Effects of 17beta-estradiol on blood-brain barrier disruption during focal cerebral ischemia in younger and older rats.

This study was performed to compare the effects of 17beta-estradiol on blood-brain barrier disruption in focal cerebral ischemia between younger and older rats. Younger (three-month-old) and older (24-month-old) ovariectomized female Fischer 344 rats were studied. In one half of each age group, a 500 microg 17beta-estradiol 21-day release pellet and in another half, a vehicle pellet was implanted 21 days before the experiments. One hour after middle cerebral artery occlusion, the transfer coefficient (Ki) of 14C-alpha-aminoisobutyric acid and the volume of 3H-dextran distribution were determined to examine the degree of blood-brain barrier disruption. In all four groups, the Ki in the ischemic cortex was higher than in the corresponding contralateral cortex. There was no significant difference in the Ki in both cortices among the groups. The volume of dextran distribution of the ischemic cortex was only greater than in the corresponding contralateral cortex in the older 17beta-estradiol-treated group, and the volume of that group was greater than the younger 17beta-estradiol-treated group (4.00 +/- 1.29 VS. 2.13 +/- 0.88 ml/100 g). After analyzing the difference in Ki between the ischemic cortex and the contralateral cortex in each animal, the difference was significantly greater in the older 17beta-estradiol-treated rats than the older vehicle-treated rats (3.40 +/- 2.10 VS. 1.26 +/- 1.44 microl/g/min). In the younger rats, however, 17beta-estradiol did not significantly affect the difference. Our data showed that 17beta-estradiol treatment failed to attenuate the BBB disruption in the cerebral ischemic cortex in the older or younger Fischer 344 rats. However, our data also suggest the possibility that 17beta-estradiol could aggravate the BBB disruption in older rats.     

Effects of 17beta-estradiol on blood-brain barrier disruption during focal ischemia in rats.

This study was performed to test whether 17beta-estradiol could attenuate the blood-brain barrier disruption caused by middle cerebral artery occlusion in the ovariectomized rats. Rats aged twelve to fourteen weeks were used in this study. Their ovaries were removed one week prior to the implantation of the pellets. For the 17beta-estradiol group, a 500 microg 17beta-estradiol 21 day-release pellet was implanted and for the control group, a vehicle pellet was implanted 21 days before the experiments. One hour after middle cerebral artery occlusion under isoflurane anesthesia, the transfer coefficient of 14C-alpha-aminoisobutyric acid (104 Daltons) and the volume of 3H-dextran (70,000 Daltons) distribution were determined to represent the degree of blood-brain barrier disruption. Blood pressures and blood gases were similar between controls and 17beta-estradiol-treated rats. In both groups, the transfer coefficient of the ischemic cortex was higher than that of the corresponding contralateral cortex (control: Ischemic Cortex 12.5 +/- 5.9 microl/g/min, Contralateral Cortex 3.0 +/- 1.6, p < 0.001; 17beta-estradiol: Ischemic Cortex 6.7 +/- 3.3 micro l/g/min, Contralateral Cortex 2.2 +/- 0.9, p < 0.01). There was no significant difference in the transfer coefficient of the contralateral cortex between these two groups. However, the transfer coefficient of the Ischemic Cortex of the 17beta-estradiol-treated animals was 46 % lower than that of the control, vehicle-treated rats (p < 0.05). The increase of the volume of 3H-dextran distribution with middle cerebral artery occlusion was significant in the vehicle-treated rats (Ischemic Cortex: 6.4 +/- 2.7 ml/100 g, Contralateral Cortex: 3.8 +/- 0.8, p < 0.01) but not in the 17beta-estradiol-treated animals. Our data suggest that chronic 17beta-estradiol treatment was effective in reducing blood-brain barrier disruption during focal ischemia in the ovariectomized rats.     

Effects of Metabotropic Glutamate Receptor Stimulation on Blood–Brain Barrier Permeability During Focal Cerebral Ischemia

This investigation was performed to evaluate whether ACPD [(1S, 3R)-1-aminocyclopentane-1, 3-dicarboxylic acid], a metabotropic glutamate receptor agonist, would enhance the degree of increase in blood-brain barrier (BBB) permeability caused by focal cerebral ischemia. In this study, male Wistar rats were placed in control (n = 7) and ACPD (n = 7) groups under isoflurane anesthesia. Twenty minutes after middle cerebral artery (MCA) occlusion, patches of 10(-5) M ACPD or normal saline were placed on the ischemic cortex (IC) for a period of 40 min. Patches were changed every 10 min. One hour after MCA occlusion, BBB permeability was determined by measuring the transfer coefficient (Ki) of [alpha-14C] aminoisobutyric acid. There were no statistical differences in systemic blood pressures and heart rates between these groups. Blood gases were within normal limits. In the control group, the Ki of ischemic cortex (IC) was 2.1 times that of the contralateral cortex (CC) (3.7+/-0.9 vs. 1.8+/-0.3 microl/g/min). In the ACPD group, the Ki of the IC was 3.3 times that of the CC (5.0+/-0.7 vs. 1.5+/-0.4 microl/g/min). The increase in Ki of the ACPD group in the ischemic cortex was significantly greater than that in the control group. There was no significant difference in the Ki of the CC between these groups. Our data suggest that activation metabotropic glutamate receptors in the cortex can further augment the increase in BBB permeability caused by focal ischemia.     

17beta-estradiol prevents blood-brain barrier disruption induced by VEGF.

We performed this study to determine how pretreatment of the ovariectomized rats with 17beta-estradiol could affect blood-brain barrier disruption caused by the vascular endothelial growth factor (VEGF), an important mediator of vascular permeability. Ovariectomized female rats aged twelve to fourteen weeks were used in the study. A 500 micro g 17beta-estradiol 21-day release pellet was implanted in the 17beta-estradiol group, and a vehicle pellet was implanted in the control group 21 days before the experiments. We performed three craniotomies under isoflurane anesthesia to expose cerebral cortices. Normal saline, 10 (- 10)M and 10 (- 9)M VEGF patches were applied on each hole for 30 min. The transfer coefficient (Ki) of (14)C-alpha-amino isobutyric acid and volume of (3)H-dextran (70,000 dalton) distribution were determined to measure the degree of BBB disruption. Ki was increased by 108 % and 138 % with 10 (- 10)M and 10 (- 9)M VEGF respectively after VEGF application in the control group (p < 0.01). However, there was no significant increase in the Ki with the VEGF application in the 17beta-estradiol group, and their values were significantly lower than the corresponding data of the control group (10 (- 10)M: - 55 %, 10 (- 9)M: - 52 %, p <0.05). The volume of dextran distribution in the control group increased by 47 % with VEGF 10 (- 9)M (p < 0.05), whereas there was no significant change in the volume of dextran distribution with VEGF application in the 17beta-estradiol group and the volume was lower than the corresponding volume of the vehicle-treated control group (10 (- 10)M: - 34 %, 10 (- 9)M: -32 %, p < 0.05). In conclusion, our study demonstrated that chronic 17beta-estradiol treatment prevented BBB disruption induced by the VEGF in the ovariectomized rats.     

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 ¹⁴C-α-aminoisobutyric acid (¹⁴C-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.     

Saturable transport of the neurokinin-1 non-peptide antagonist LY303870 across the rat blood-brain barrier after intravenous administration

LY303870 (LY) is a non-peptide neurokinin-1 receptor antagonist that has effects on the brain after peripheral administration. We determined whether LY given by intravenous (iv) injection can cross the blood-brain barrier (BBB). Multiple-time regression analysis showed the unidirectional influx rate (Ki) from blood to brain for LY labeled with tritium to be 6.41+/-0.85 microl/g-min and influx was inhibited by unlabeled LY. HPLC and mass spectrometry showed LY was stable in blood and brain. LY reached a brain/serum ratio of 190+/-12 microl/g with about 0.07% of the injected dose entering each gram of brain. These results show that LY is transported across the BBB from serum into brain by a saturable system.     

Efflux of a suppressive neurotransmitter, GABA, across the blood-brain barrier

In this study, GABA efflux transport from brain to blood was estimated by using the brain efflux index (BEI) method. [3H]GABA microinjected into parietal cortex area 2 (Par2) of the rat brain was eliminated from the brain with an apparent elimination half-life of 16.9 min. The blood-brain barrier (BBB) efflux clearance of [3H]GABA was at least 0.153 mL/min/g brain, which was calculated from the elimination rate constant (7.14 x 10(-2) x min(-1)) and the distribution volume in the brain (2.14 mL/g brain). Direct comparison of the apparent BBB influx clearance [3H]GABA (9.29 microL/min/g brain) and the apparent efflux clearance (153 microL/min/g brain) indicated that the efflux clearance was at least 16-fold greater than the influx clearance. In order to reduce the effect of metabolism in the neuronal cells following intracerebral microinjection, we determined the apparent efflux of [3H]GABA in the presence of nipecotic acid, a GABA transport inhibitor in parenchymal cells, using the BEI method. Under such conditions, the elimination of [3H]GABA across the BBB showed saturation and inhibition by probenecid in the presence of nipecotic acid. Furthermore, the uptake of [3H]GABA by MBEC4 cells was inhibited by GABA, taurine, beta-alanine and nipecotic acid in a concentration-dependent manner. It is likely that GABA inhibits the first step in the abluminal membrane uptake by brain endothelial cells, and that probenecid selectively inhibits the luminal membrane efflux transport process from the brain capillary endothelial cells based on the in vivo and in vitro evidence. The BBB acts as the efflux pump for GABA to reduce the brain interstitial fluid concentration.     

GABAergic modulation of ventilation and peak oxygen consumption in obese Zucker rats.

Obesity is often associated with a reduced ventilatory response and a decreased maximal exercise capacity. GABA is a major inhibitory neurotransmitter in the mammalian central nervous system. Altered GABAergic mechanisms have been detected in obese Zucker rats and implicated in their hyperphagic response. Whether altered GABAergic mechanisms also contribute to regulate ventilation and influence exercise capacity in obese Zucker rats is unknown and formed the basis of the present study. Eight lean [317 +/- 18 (SD) g] and eight obese (450 +/- 27 g) Zucker rats were studied at 12 wk of age. Ventilation at rest and ventilation during hypoxic (10% O(2)) and hypercapnic (4% CO(2)) challenges were measured by the barometric method. Peak O(2) consumption (VO(2 peak)) in response to a progressive treadmill test to exhaustion was measured in a metabolic treadmill. Ventilation and VO(2 peak) were assessed after administration of equal volumes of DMSO (vehicle) and the GABA(A) receptor antagonist bicuculline (1 mg/kg). In lean animals, bicuculline administration had no effect on ventilation and VO(2 peak). In obese rats, bicuculline administration significantly (P < 0.05) increased resting ventilation (465 +/- 53 and 542 +/- 72 ml. kg(-1). min(-1) for control and bicuculline, respectively), ventilation during exposure to hypoxia (899 +/- 148 and 1,038 +/- 83 ml. kg(-1). min(-1) for control and bicuculline, respectively), and VO(2 peak) (62 +/- 3.7 and 67 +/- 3.5 ml. kg(-0.75). min(-1) for control and bicuculline, respectively). However, in obese Zucker rats, ventilation in response to hypercapnia did not change after bicuculline administration (608 +/- 96 vs. 580 +/- 69 ml. kg(-1). min(-1)). Our findings indicate that endogenous GABA depresses ventilation and limits exercise performance in obese Zucker rats.     

Role of Hydrogen Sulfide in Early Blood-Brain Barrier Disruption following Transient Focal Cerebral Ischemia

We determined the role of endogenous hydrogen sulfide (H₂S) in cerebral vasodilation/hyperemia and early BBB disruption following ischemic stroke. A cranial window was prepared over the left frontal, parietal and temporal cortex in mice. Transient focal cerebral Ischemia was induced by directly ligating the middle cerebral artery (MCA) for two hours. Regional vascular response and cerebral blood flow (CBF) during ischemia and reperfusion were measured in real time. Early BBB disruption was assessed by Evans Blue (EB) and sodium fluorescein (Na-F) extravasation at 3 hours of reperfusion. Topical treatment with DL-propargylglycine (PAG, an inhibitor for cystathionine γ-lyase (CSE)) and aspartate (ASP, inhibitor for cysteine aminotransferase/3-mercaptopyruvate sulfurtransferase (CAT/3-MST)), but not O-(Carboxymethyl)hydroxylamine hemihydrochloride (CHH, an inhibitor for cystathionine β-synthase (CBS)), abolished postischemic cerebral vasodilation/hyperemia and prevented EB and Na-F extravasation. CSE knockout (CSE^-/-) reduced postischemic cerebral vasodilation/hyperemia but only inhibited Na-F extravasation. An upregulated CBS was found in cerebral cortex of CSE^-/- mice. Topical treatment with CHH didn’t further alter postischemic cerebral vasodilation/hyperemia, but prevented EB extravasation in CSE^-/- mice. In addition, L-cysteine-induced hydrogen sulfide (H₂S) production similarly increased in ischemic side cerebral cortex of control and CSE^-/- mice. Our findings suggest that endogenous production of H₂S by CSE and CAT/3-MST during reperfusion may be involved in postischemic cerebral vasodilation/hyperemia and play an important role in early BBB disruption following transient focal cerebral ischemia.     

Involvement of central GABAergic neurons in basal and diurnal changes of tuberoinfundibular dopaminergic neuronal activity and prolactin secretion

Central administration of gamma-aminobutyric acid (GABA) has been shown to stimulate the secretion of prolactin (PRL). Whether GABA acts via dopamine, the major PRL-inhibiting hormone, and which GABA receptor type(s) is involved have not been ascertained. Both GABA(A) and GABA(B) receptor agonists and/or antagonists were administered centrally in this study and their effects on both basal and diurnal changes of tuberoinfundibular dopaminergic (TIDA) neuronal activity were determined by measuring the concentration of 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence (ME). Serum PRL level was determined by RIA. Ovariectomized, estrogen-primed Sprague-Dawley rats implanted with intracerebroventricular (icv) cannulae were used. Muscimol (1 ng/3 microl/rat, icv), a GABA(A) receptor agonist, but not baclofen (1-100 ng/3 microl/rat, icv), a GABA(B) receptor agonist, injected in the morning significantly lowered and elevated ME DOPAC and serum PRL levels, respectively at 15 and 30 min. Lower and higher doses of muscimol were not effective. The effects of muscimol could also be prevented by co-administration of bicuculline (0.1-10 ng/3 microl, icv), a GABA(A) receptor antagonist. When bicuculline (10-500 ng/3 microl, icv) was given in the afternoon (at 1500 h), it significantly reversed the lowered ME DOPAC level in the afternoon and prevented the concurrent PRL surge. We conclude that endogenous GABA acting through GABA(A) receptors may play a significant role in the control of basal and diurnal changes of TIDA neuronal activity, and in turn, PRL secretion.     

Brain-to-blood efflux transport of estrone-3-sulfate at the blood-brain barrier in rats

Efflux transport of estrogens such as estrone-3-sulfate (E1S), and estrone (E1) across the blood-brain barrier (BBB) was evaluated using the Brain Efflux Index (BEI) method. The apparent BBB efflux rate constant (Keff) of [3H]E1S, and [3H]E1 was 6.63 x 10(-2) +/- 0.77 x 10(-2) min(-1), and 6.91 x 10(-2) +/- 1.23 x 10(-2) min(-1), respectively. The efflux transport of [3H]E1S from brain across the BBB was a saturable process with Michaelis constant (Km) of 96.0 +/- 34.4 microM and 93.4 +/- 22.0 microM estimated by two different methods. By determining [3H]E1S metabolites using high performance liquid chromatography (HPLC) after intracerebral injection, significant amounts of [3H]E1S were found in the jugular venous plasma, providing direct evidence that most of [3H]E1S is transported from brain across the BBB in intact form. To compare the apparent efflux clearance across the BBB of E1S with that of E1, the brain distribution volume of E1S and E1 was estimated using the brain slice uptake method. The apparent efflux clearance of [3H]E1S was determined to be 74.9 +/- 3.8 microl/(min x g brain) due to the distribution volume of 1.13 +/- 0.06 ml/g brain. By contrast, the apparent efflux clearance of E1 was more than 227 +/- 3 microl/(min x g brain), since the distribution volume of [3H]E1 at 60 min was 3.28 +/- 0.13 ml/g. The E1S efflux transport process was inhibited by more than 40% by coadministration of bile acids (taurocholate, and cholate), and organic anions (sulfobromophthalein, and probenecid), whereas other organic anions did not affect the E1S efflux transport. The [3H]E1S efflux was significantly reduced by 48.6% after preadministration of 5 mM dehydroepiandrosterone sulfate. These results suggest that E1S is transported from brain to the circulating blood across the BBB via a carrier-mediated efflux transport system.     

Effects of 17beta-estradiol and flutamide on splenic macrophages and splenocytes after trauma-hemorrhage

Since splenic immune functions are depressed in metestrus females following trauma-hemorrhage, we hypothesized that administration of the androgen receptor antagonist flutamide at the onset of resuscitation will maintain the immune function of the spleen following trauma-hemorrhage. Female C57BL6/J mice (metestrus state, 8-12 weeks old), underwent laparotomy and hemorrhagic shock (35.0+/-5.0 mm Hg for 90 min) and received 17beta-estradiol (50 microg/25 g), flutamide (625 microg/25 g) or 17beta-estradiol+flutamide. Four hours after resuscitation, the in vitro productive capacity of different cytokines (TNF-alpha, IL-6, IL-10, and IFN-gamma) by splenic MPhi and splenocytes were determined by flow cytometry. A significantly decreased cytokine production by both splenocytes and splenic MPhi was observed following trauma-hemorrhage compared to shams. Administration of 17beta-estradiol, flutamide and 17beta-estradiol+flutamide following trauma-hemorrhage resulted in a significant increase in the in vitro IL-6 release by splenic MPhi. The TNF-alpha productive capacity, however, was only restored by 17beta-estradiol and 17beta-estradiol+flutamide administration following trauma-hemorrhage. No significant effect of either treatment was observed with regard to the suppressed splenic MPhi IL-10 release. Anti-CD3 stimulation, administration of 17beta-estradiol and 17beta-estradiol+flutamide, but not the administration of flutamide alone resulted in a significant increased release of TNF-alpha, IL-6 and IFN-gamma compared to vehicle-treated animals. No significant effect of either treatment was found on IL-10 productive capacity. These results collectively suggest that flutamide administration following trauma-hemorrhage in females has beneficial effects on splenic immune function. However, flutamide administration in combination with estrogen does not provide any significant, additional effects over 17beta-estradiol administration alone.     

Endothelial protective and antishock effects of a selective estrogen receptor modulator in rats

This study investigated whether idoxifene, a selective estrogen receptor modulator (SERM), exerted protective effects against ischemia-reperfusion-induced shock. Ovariectomized rats were treated with vehicle, idoxifene, or 17beta-estradiol for 4 days. Rats were subjected to splanchnic artery occlusion (SAO) followed by reperfusion (SOA/R). In vehicle-treated rats, SAO/R resulted in hypotension, hemoconcentration, increased plasma tumor necrosis factor (TNF)-alpha levels, intestinal neutrophil accumulation, and endothelial dysfunction. 17beta-Estradiol treatment increased plasma estradiol concentration and reduced SAO/R-induced tissue injury. Idoxifene treatment had no effect on plasma estradiol concentration but reduced SAO/R-induced hemoconcentration (+8.8 +/- 1.3 vs. +14 +/- 1.3% in the vehicle group, P < 0.01), TNF-alpha production (98 +/- 3.2 vs. 214 +/- 13 pg/ml, P < 0.01), and neutrophil accumulation (0.025 +/- 0.005 vs. 0.047 +/- 0.005 U/g protein, P < 0.01). It also improved endothelial function, prolonged survival time (172 +/- 3.5 vs. 147 +/- 8 min, P < 0.01), and increased survival rate (69 vs. 23%, P < 0.01). Moreover, treatment with 17beta-estradiol or idoxifene in vivo reduced TNF-alpha-induced endothelial dysfunction in vitro. Taken together, these results demonstrated that idoxifene exerted estrogen-like, endothelial-protective, and antishock effects in ovariectomized rats, suggesting that SERMs have therapeutic potential in tissue injury resulting from ischemia-reperfusion.     

Subcellular distribution and properties of progesterone (delta4-steroid) 5alpha-reductase in rat medial basal hypothalamus.

The subcellular distribution and properties of rat hypothalamic progesterone 5 alpha-reductase, which accelerates the conversion of progesterone to 5 alpha-pregnane-3,20-dione, have been investigated by utilizing 3H-labeled substrate and a reverse isotopic dilution assay system. The enxymic activity was associated primarily with a cell debris-membranes fraction deribed from the 100 x g pellet. This fraction contained mainly membrane-like particulates and was free of nuclei. Little or no activity was associated with the purified nuclei. The hypothalamic 5 alpha-reductase was stimulated by NADPH but not by NADH. The reaction proceeded optimally over a pH range of 6.0 to 7.2 and at a temperaturhe substrate specificity of the enzyme for other delta 4-3-ketosteroids and the ability of these steroids to inhibit the 5 alpha reduction of [1,2-3H]progesterone as well as the effect of 17 beta-estradiol were also studied. 20 alpha-hydroxypregn-4-en-3-one was more reactive that progesterone, while testosterone was the least reactive. The estimated Km for 20 alpha-hydroxypregn-4-en-3-one was 8.6 +/- 1.9 x 10(-7) M, and for testosterone, 1.6 +/- 1.4 x 10(-5) M. The inhibition studies indicate that 20 alpha-hydroxypregn-4-en-3-one and 17 beta-estradiol are competitive and noncompetitive inhibitors, respectively, of the 5 alpha reduction of progesterone with Ki of 6.0 +/- 3.0 x 10(-8) M for 20 alpha-hydroxypregn-4-en-3-one and Kii (intercept inhibition constant) of 2.6 +/- 0.7 x 10(-5) M and Kis (slope inhibition constant) of 3.6 +/- 0.6 x 10(-5) M for 17 beta-estradiol. Testosterone is a poor competitive inhibitor of the reaction.     

Microdialysis and the measurement of muscle interstitial K+ during rest and exercise in humans.

The purpose of this study was to examine whether microdialysis and the internal reference thallium-201 ((201)Tl) could accurately measure muscle interstitial K+ (Ki+) before, during, and after exercise. The relative loss of (201)Tl and simultaneous relative recovery of K+ were measured in vitro for 12 microdialysis probes that were bathed in Ringer acetate medium and perfused at various flows (3-10 microl/min). (201)Tl loss was linearly related to K+ recovery, and their level of agreement was not different from zero. Microdialysis and (201)Tl were then used to measure Ki+ in the gastrocnemius medialis muscle of four humans during rest and static plantar flexion exercise. At rest, Ki+ was 3.9-4.3 mmol/l when the perfusate flow was 2 or 5 microl/min. During exercise, Ki+ increased from 6.9 +/- 0.4 to 7.5 +/- 0.3 mmol/l at low to high intensity and declined to 5.2 +/- 0.3 mmol/l after exercise. These results suggest that large changes in Ki+ in human skeletal muscle can be accurately measured by using microdialysis and (201)Tl.     

Reversibility of Nimodipine Binding to Brain in Transient Cerebral Ischemia

Using autoradiography, we have measured the in vivo binding of [3H]nimodipine to brain in a rat model of reversible cerebral ischemia. Ischemia was induced by simultaneous occlusion of the middle cerebral artery (MCA) and ipsilateral common carotid artery by microaneurysm clips. Rats were studied after 15 min of ischemia (ischemic group) or after 45 min of reperfusion following 15 min of ischemia (reperfused group). Regional cerebral blood flow (CBF) was determined autoradiographically using [14C]iodoantipyrine in both ischemic (n = 6) and reperfused (n = 6) groups. During ischemia blood flow in the territory of the MCA was depressed and recovered to normal only in the distal territory of the MCA following reperfusion. [3H]Nimodipine binding in the ischemic group (n = 12) was elevated in ischemic brain regions and declined significantly (p < 0.01) in these regions in the reperfused group (n = 11). The ratio of the volume of cortex showing increased binding to the total volume of the forebrain was 0.113 +/- 0.025 (mean +/- SD) in the ischemic group and declined to 0.080 +/- 0.027 following reperfusion (p < 0.005). In general, infarct was only observed in regions showing persistent elevation of nimodipine binding following reperfusion as determined by histology performed in a separate group of rats (n = 8) after 24 h of reperfusion. We conclude that increased nimodipine binding to ischemic tissue is initially reversible with prompt reestablishment of CBF and is a sensitive indicator of early and reversible ischemia-induced cerebral dysfunction.     

Acute changes in intermediary metabolism in cerebellum and contralateral hemisphere following middle cerebral artery occlusion in rat

Focal ischemia leads to functional deafferentation of regions connected to the ischemic area via fiber tracts. Using i.v. administration of ¹³C-labeled glucose and acetate combined with ex vivo ¹³C MR spectroscopy and HPLC of brain extracts we identify the effect of middle cerebral artery occlusion (MCAO) on neurotransmitter synthesis and turnover, and on neuro-astrocytic interactions in the non-ischemic cerebellum and in contralesional lateral caudoputamen plus lower parietal cortex (LPC), and upper frontoparietal cortex (UFPCx) in the rat after 30, 60, 120 and 240 min of ischemia. In all regions, there was a significant persisting loss of glutamate, and in LCP and UFPCx also of glutamine, but only in LCP was GABA reduced at all times. Metabolism and blood flow were uncoupled in all regions. In cerebellum, glucose metabolism as well as utilization of intermediates derived from astrocytic tricarboxylic acid cycle activity were significantly decreased at all times in both glutamatergic and GABAergic neurons. In LCP and UFPCx, there were normal or increased enrichment in glutamate and GABA from glucose. Glutamate derived from astrocytic acetate metabolism was increased, but GABA synthesis from acetate was initially impaired. The results showed that both the type of afferent connection, i.e., glutamatergic and/or GABAergic, and local cytoarchitecture, determined the effect MCAO had on metabolic activity in the non-ischemic regions. In conclusion, it was primarily excitatory input into non-ischemic regions that was affected by MCAO, perhaps enabling resetting of the excitatory/inhibitory balance, aiding reshaping of the receptive fields and thus facilitating recovery.     

Evidence that GAD65 mediates increased GABA synthesis during intense neuronal activity in vivo

In this study we tested the hypothesis that the 65-kDa isoform of glutamate decarboxylase (GAD(65)) mediates activity-dependent GABA synthesis as invoked by seizures in anesthetized rats. GABA synthesis was measured following acute GABA-transaminase inhibition by gabaculine using spatially localized (1)H NMR spectroscopy before and after bicuculline-induced seizures. Experiments were conducted with animals pre-treated with vigabatrin 24 h earlier in order to reduce GAD(67) protein and also with non-treated controls. GAD isoform content was quantified by immunoblotting. GABA was higher in vigabatrin-treated rats compared to non-treated controls. In vigabatrin-treated animals, GABA synthesis was 28% lower compared to controls [p < 0.05; vigabatrin-treated, 0.043 +/- 0.011 micromol/(g min); non-treated, 0.060 +/- 0.014 micromol/(g min)] and GAD(67) was 60% lower. No difference between groups was observed for GAD(65). Seizures increased GABA synthesis in both control [174%; control, 0.060 +/- 0.014 micromol/(g min) vs. seizures, 0.105 +/- 0.043 micromol/(g min)] and vigabatrin-treated rats [214%; control, 0.043 +/- 0.011 micromol/(g min); seizures, 0.092 +/- 0.018 micromol/(g min)]. GAD(67) could account for at least half of basal GABA synthesis but only 20% of the two-fold increase observed in vigabatrin-treated rats during seizures. The seizure-induced activation of GAD(65) in control cortex occurs concomitantly with a 2.3-fold increase in inorganic phosphate, known to be a potent activator of apoGAD(65)in vitro. Our results are consistent with a major role for GAD(65) in activity-dependent GABA synthesis.     

Brain to blood efflux transport of adenosine: blood-brain barrier studies in the rat

The blood-brain barrier (BBB) efflux transport of [(14)C] adenosine was studied using the brain efflux index (BEI) technique. BEI increased linearly over the first 2 min after injection, with deviation from linearity thereafter; 90.12 +/- 1.5% of the injected [(14)C] radioactivity remained within the brain after 20 min. The remaining tracer appears to be mainly intracellular, trapped by phosphorylation, as an almost linear increase of BEI over 20 min was observed after intracerebral injection of [(14)C] adenosine together with 5-iodo tubercidin. The BBB efflux clearance of [(14)C] radioactivity was estimated to be 27.62 +/- 5.2 micro L/min/g, almost threefold higher than the BBB influx clearance estimated by the brain uptake index technique. High-performance liquid chromatography (HPLC) analysis of blood plasma collected from the jugular vein after the intracerebral injection revealed metabolic breakdown of [(14)C] adenosine into nucleobases. The BBB efflux transport was saturable with apparent K(m) = 13.22 +/- 1.75 micro m and V(max) = 621.07 +/- 71.22 pmole/min/g, which indicated that BBB efflux in vivo is 6.2-12p mole/min/g, negligible when compared to the reported rate of adenosine uptake into neurones/glia. However, these kinetic parameters also suggest that under conditions of elevated ISF adenosine in hypoxia/ischaemia, BBB efflux transport could increase up to 25% of the uptake into neurones/glia and become an important mechanism to oppose the rise in ISF concentration. HPLC-fluorometry detected 93.6 +/- 5.25 nm of adenosine in rat plasma, which is 17- to 220-fold lower than the reported K(m) of adenosine BBB influx in rat. Together with the observed rapid degradation inside endothelial cells, this indicated negligible BBB influx of intact adenosine under resting conditions. Cross-inhibition studies showed that unlabelled inosine, adenine and hypoxanthine caused a decrease in BBB efflux of [(14)C] radioactivity in a concentration-dependent manner, with K(i) of 16.7 +/- 4.88, 65.1 +/- 14.1 and 71.1 +/- 16.9 micro m, respectively. This could be due to either competition of unlabelled molecules with [(14)C] adenosine or competition with its metabolites hypoxanthine and adenine for the same transport sites.     

5'-Adenosine monophosphate and adenosine metabolism, and adenosine responses in mouse, rat and guinea pig heart.

We examined myocardial 5'-adenosine monophosphate (5'-AMP) catabolism, adenosine salvage and adenosine responses in perfused guinea pig, rat and mouse heart. MVO(2) increased from 71+/-8 microl O(2)/min per g in guinea pig to 138+/-17 and 221+/-15 microl O(2)/min per g in rat and mouse. VO(2)/beat was 0.42+/-0.03, 0.50+/-0.03 and 0.55+/-0.04 microl O(2)/g in guinea pig, rat and mouse, respectively. Resting and peak coronary flows were highest in mouse vs. rat and guinea pig, and peak ventricular pressures and Ca(2+) sensitivity declined as heart mass increased. Net myocardial 5'-AMP dephosphorylation increased significantly as mass declined (3.8+/-0.5, 9.0+/-1.4 and 11.0+/-1.6 nmol/min per g in guinea pig, rat and mouse, respectively). Despite increased 5'-AMP catabolism, coronary venous [adenosine] was similar in guinea pig, rat and mouse (45+/-8, 69+/-10 and 57+/-14 nM, respectively). Comparable venous [adenosine] was achieved by increased salvage vs. deamination: 64%, 41% and 39% of adenosine formed was rephosphorylated while 23%, 46%, and 50% was deaminated in mouse, rat and guinea pig, respectively. Moreover, only 35-45% of inosine and its catabolites derive from 5'-AMP (vs. IMP) dephosphorylation in all species. Although post-ischemic purine loss was low in mouse (due to these adaptations), functional tolerance to ischemia decreased with heart mass. Cardiovascular sensitivity to adenosine also differed between species, with A(1) receptor sensitivity being greatest in mouse while A(2) sensitivity was greatest in guinea pig. In summary: (i) cardiac 5'-AMP dephosphorylation, VO(2), contractility and Ca(2+) sensitivity all increase as heart mass falls; (ii) adaptations in adenosine salvage vs. deamination limit purine loss and yield similar adenosine levels across species; (iii) ischemic tolerance declines with heart mass; and (iv) cardiovascular sensitivity to adenosine varies, with increasing A(2) sensitivity relative to A(1) sensitivity in larger hearts.