Journal of Cerebral Blood Flow & Metabolism

α[C-11]Methyl-l-tryptophan (AMT) is an analog of tryptophan used with positron emission tomography for the measurement of serotonin synthesis in humans. Several attempts have been made to estimate the serotonin synthesis rate from plasma and brain kinetic data of AMT using the same model as that applied for the measurement of the glucose metabolic rate with 2-deoxyglucose. However, although AMT is similar to 2-deoxyglucose with regard to an irreversible pool of tracer uptake, there are important differences between the two tracers and how the model can be applied. These differences include transport at the blood-brain barrier and the presence of a large unmetabolized pool of AMT, precluding the method from providing the absolute serotonin synthesis rate. Despite this limitation, the unidirectional uptake rate constant (K-complex) values have been found to be stable within an individual, and the rank order of regional brain values for K-complex are consistent with the rank order for serotonin content in human brain. Furthermore, changes in K-complex with age, gender, and disease states are consistent with previously reported biochemical measurements of serotonin in brain tissue. The authors suggest, therefore, that the K-complex is an index of serotonin synthesis which they have termed the “serotonin synthesis capacity.” The authors argue that AMT is a useful tracer for study of serotonergic mechanisms, and under certain pathologic states, of metabolism by means of the kynurenine pathway.

Autoradiographic measurement of local cerebral blood flow (CBF) with [¹⁴C]iodoantipyrine (IAP) is limited in mice by the difficulty in cannulating vessels and the blood loss for repeated blood sampling. The authors modified and validated the method to measure local CBF with [¹⁴C]IAP in mice by combining intraperitoneal tracer application with a single blood sampling from the heart at the end of the experiment. Experiments were carried out in male SV129 mice under halothane anesthesia. After intraperitoneal administration of 15 μCi [¹⁴C]IAP, arterial blood samples were collected repeatedly and anesthetized animals were immersed in liquid nitrogen. In addition, frozen blood from the heart was sampled to obtain the final blood [¹⁴C]radioactivity. Correlation analysis between the sampling time and [¹⁴C] radioactivity of the arterial blood revealed a highly significant linear relationship (P < 0.001, r = 0.978) and a lag time of the [¹⁴C]tracer in arterial blood of 3.3 ± 0.6 seconds. [¹⁴C]radioactivity of the final arterial blood sample (444 ± 264 nCi/mL) was almost equal to that of the heart blood (454 ± 242 nCi/mL), and the absolute difference in each animal was 3.3 ± 4.2% (mean ± SD). The convolution integrals for the CBF calculation were determined either by integrating the radioactivity of individual arterial blood samples or by assuming a linear rise from [¹⁴C]tracer lag time after intraperitoneal [¹⁴C]IAP injection to the value measured in the blood sample from the frozen heart. Regional flow values calculated by the two methods differed by less than 11 % (not significant). This method allows the quantitative measurement of local CBF in anesthetized mice without any vessel catheterization and will make mutant mice a more powerful tool to elucidate the molecular mechanisms of brain injuries by combining flow studies with molecular-biological methods.

A short duration of ischemia (i.e., ischemic preconditioning) results in significant brain protection to subsequent severe ischemic insult. Because previous studies suggest that tumor necrosis factor-α (TNF-α) plays a role in both promoting ischemic damage and neuroprotection, the present work aimed to evaluate the expression of TNF-α mRNA in an established model of ischemic preconditioning using a transient 10-minute occlusion of the middle cerebral artery. Because the level of TNF-α mRNA expression in the brain was too low to be consistently detected by Northern technique, a real-time polymerase chain reaction method was applied to quantitate the absolute copy number of TNF-α transcript in rat brain after the preconditioning procedure. TNF-α mRNA was induced in the ipsilateral cortex as early as 1 hour (27 ± 1 copies of mRNA per microgram of tissue compared to 11 ± 3 copies in sham-operated samples) after preconditioning, reached a peak level at 6 hours (49 ± 10 copies of transcript, n = 4, P < 0.01), and persisted up to 2 days. These data not only demonstrate the utility of real-time polymerase chain reaction for sensitive and accurate measurement of mRNA expression in normal and injured tissues but also suggest a potential role of TNF-α in the phenomenon of ischemic preconditioning.

With use of magnetic resonance imaging (MRI), the effects of early and delayed treatment of embolic stroke in rat with recombinant tissue plasminogen activator (rt-PA) were investigated. Rats with embolic stroke were treated with rt-PA at 1 (n = 9) or 4 (n = 7) hours after stroke onset or were untreated (n = 15). Diffusion-weighted imaging, perfusion-weighted imaging, and T2-weighted imaging were performed before and after embolization from 1 hour to 7 days. No significant differences were detected in the relative areas with low cerebral blood flow (CBF), apparent diffusion coefficient of water (ADC_w), and T2 between the 4-hour treated group and the untreated group. Significant decreases in the average relative areas with low CBF were detected in the 1-hour treated group from 4 to 48 hours after embolization as compared with the untreated group. The increase in T2 in the 1-hour treated group was significantly lower than in the untreated and 4-hour treated groups. A significant increase in ADC_w was detected in the 1-hour treated group at 3 and 24 hours after embolization as compared with the untreated and 4-hour treated groups. Secondary embolization was detected by both MRI and laser scanning confocal microscopy. The data suggest that MRI can detect the efficacy of rt-PA treatment and secondary ischemic damage.

A reduction in the apparent diffusion coefficient (ADC) of water measured by magnetic resonance imaging (MRI) has been shown to occur early after cerebrovascular occlusion. This change may be a useful indicator of brain tissue adversely affected by inadequate blood supply. The objective of this study was to test the hypothesis that loss of membrane ion homeostasis and depolarization can occur simultaneously with the drop in ADC. Also investigated was whether elevation of extracellular glutamate ([GLU]_e) would occur before ADC changes. High-speed MRI of the trace of the diffusion tensor (15-second time resolution) was combined with simultaneous recording of the extracellular direct current (DC) potential and on-line [GLU]_e from the striatum of the anesthetized rat. After a control period, data were acquired during remote middle cerebral artery occlusion for 60 minutes, followed by 30 minutes of reperfusion, and cardiac arrest-induced global ischemia. After either focal or global ischemia, the ADC was reduced by 10 to 25% before anoxic depolarization occurred. After either insult, the time for half the maximum change in ADC was significantly shorter than the corresponding DC potential parameter (P < 0.05). The [GLU]_e remained at low levels during the entire period of varying ADC and DC potential and did not peak until much later after either ischemic insult. This study demonstrates that ADC changes can occur before membrane depolarization and that high [GLU]_e has no involvement in the early rapid ADC decrease.

Magnetic resonance imaging (MRI) was utilized to obtain absolute estimates of regional brain water content (W), and results were compared with those obtained with conventional wet/dry measurements. In total, 31 male Long-Evans rats were studied and divided into two groups based on the surgical procedures used to induce cerebral focal ischemia: suture (n = 18) and three-vessel ligation (TVL; n = 13) groups. Both relative spin density and T1 were extracted from the acquired MR images. After correcting for radiofrequency field inhomogeneities, T2* signal decay, and temperature effects, in vivo regional brain water content, in absolute terms, was obtained by normalizing the measured relative brain spin density of animals to that of a water phantom. A highly linear relationship between MR-estimated brain water content based on the normalized spin density and wet/dry measurements was obtained with slopes of 0.989 and 0.986 for the suture (r = 0.79) and TVL (r = 0.83) groups, respectively. Except for the normal subcortex of the TVL group (P < 0.02) and the normal hemisphere of the suture group (P < 0.003), no significant differences were observed between MR-estimated and wet/dry measurements of brain water content. In addition, a highly linear relationship between MR-measured R1 (= 1/T1) and 1/W of wet/dry measurements was obtained. However, slopes of the linear regression lines in the two groups were significantly different (P < 0.02), indicating that different R1 values were associated with the same water content depending on the model. These results show that an absolute measurement of in vivo regional brain water content can be obtained with MRI and potentially serves as a noninvasive means to monitor different therapeutic interventions for the management of brain edema subsequent to stroke and head trauma.

Transient middle cerebral artery (MCA) occlusion results in substantially smaller cortical infarcts than permanent MCA occlusion if reperfusion is initiated within the first few hours. Only little information is available on the long-term functional outcome of the cortical regions “salvaged” by early reperfusion. To address this issue we examined basic electrophysiologic parameters in vitro using standard extracellular recording techniques at 7 and 28 days after transient MCA occlusion (1- and 2-hour ischemia) in rats. Both neocortical areas ipsi- and contralateral to MCA occlusion were systematically mapped to delineate the extent of periinfarct and remote alterations. In the periinfarct region we found a significant reduction of field potential amplitudes up to 3 mm when measuring from the infarct border at 7 days and up to 7 mm at 28 days. Paired-pulse inhibition, an indicator of GABAergic transmission, was only moderately impaired in this region at 7 days and not significantly different from control at 28 days. Remote effects were observed both ipsi- and contralaterally. Ipsilaterally they were restricted to a region close to the midline (presumably motor cortex) and were most likely attributable to the degeneration of corticostriatal connections. The extent of the contralateral excitability changes was clearly related to the size of the neocortical infarcts with large infarcts resulting in the widespread reduction of field potential amplitudes and an impairment of paired-pulse inhibition. The results show that there is a relatively large periinfarct region with decreased overall excitability after transient MCA occlusion which is likely to have a profound effect on perilesional processes involved in functional recovery. Remote excitability changes may contribute to the functional deficit and are probably related to deafferentation.

The proinflammatory cytokine tumor necrosis factor (TNF) is known to be expressed in brain ischemia; however, its cellular and temporal appearance is not fully settled. In this study, nonradioactive in situ hybridization for murine TNF mRNA was performed on brain sections from adult C57×129 mice at 6 hours, 12 hours, 24 hours, 2 days, 5 days, or 10 days (six to eight mice per group) after induction of permanent focal cerebral ischemia. Cortical infarct volumes were estimated, and TNF mRNA-expressing cells were counted within the infarct and infarct border using Cast-Grid analysis. At 12 hours, a peak of 19.2 ± 5.1 TNF mRNA-expressing cells/mm² was counted, contrasting two to three times lower values at 6 and 24 hours (6.4 ± 4.6 and 9.2 ± 3.4 cells/mm², respectively) and <2 cells/ mm² at 48 hours and later stages. The TNF mRNA-expressing cells were distributed along the entire rostrocaudal axis of the cortical infarcts and occasionally within the caudate putamen. At all time points, TNF mRNA colocalized with Mac-1-positive microglia/macrophages but not with Ly-6G (Gr-1)-positive polymorphonuclear leukocytes. Similarly, combined in situ hybridization for TNF mRNA and immunohistochemistry for glial fibrillary acidic protein at 12 and 24 hours revealed no TNF mRNA-expressing astrocytes at these time points. Translation of TNF mRNA into bioactive protein was demonstrated in the neocortex of C57Bl/6 mice subjected to permanent middle cerebral artery occlusion. In summary, this study points to a time-restricted microglial/macrophage production of TNF in focal cerebral ischemia in mice.

Administration of the selective calpain inhibitor AK295 has been shown to attenuate motor and cognitive dysfunction after brain trauma in rats. To explore mechanisms underlying the behavioral efficacy of posttraumatic calpain inhibition, we investigated histologic consequences of AK295 administration. Anesthetized Sprague-Dawley rats received lateral fluid percussion brain injury of moderate severity (2.2 to 2.4 atm) or served as uninjured controls. At 15 minutes after injury, animals were randomly assigned to receive a 48-hour infusion of either 2 mmol/L AK295 (120 to 140 mg/kg) or saline via the carotid artery. At 48 hours and 1 week after injury, spectrin fragments generated specifically by calpain were detected by Western blotting and immunohistochemistry, respectively, in saline-treated, brain-injured animals. Interestingly, equivalent spectrin breakdown was observed in AK295-treated animals when cortical and hippocampal regions were evaluated. Similarly, administration of the calpain inhibitor did not attenuate cortical lesion size or the numbers of apoptotic cells in the cortex, subcortical white matter, or hippocampus, as verified by terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick-end labeling and morphology, at 48 hours after injury. These data suggest that an overt reduction in spectrin proteolysis, cortical lesion, or apoptotic cell death at 48 hours or 1 week is not required for behavioral improvements associated with calpain inhibition by AK295 after experimental brain injury in rats.

To investigate the chronic effects of a novel thyrotropin-releasing hormone analog, JTP-2942 (N^α-[(1S, 2R)-2-methyl-4-oxocyclopentylcarbonyl]-l-histidyl-l-prolinamide monohydrate), on behavioral changes after stroke, the authors examined its effects on motor and neurologic deficits using a middle cerebral artery (MCA) occlusion model in rats. A left MCA was permanently occluded at a proximal site. From 1 week after occlusion, JTP-2942 was intravenously administered once a day for 4 weeks. Sensorimotor performance was evaluated weekly for 10 weeks after the occlusion. The ability of the rat to maintain its body position on an inclined plane and neurologic examination based on hemiparesis and abnormal posture were examined. After all behavioral examinations were completed, the degree of shrinkage of the left hemisphere was measured. The ability of MCA-occluded rats to maintain body position on an inclined plane in the left-headed position was significantly lower than that of sham-operated rats throughout the test period. JTP-2942 gradually improved this deficit dose dependently, and a dose of 0.03 mg/kg of JTP-2942 significantly improved performance to the levels of the sham-operated rats. Neurologic deficits were also observed in MCA-occluded rats. JTP-2942 also significantly improved these deficits dose dependently. On the other hand, CDP-choline (500 mg/kg, administered intravenously), a therapeutic agent for the disturbance of consciousness and hemiparesis after stroke, improved neurologic deficits but did not affect the motor deficits measured using the inclined plane. It is noteworthy that the effects of JTP-2942 on these deficits were observed 4 weeks after cessation of drug administration. Furthermore, there was no difference in the degree of shrinkage of the cerebrum among the MCA-occluded groups. In the present study, long-lasting improving effects of JTP-2942 on the impairment of motor and neurologic functions were observed in rats with MCA occlusion, which continued after cessation of drug administration and which were not attributable to a reduction in ipsilateral cerebral shrinkage. It is considered that the effect of JTP-2942 on functional recovery is attributable to the activation of substitutive functions such as neuronal reconstruction. These pharmacologic properties of JTP-2942 may be of interest for the treatment of patients with motor and neurologic deficits during the chronic or subacute phase of stroke.

Hypoglycemia can cause brain dysfunction, brain injury, and death. The present study seeks to broaden current information regarding mechanisms of hypoglycemic brain injury by investigating a novel etiology. The cat's high resistance to brain injury from hypoglycemia suggested that additional influences such as respiratory depression might play a facilitating role. Three groups of cats were exposed to fasting and insulin-induced hypoglycemia (HG; n = 6), euglycemic respiratory depression (RD; n = 5), and combined hypoglycemic respiratory depression (HG/RD; n = 10). The HG animals were maintained at < 1.5 mmol (mean 1 mmol) serum glucose concentration for 2 to 6.6 hours. The respiratory depression was associated with Pao₂ and Paco₂ values of ~50 mm Hg for 1 hour and of ~35 and ~75 mm Hg, respectively, for the second hour. Magnetic resonance diffusion-weighted imaging estimated brain energy state before, during, and after hypoglycemia. The hypoglycemic respiratory depression exposures were terminated either to euglycemia (n = 4) or to hyperglycemia (n = 6). Brain injury was assessed after 5 to 7 days of survival. Cats exposed to hypoglycemia alone maintained unchanged diffusion coefficients; that is, they lacked evidence of brain energy failure and all six remained brain-intact. Only 1 of 5 euglycemic RD but 10 of 10 HG/RD cats developed brain damage (HG and RD vs. HG/RD, P < 0.01). This difference in brain injury rates suggests injury potentiation by hypoglycemia and respiratory depression acting together. Three injury patterns emerged, including activation of microglia, selective neuronal necrosis, and laminar cortical necrosis. Widespread activation of microglia suggesting damage to neuronal cell processes affected all damaged brains. Selective neuronal necrosis affecting the cerebral cortex, hippocampus, and basal ganglia was observed in all but one case. Instances of laminar cortical necrosis were limited to cats exposed to hypoglycemic respiratory depression treated with hyperglycemia. Thus, treatment with hyperglycemia compared with euglycemia after hypoglycemic respiratory depression exposures significantly increased the brain injury scores (24 ± 6 vs. 13 ± 2 points; P < 0.05). This new experimental hypoglycemia model's contribution lies in recognizing additional factors that critically define the occurrence of hypoglycemic brain injury.

The delayed death of CA1 neurons after global brain ischemia is associated with induction of apoptosis genes and is inhibited by protein synthesis inhibitors, suggesting that the degeneration of CA1 pyramidal neurons is an active process that requires new gene expression. The transient global ischemia model has been extensively used to identify enzymes and other proteins underlying delayed neuronal cell death. The expression of protein kinase C (PKC) subspecies after 20 minutes of global brain ischemia produced by a four-vessel occlusion model in the rat was studied. From the multiple PKC subspecies studied, only PKCδ mRNA was significantly up-regulated in CA1 pyramidal neurons at 24 hours and in activated microglia at 3 to at least 7 days after ischemia. The induction of PKCδ mRNA was also found in the cortex at 8 hours and 3 days after ischemia. This cortical but not hippocampal induction was regulated by an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/kainate receptor antagonist, 6-nitro-7-sulfamobenzo[f]quinoxaline-2,3-dione, and glucocorticoids. An N-methyl-d-aspartate receptor antagonist, MK-801, was without effect on the induction of PKCδ subspecies. The selective and prolonged induction of the PKCδ mRNA and protein first in CA1 pyramidal neurons and at a later stage in activated microglia suggests that the PKCδ isozyme may take part in regulation of the delayed death of CA1 neurons after transient global brain ischemia.

The hypotheses that cerebral embolic events lead to repetitive episodes of cortical spreading depression (CSD) and that these propagating waves trigger the expression of c-fos, brain-derived neurotrophic factor (BDNF), glial fibrillary acidic protein (GFAP), and heat shock protein 70 (HSP70) mRNA were tested. Wistar rats underwent photochemically induced right common carotid artery thrombosis (CCAT) (n = 18) or sham (n = 8) procedures. In a subgroup of rats (n = 5), laser-Doppler flowmetry probes were placed overlying the right parietal cortex to record CSD-like changes in cortical blood flow during the initial 2-hour postinjury period. Rats were killed by decapitation at 2 or 24 hours after CCAT, and brains were processed for in situ localization of the gene expression. Two to five intermittent transient hyperemic episodes lasting 1 to 2 minutes were recorded ipsilaterally after CCAT. At 2 hours after CCAT, the widespread expression of c-fos and BDNF mRNAs was observed throughout the ipsilateral cerebral cortex. Pretreatment with the N-methyl-d-aspartate receptor blocker MK-801 (2 mg/kg) 1 hour before CCAT reduced the expression of BDNF mRNA expression at 2 hours. At 24 hours after CCAT, increased expression of GFAP mRNA was present in cortical and subcortical regions. In contrast, multifocal regions of HSP70 expression scattered throughout the thrombosed hemisphere were apparent at both 2 and 24 hours after injury. These data indicate that thromboembolic events lead to episodes of CSD and time-dependent alterations in gene expression. The ability of embolic processes to induce widespread molecular responses in neurons and glia may be important in the pathogenesis of transient ischemic attacks and may influence the susceptibility of the postembolic brain to subsequent insults including stroke.

Recent findings in animals emphasize that experimental ischemic brain damage can be strikingly reduced by estrogen; however, the neuroprotective mechanisms are not well understood. It was hypothesized that estrogen signaling via cognate estrogen receptors (ERs) within the vasculature is an important aspect of cerebral ischemic protection in the female brain, in part by amplifying intraischemic cerebral blood flow (CBF). In the present study, the hypothesis that chronic treatment with the pure ER antagonist ICI182,780 (ICI) would increase ischemic brain damage by a blood flow-mediated mechanism was investigated. Adult C57Bl/6J mice were pretreated with either subcutaneous ICI (100 μg/day) or oil/ ethanol vehicle for 1 week before 2 hours of middle cerebral artery occlusion (MCAO) and 22 hours of reperfusion. End-ischemic regional CBF was evaluated in additional cohorts using [¹⁴C]iodoantipyrine autoradiography. Infarction volume as measured by cresyl violet histology was greater in the striatum of ICI-treated females (70 ± 3% of contralateral striatum vs. 40 ± 12% in vehicle-treated females). Cortical injury was not enhanced relative to control animals (39 ± 6% of contralateral cortex in ICI group vs. 27 ± 8% in vehicle-treated group). Physiologic variables and ischemic reduction of the ipsilateral cortical laser-Doppler flow signal were similar between groups. Further, ICI treatment did not alter end-ischemic cortical or striatal CBF. The deleterious effect of ICI was limited to females, as there were no differences in stroke damage or CBF between male treatment groups. These data suggest that estrogen inhibits ischemic brain injury in striatum of the female by receptor-mediated mechanisms that are not linked to preservation of intraischemic CBF.

Excitotoxicity is implicated in the pathogenesis of several neurologic diseases, such as chronic neurodegenerative diseases and stroke. Recently, it was reported that excitotoxicity has a relationship to apoptotic neuronal death, and that the mitochondrial toxin, 3-nitropropionic acid (3-NP), could induce apoptosis in the striatum. Although striatal lesions produced by 3-NP could develop through an excitotoxic mechanism, the exact relationship between apoptosis induction and excitotoxicity after 3-NP treatment is still not clear. The authors investigated the role of excitotoxicity and oxidative stress on apoptosis induction within the striatum after intraperitoneal injection of 3-NP. The authors demonstrated that removal of the corticostriatal glutamate pathway reduced superoxide production and apoptosis induction in the denervated striatum of decorticated mice after 3-NP treatment. Also, the N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801, prevented apoptosis in the striatum after 3-NP treatment for 5 days, whereas the non-NMDA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, was ineffective. The authors also evaluated the initial type of neuronal death by 3-NP treatment for different durations from 1 to 5 days. In early striatal damage, apoptotic neuronal death initially occurred after 3-NP treatment. Our data show that excitotoxicity related to oxidative stress initially induces apoptotic neuronal death in mouse striatum after treatment with 3-NP.

Matrix metalloproteinases (MMPs), a family of proteolytic enzymes which degrade the extracellular matrix, are implicated in blood—brain barrier disruption, which is a critical event leading to vasogenic edema. To investigate the role of reactive oxygen species (ROS) in the expression of MMPs in vasogenic edema, the authors measured gelatinase activities before and after cold injury (CI) using transgenic mice that overexpress superoxide dismutase-1. A marked induction of pro-gelatinase B (pro-MMP-9) was seen 2 hours after CI and was maximized at 12 hours in wild-type mice. The pro-MMP-9 level was significantly lower in transgenic mice 4 hours (P < 0.001) and 12 hours (P < 0.05) after CI compared to wild-type mice. The activated MMP-9 was detected from 6 to 24 hours after injury. A mild induction of pro-gelatinase A (pro-MMP-2) was seen at 6 hours and was sustained until 7 days. In contrast, the activated form of MMP-2 appeared at 24 hours, was maximized at 7 days, and was absent in transgenic mice. Western blot analysis showed that the tissue inhibitors of metalloproteinases were not modified after CI. The results suggest that ROS production after CI may contribute to the induction and/or activation of MMPs and could thereby exacerbate endothelial cell injury and the development of vasogenic edema after injury.

The neurotrophins and the tyrosine kinase (Trk) B receptor may play a protective role in the pathophysiology of cerebral ischemia. In this study, the authors investigated whether reducing endogenous expression of TrkB-binding neurotrophins modifies the susceptibility to ischemic injury after 1-hour middle cerebral artery occlusion followed by 23 hours of reperfusion in a filament middle cerebral artery occlusion model. Mice lacking both alleles for neurotrophin-4 (nt4^−/−) or deficient in a single allele for brain-derived neurotrophic factor (bdnf+/−) exhibited larger cerebral infarcts compared to wild-type inbred 129/SV_jae mice (68% and 91%, respectively, compared to controls). Moreover, lesions were larger (21%) in nt4^−/− mice after permanent middle cerebral artery occlusion. Hence, expression of both NT4 and BDNF, and by inference the TrkB receptor, confers resistance to ischemic injury.

Slices from control C57, mdx, and mdx^3cv mice were made hypoxic until both field excitatory postsynaptic potential (fEPSP) and presynaptic afferent volley (AV) disappeared (H1). After reoxygenation and recovery of fEPSP, a second and longer hypoxic test (H2) lasted 3 minutes beyond the time required to block AV. When slices were kept in 10 mmol/L glucose, H1 abolished AV 37 and 19% earlier in slices from mdx and mdx^3cv mutants than in control slices (where H1 = 12 ± 4.6 minutes, mean ± SD). During H2 or when slices were kept in 4 mmol/L glucose, AV vanished even more quickly, but the times to block did not differ significantly between slices from controls and mutants. After reoxygenation, AV fully recovered in most slices. Rates of blockade of fEPSPs were comparable in all slices, and most fEPSPs recovered fully after H1. But even in the presence of 10 mmol/L glucose, the second hypoxia suppressed fEPSPs irreversibly in some slices: 2 of 10 from control, 3 of 7 from mdx, and 1 of 6 from mdx^3cv mice. Most slices in 4 mmol/L glucose showed no recovery at all: six of seven from control, three of five from mdx, and four of five from mdx^3cv mice. Thus, slices from mdx mice were more susceptible than other slices to irreversible hypoxic failure when slices were kept in 10 mmol/L glucose, but they were less susceptible than other slices when kept in 4 mmol/L glucose. In conclusion, the lack of full-length dystrophin (427 kDa) predisposes to quicker loss of nerve conduction in slices from mdx and mdx^3cv mutants and improved posthypoxic recovery of fEPSPs in 4 mmol/L glucose in slices from mdx but not mdx^3cv mutants, perhaps because the 70-kDa and other C-terminal isoforms are still present in mdx mice.

It was evaluated whether postischemic neurodegeneration is apoptosis and occurs with alterations in phosphoinositide-linked metabotropic glutamate receptors (mGluRs) and their associated signaling pathways. A dog model of transient global incomplete cerebral ischemia was used. The CA1 pyramidal cells and cerebellar Purkinje cells underwent progressive delayed degeneration. By in situ end-labeling of DNA, death of CA1 and Purkinje cells was greater at 7 days than 1 day after ischemia, whereas death of granule neurons in dentate gyrus and cerebellar cortex was greater at 1 than at 7 days. Ultrastructurally, degenerating CA1 pyramidal neurons and cerebellar Purkinje cells were necrotic; in contrast, degenerating granule neurons were apoptotic. In agarose gels of regional DNA extracts, random DNA fragmentation coexisted with internucleosomal fragmentation. By immunoblotting of regional homogenates, mGluR1α, mGluR5, phospholipase Cβ (PLCβ), and Gα_q/11 protein levels in hippocampus at 1 and 7 days after ischemia were similar to control levels, but in cerebellar cortex, mGluR1α and mGluR5 were decreased but PLCβ was increased. By immunocytochemistry, mGluR and PLCβ immunoreactivity dissipated in CA1 and cerebellar Purkinje cell/ molecular layers, whereas immunoreactivities for these proteins were enhanced in granule neurons. It was concluded that neuronal death after global ischemia exists as two distinct, temporally overlapping forms in hippocampus and cerebellum: necrosis of pyramidal neurons and Purkinje cells and apoptosis of granule neurons. Neuronal necrosis is associated with a loss of phosphoinositide-linked mGluR transduction proteins, whereas neuronal apoptosis occurs with increased mGluR signaling.

The brain endothelial large neutral amino acid carrier (l-system) is well suited for facilitated drug transport to the brain because of its high transport capacity and relatively broad structural substrate tolerance. The authors have examined the potential of this transporter for central nervous system (CNS) delivery of a new family of compounds derived from the large neutral amino acid phenylglycine. These compounds are highly selective for specific isoforms of metabotropic glutamate receptors (mGluRs) but will only become effective therapeutics for CNS diseases such as ischemic disorders, stroke, and epilepsy if they can effectively cross the blood-brain barrier. Using the immortalized rat brain endothelial cell line RBE4 as in vitro blood-brain barrier model, the authors have studied the interaction of phenylglycine and selected derivatives with the l-system-mediated transport of l-[³H]-histidine. The transport of l-histidine was characteristic of the l-system in vivo with the following kinetic parameters: K_m 135 ± 18 μmol/L, V_max 15.3 ± 1.13 nmol/min/mg protein, and K_D 2.38 ± 0.84 μL/min/mg protein. The affinities of the l-system for phenylglycine and the derivatives investigated increased in the order S-4-carboxy-phenylglycine (K_i = 16 mmol/L) < R-phenylglycine (2.2 mmol/L) < S-3-hydroxy-phenylglycine (48 μmol/L) < S-phenylglycine (34 μmol/L), suggesting that a negative charge at the side chain or R-configuration is detrimental for carrier recognition, whereas neutral side chain substituents are well tolerated. The authors have further shown (1) that the mode of interaction with the l-system of S-phenylglycine and S-3-hydroxy-phenylglycine is competitive, and (2) that the transporter carries these two agents into the cell as shown by high-performance liquid chromatography (HPLC) analysis of the RBE4 cell contents. The study provides the first evidence for the potential of S-phenylglycine derivatives for carrier-mediated delivery to the CNS and outlines the substrate specificity of the l-system at the blood-brain barrier for this class of mGluR ligands. As the affinities of S-phenylglycine and S-3-hydroxy-phenylglycine for the l-system carrier are even higher than those of some natural substrates, these agents should efficiently enter CNS via this route. Possible strategies for a synergistic optimization of phenylglycine-derived therapeutics with respect to desired activity at the CNS target combined with carrier-mediated delivery to overcome the blood-brain barrier are discussed.

Both thrombin and plasmin induce contraction of brain endothelial cells, which may increase capillary permeability thereby leading to disruption of the blood-brain barrier. Identification of thrombin receptors, as well as the influence of plasmin on their activation, in capillary endothelial cells and astrocytes are therefore essential for understanding injury-related actions of thrombin in the brain. Using the reverse transcriptase-polymerase chain reaction method, the present study shows that primary cultures of rat brain capillary endothelial (RBCE) cells and astrocytes derived from rat brain express two different thrombin receptors. The first is proteolytically activated receptor (PAR)-1, the receptor responsible for the vast majority of the thrombin's cellular activation functions; the second is PAR-3, a receptor described to be essential for normal responsiveness to thrombin in mouse platelets. In addition to these thrombin receptors, the mRNA (messenger RNA) for PAR-2, a possible trypsin receptor, was also identified. Functional significance of thrombin receptors was indicated by changes in [Ca²⁺]_i in response to thrombin, as measured by FURA-2 fluorescence in RBCE cells. Thrombin as low as 4 nmol/L induced an abrupt increase in [Ca²⁺]_i whereas, upon addition of active site-blocked thrombin or plasmin, [Ca²⁺]_i remained unchanged. The [Ca²⁺]_i signal attributable to thrombin was smaller in a low Ca²⁺-containing medium, indicating that an influx of Ca²⁺ from the extracellular medium makes a contribution to the overall [Ca²⁺]_i rise. The amplitude of the transient [Ca²⁺]_i signal was dependent on the concentration of thrombin, and repeated application of the enzyme caused an essentially complete and long-term desensitization of the receptor. The PAR-1 agonist peptide SFLLRN also elicited a transient increase in [Ca²⁺]_i. After activation by SFLLRN, cells showed a diminished response to thrombin, but the response was not absent, indicating that PAR-3 might contribute to the generation of the [Ca²⁺]_i signal. Pretreatment of RBCE cells with 100 nmol/L plasmin completely prevented [Ca²⁺]_i rise attributable to thrombin. These data show that RBCE cells and astrocytes express at least two receptors for thrombin, PAR-1 and PAR-3, and probably both receptors are involved in thrombin-induced [Ca²⁺]_i signals. Plasmin itself does not elevate [Ca²⁺]_i but prevents the activation of receptors by thrombin.

Results from pharmacological studies have suggested that presynaptic N-type Ca²⁺ channels play an important role in regulating neuronal Ca²⁺ influx and transmitter nitric oxide (NO) release in isolated cerebral arteries. However, the presence of N-type Ca²⁺ channels in cerebral perivascular nerves has not been directly demonstrated. As a major source of cerebral perivascular NOergic innervation is the sphenopalatine ganglion (SPG), adult rat SPGs were cultured and examined by whole-cell patch-clamp technique. One week after growing in the culture medium, significant neurite outgrowth from the SPG neuronal cells was observed. Both soma and neurites of these cells were immunoreactive for N-type Ca²⁺ channels, transmitter-synthesizing enzymes (choline acetyltransferase and NO synthase), and several neuropeptides (vasoactive intestinal peptide, neuropeptide Y, calcitonin gene-related peptide, substance P, and pituitary adenylate cyclase-activating peptide-38) that had been found in cerebral perivascular nerves in whole-mount vascular preparations. In current-clamp recordings, injection of a small depolarizing current caused action potential firing. In voltage-clamp recordings, the fast inward currents were blocked by tetrodotoxin and outward currents by tetraethylammonium, which is typical for neurons. Most Ca²⁺ currents isolated by blockade of sodium and potassium currents were blocked by ω-conotoxin, indicating that N-type Ca²⁺ channels are the dominant voltage-dependent Ca²⁺ channels regulating Ca²⁺ influx during membrane depolarization of SPG neurons. The ability to culture postganglionic SPG neurons provides an opportunity to directly study the electrophysiological and pharmacological properties of these neurons.

The relationship between local rates of cerebral glucose utilization (lCMR_glc) and glucose transporter expression was examined during physiologic activation of the hypothalamoneurohypophysial system. Three days of water deprivation, which is known to activate the hypothalamoneurohypophysial system, resulted in increased lCMR_glc and increased concentrations of GLUT1 and GLUT3 in the neurohypophysis; mRNA levels of GLUT1 and GLUT3 were decreased and increased, respectively. Water deprivation also increased lCMR_glc in the hypothalamic supraoptic and paraventricular nuclei; mRNA levels of GLUT1 and GLUT3 appeared to increase in these nuclei, but the changes did not achieve statistical significance. Restoration of water for 3 to 7 days reversed all observed changes in GLUT expression (protein and mRNA); restoration of water also reversed changes in lCMR_glc in both the neurohypophysis and the hypothalamic nuclei. These results indicate that under conditions of neural activation and recovery, changes in lCMR_glc and the levels of GLUT1 and GLUT3 are temporally correlated in the neurohypophysis and raise the possibility that GLUT1 and GLUT3 transporter expression may be regulated by chronic changes in functional activity. In addition, increases in the expression of GLUT5 mRNA in the neurohypophysis after dehydration provide evidence for involvement of microglial activation.

The close correspondence between neural activity in the brain and cerebral blood flow (CBF) forms the basis for modern functional neuroimaging methods. Yet, the temporal characteristics of hemodynamic changes induced by neuronal activity are not well understood. Recent optical imaging observations of the time course of deoxyhemoglobin (HbR) and oxyhemoglobin have suggested that increases in oxygen consumption after neuronal activation occur earlier and are more spatially localized than the delayed and more diffuse CBF response. Deoxyhemoglobin can be detected by blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI). In the present study, the temporal characteristics of CBF and BOLD changes elicited by somatosensory stimulation in rat were investigated by high-field (9.4 T) MRI. With use of high-temporal-resolution fMRI, it was found that the onset time of the CBF response in the somatosensory cortex was 0.6 ± 0.4 seconds (n = 10). The CBF changes occurred significantly earlier than changes in HbR concentration, which responded after 1.1 ± 0.3 seconds. Furthermore, no early increases in HbR (early negative BOLD signal changes) were observed. These findings argue against the occurrence of an early loss of hemoglobin oxygenation that precedes the rise in CBF and suggest that CBF and oxygen consumption increases may be dynamically coupled in this animal model of neural activation.

Geometric isomers of radioiodinated l-meta-tyrosine, 6-[I-125]iodo-and 4-[I-125]iodo-l-meta-tyrosine (6-I-l-mTyr, 4-I-l-mTyr) were separated by high-performance liquid chromatography. Both 6-I- and 4-I-l-mTyr had high energy-dependent brain accumulation. 6-I- and 4-I-l-mTyr were also metabolically stable and were rapidly excreted through the urine. 6-I-l-mTyr had a predilection for the cerebral aromatic l-amino acid decarboxylase (DOPA decarboxylase), the final enzyme of dopamine biosynthesis. 6-Radioiodinated l-mTyr is a new radiopharmaceutical that can be both useful in assessing cerebral amino acid transport mechanism and quantifying metabolically active DOPA decarboxylase.