victims, it is important to investigate BBB disruption in the early stage of cerebral ischemia. We compared the degree of BBB disruption in diabetic and in control rats at two hours of reperfusion after one hour of middle cerebral artery (MCA) occlusion with or without inhibition of mTOR. Two weeks after streptozotocin ip to induce diabetes, MCA occlusion was performed. In half of the rats, an mTOR inhibitor, rapamycin was given for 2days before MCA occlusion. After one hour of MCA occlusion and two hours of the reperfusion, the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid was determined to quantify degree of BBB disruption. Ischemia-reperfusion increased the Ki in the control animals. Streptozotocin increased the Ki in the ischemic-reperfused (IR-C, +22%) as well as in the contralateral cortex (CC, +40%). Rapamycin decreased the Ki in the IR-C (-32%) as well as in the CC (-26%) in the control rats. However, rapamycin did not affect Ki in the IR-C or in the CC in the diabetic rats. Our data demonstrated a greater BBB disruption in diabetes in the ischemic as well as non-ischemic cortex even in the early stage of cerebral ischemia-reperfusion and that acute administration of rapamycin did not significantly affect BBB permeability in diabetes. From our quantitative analysis of BBB disruption, the vulnerability of BBB in diabetes has been emphasized in the early stage of cerebral ischemia-reperfusion and a less important role of the mTOR pathway is suggested in altering BBB permeability in diabetes.

Blood -brain barrier disruption was less under isoflurane than pentobarbital anesthesia via a PI3K/Akt pathway in early cerebral ischemia.

One of the important factors altering the degree of blood-brain barrier (BBB) disruption in cerebral ischemia is the anesthetic used. The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway has been reported to be involved in modulating BBB permeability and in isoflurane induced neuroprotection. This study was performed to compare the degree of BBB disruption in focal cerebral ischemia under isoflurane vs pentobarbital anesthesia and to determine whether inhibition of PI3K/Akt would affect the disruption in the early stage of focal cerebral ischemia. Permanent middle cerebral artery (MCA) occlusion was performed in rats under 1.4% isoflurane or pentobarbital (50mg/kg i.p.) anesthesia with controlled ventilation. In half of each group LY294002, which is a PI3K/Akt inhibitor, was applied on the ischemic cortex immediately after MCA occlusion. After one hour of MCA occlusion, the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid (14C-AIB) was determined to quantify the degree of BBB disruption. MCA occlusion increased the Ki both in the isoflurane and pentobarbital anesthetized rats. However, the value of Ki was lower under isoflurane (11.5±6.0µL/g/min) than under pentobarbital (18.3±7.1µL/g/min) anesthesia. The Ki of the contralateral cortex of the pentobarbital group was higher (+74%) than that of the isoflurane group. Application of LY294002 on the ischemic cortex increased the Ki (+99%) only in the isoflurane group. The degree of BBB disruption by MCA occlusion was significantly lower under isoflurane than pentobarbital anesthesia in the early stage of cerebral ischemia. Our data demonstrated the importance of choice of anesthetics and suggest that PI3K/Akt signaling pathway plays a significant role in altering BBB disruption in cerebral ischemia during isoflurane but not during pentobarbital anesthesia.

Hypoxic Preconditioning Increases Blood-Brain Barrier Disruption in the Early Stages of Cerebral Ischemia.

Even though hypoxic preconditioning has been reported to produce neuroprotection, its effect on blood-brain barrier (BBB) disruption in the early stages of cerebral ischemia within the therapeutic window is not clear. Since hypoxic preconditioning increases expression of vascular endothelial growth factor (VEGF) that modulates vascular permeability, the effects of hypoxic preconditioning and VEGF on BBB permeability were investigated after one hour of focal cerebral ischemia. Rats were exposed to 8% of oxygen for two hours or room air and then 24 hours later, permanent middle cerebral artery (MCA) occlusion was performed. In some of the hypoxic preconditioned rats, a VEGF-A antibody was applied to the ischemic cortex one hour before MCA occlusion. One hour after MCA occlusion, the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid was determined to measure the degree of BBB disruption. MCA occlusion increased the Ki when compared with the contralateral cortex (14.1 ± 4.0 vs 4.2 ± 1.9 µL/g/min, p < 0.0001). Hypoxic preconditioning further increased the Ki in the ischemic cortex when compared with the control rats (25.1 ± 8.7 µL/g/min, p < 0.01). Application of VEGF antibody to the ischemic cortex of the hypoxic preconditioned animals reduced the Ki to the level of the control rats (13.6 ± 5.1 µL/g/min, p < 0.01). Our data demonstrated that hypoxic preconditioning increased BBB disruption through a VEGF related pathway and suggest the possibility of aggravation of brain edema by hypoxic preconditioning in the early stages of cerebral ischemia.

Effects of rapamycin pretreatment on blood-brain barrier disruption in cerebral ischemia-reperfusion.

The mammalian target of rapamycin (mTOR) pathway is essential in neuronal survival and repair in cerebral ischemia. Decreases in blood-brain barrier (BBB) disruption are associated with a decrease in neuronal damage in cerebral ischemia. This study was performed to investigate how pre-inhibition of the mTOR pathway with rapamycin would affect BBB disruption and the size of the infarcted cortical area in the early stage of focal cerebral ischemia-reperfusion using quantitative analysis of BBB disruption. Rats were treated with 20mg/kg of rapamycin i.p. once a day for 2days (Rapamycin Group) or vehicle (Control Group) before transient middle cerebral artery (MCA) occlusion. After one hour of MCA occlusion and two hours of reperfusion, the transfer coefficient (Ki) of (14)C-α-aminoisobutyric acid ((14)C-AIB) to measure the degree of BBB disruption and the size of the cortical infarct were determined. Ischemia-reperfusion increased the Ki in the Rapamycin treated (+15%) as well as in the untreated control group (+13%). However, rapamycin pretreatment moderately decreased Ki in the contralateral (-30%) as well as in the ischemic-reperfused (-29%) cortex when compared with the untreated control group. Rapamycin pretreatment substantially increased the percentage of cortical infarct compared with the control group (+56%). Our data suggest that activation of mTOR pathway is necessary for neuronal survival in the early stage of cerebral ischemia-perfusion and that the reason for the enlarged cortical infarct by rapamycin pretreatment may be related to its non-BBB effects on the mTOR pathway.

Restoration of Normal Cerebral Oxygen Consumption with Rapamycin Treatment in a Rat Model of Autism-Tuberous Sclerosis.

Tuberous sclerosis (TSC) is associated with autism spectrum disorders and has been linked to metabolic dysfunction and unrestrained signaling of the mammalian target of rapamycin (mTOR). Inhibition of mTOR by rapamycin can mitigate some of the phenotypic abnormalities associated with TSC and autism, but whether this is due to the mTOR-related function in energy metabolism remains to be elucidated. In young Eker rats, an animal model of TSC and autism, which harbors a germ line heterozygous Tsc2 mutation, we previously reported that cerebral oxygen consumption was pronouncedly elevated. Young (4 weeks) male control Long-Evans and Eker rats were divided into control and rapamycin-treated (20 mg/kg once daily for 2 days) animals. Cerebral regional blood flow ((14)C-iodoantipyrine) and O2 consumption (cryomicrospectrophotometry) were determined in isoflurane-anesthetized rats. We found significantly increased basal O2 consumption in the cortex (8.7 ± 1.5 ml O2/min/100 g Eker vs. 2.7 ± 0.2 control), hippocampus, pons and cerebellum. Regional cerebral blood flow and cerebral O2 extractions were also elevated in all brain regions. Rapamycin had no significant effect on O2 consumption in any brain region of the control rats, but significantly reduced consumption in the cortex (4.1 ± 0.3) and all other examined regions of the Eker rats. Phosphorylation of mTOR and S6K1 was similar in the two groups and equally reduced by rapamycin. Thus, a rapamycin-sensitive, mTOR-dependent but S6K1-independent, signal led to enhanced oxidative metabolism in the Eker brain. We found decreased Akt phosphorylation in Eker but not Long-Evans rat brains, suggesting that this may be related to the increased cerebral O2 consumption in the Eker rat. Our findings suggest that rapamycin targeting of Akt to restore normal cerebral metabolism could have therapeutic potential in tuberous sclerosis and autism.

Inhibition of neuronal nitric oxide synthase improves microregional O2 balance in cerebral ischemia-reperfusion.

Objectives Return of regional cerebral blood flow (rCBF) in focal cerebral ischaemia may not ensure proper distribution of blood flow to meet metabolic demand. This study was performed to determine how inhibition of neuronal nitric oxide synthase (NOS) during ischaemia-reperfusion would affect microregional O2 supply/consumption balances and their variation. Methods Twenty minutes before middle cerebral artery (MCA) occlusion, a NOS inhibitor 7-nitroindazole (7-NI) 50 mg/kg ip (7-NI group) or vehicle (control group) was administered. At 1 hour of ischaemia and 2 hours of reperfusion, rCBF, the size of cortical infarct and arteriolar and venular O2 saturations (20-60 µm in diameter) using cryomicrospectrophotometry were determined. Results Ischaemia-reperfusion decreased the average venular O2 saturation and the ratio of O2 supply/consumption. But, 7-NI treatment improved the average O2 supply/consumption ratio and venular O2 saturation (57.6 ± 1.3 vs 52.0 ± 3.8%) in ischaemia-reperfusion. The heterogeneity of venular O2 saturations reported as coefficient of variation (CV = 100 × SD/mean) was much smaller in the 7-NI than the control group (8.8 vs 15.5). The number of veins with low O2 saturation ( < 50%) was also smaller with the 7-NI (4/70) than the control group (18/70). The size of cortical infarct was smaller with 7-NI treatment. Discussion Our data suggest that inhibition of neuronal NOS by 7-NI improved microregional O2 balance in the ischaemic-reperfused cortex (IR-C). The improvement in microregional O2 balance could be one of the contributing factors to the reduced size of cortical infarct.

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.

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.