bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct Physiologic Properties of Microglia and Blood-Borne Cells in Rat Brain Slices After Permanent Middle Cerebral Artery Occlusion
Authors: Lyons Susan A. , Pastor Andrea , Ohlemeyer Carsten , Kann Oliver , Wiegand Frank , Prass Konstantin , Knapp Felix , Kettenmann Helmut , Dirnagl Ulrich
Published: November 2003

The authors investigated the time course of leukocyte infiltration compared with microglial activation in adult rat brain slices after permanent middle cerebral artery occlusion (MCAO). To distinguish peripheral leukocytes from microglia, the blood cells were prelabeled in vivo with Rhodamine 6G (Rhod6G) IV before induction of ischemia. At specific times after infarct, invading leukocytes, microglia, and endothelial cells were labeled in situ with isolectin (IL)B4-FITC (ILB4). Six hours after MCAO only a few of the ILB4+ cells were colabeled by Rhod6G. These cells expressed the voltage-gated inwardly and outwardly rectifying K+ currents characteristic of macrophages. The majority of the ILB4+ cells were Rhod6G− and expressed a lack of voltage-gated channels, recently described for ramified microglial cells in brain slices, or exhibited only an inward rectifier current, a unique marker for cultured (but unstimulated) microglia. Forty-eight hours after MCAO, all blood-borne and the majority of Rhod6G− cells expressed outward and inward currents indicating that the intrinsic microglial population exhibited physiologic features of stimulated, cultured microglia. The ILB4+/Rhod6G− intrinsic microglial population was more abundant in the border zone of the infarct and their morphology changed from radial to ameboid. Within this zone, the authors observed rapidly migrating cells and recorded this movement by time-lapse microscopy. The current findings indicate that microglial cells acquire physiologic features of leukocytes at a later time point after MCAO.
Alteration of Cyclic Adenosine Monophosphate Response Element Binding Protein in Rat Brain After Hypoglycemic Coma
Authors: Ouyang Yi-Bing , He Qing-Ping , Zhang Xiao-Hu , Wang Gui-Xia , Siesjö Bo K. , Hu Bing-Ren
Published: November 2003

In the current study, the temporal and regional changes of the transcription factor cyclic adenosine monophosphate response element binding protein (CREB) were investigated in rat brains subjected to 30 minutes of hypoglycemic coma followed by varied periods of recovery using Western blot and confocal microscopy. The total amount of CREB was not altered in any area examined after coma. The level of the phosphorylated form of CREB decreased during coma but rebounded after recovery. In the relatively resistant areas, such as the inner layers of the neocortex and the inner and outer blades of the dentate gyrus (DG), phospho-CREB increased greater than the control level after 30 minutes of recovery and continued to increase up to 3 hours of recovery. In contrast, little or no increase of phospho-CREB was observed during the recovery period in the outer layers of the neocortex and at the tip of the DG, that is, regions that are selectively vulnerable to hypoglycemic insults. The current findings suggest that a neuroprotective signaling pathway may be more activated in the resistant regions than in the vulnerable ones after hypoglycemic coma.
Expression of Large Amino Acid Transporter LAT1 in Rat Brain Endothelium
Authors: Duelli Roman , Enerson Bradley E. , Gerhart David Z. , Drewes Lester R.
Published: November 2003

The expression of the large amino acid transporter, LAT1, was investigated in brain of adult Long-Evans rats. The LAT1 transcript was readily detected in brain microvessels and choroid plexus by reverse transcription polymerase chain reaction analysis using three different gene specific primer pairs. A polyclonal affinity purified antibody against the N-terminus of LAT1 was generated in chickens and used in immunoblot and immunocytochemical analyses of brain tissue sections of adult rats. On immunoblots, the antibody detected a peptide-inhibitable 45 kDa band in a rat brain microvessel membrane preparation. It also identified the same protein band in membrane preparations of different brain structures, as well as in heart and testis, whereas the protein was absent or only faintly detectable in muscle, kidney, and liver. In brain sections, the antibody intensely labeled the luminal and abluminal membranes of brain microvessel endothelial cells in all brain areas examined including cerebral cortex, cerebellum, hippocampus, and in gray and white matter regions. These results suggest that LAT1 is involved in transcellular transport and may play an important role in large, neutral amino acid transfer across the blood–brain barrier.
Functional Interdependence and Colocalization of Endothelial Nitric Oxide Synthase and Heat Shock Protein 90 in Cerebral Arteries
Authors: Khurana Vini G. , Feterik Kristian , Springett Margaret J. , Eguchi Daihiko , Shah Vijay , Katusic Zvonimir S.
Published: November 2003

Heat shock protein 90 (HSP90), an essential component of several signal transduction systems, participates in the activation of endothelial nitric oxide synthase (eNOS) in cells. The objective of the current study was to determine if HSP90 and eNOS were functionally interdependent and colocalized in the cerebral circulation. The authors used isometric force recording, cyclic 3′5′-guanosine monophosphate (cGMP) radioimmunoassay (RIA), and immunogold electron microscopy (EM) to study canine basilar artery. They found that geldanamycin (0.1 to 10 μg/mL), a selective HSP90 inhibitor, caused concentration-dependent contractions in arterial rings (n = 6 dogs). Contractions to geldanamycin were unaffected by a cyclooxygenase inhibitor, indomethacin (10 μmol/L; P < 0.05, n = 6). Functional evidence for interaction between HSP90 and nitric oxide (NO)-mediated signaling included observations that the contractile effect of geldanamycin was the following: (1) endothelium-dependent, (2) abolished by Ng-nitro-l-arginine methylester (L-NAME; 0.3 mmol/L), and (3) nonadditive with the contractile effect of this NOS inhibitor ( P < 0.01, n = 6 for each). Furthermore, RIA showed significant reduction in cGMP levels in arteries treated with geldanamycin (3 μg/mL; P < 0.02, n = 8), whereas immunogold EM demonstrated areas of colocalization of HSP90 and eNOS selectively in the cytoplasm of endothelial cells. The current findings suggest that in cerebral arteries, endothelial HSP90 plays an important role in modulation of basal NO-mediated signaling. This interaction may be particularly important in stress-induced up-regulation of HSP90 with subsequent alteration of vasomotor function.
Influence of the Endothelial Glycocalyx on Cerebral Blood Flow in Mice
Authors: Vogel J. , Sperandio M. , Pries A.R. , Linderkamp O. , Gaehtgens P. , Kuschinsky W.
Published: November 2003

The endothelial surface layer (glycocalyx) of cerebral capillaries may increase resistance to blood flow. This hypothesis was investigated in mice by intravenous administration of heparinase (2500 IU/kg body weight in saline), which cleaves proteoglycan junctions of the glycocalyx. Morphology was investigated by transmission electron microscopy. Cerebral perfusion velocity was recorded before and during heparinase or saline treatment using laser–Doppler flowmetry. In addition, cerebral blood flow (CBF) was measured 10 minutes after heparinase or saline treatment using the iodo[14C]antipyrine method. Laser–Doppler flowmetry and CBF measurements were performed during normocapnia and severe hypercapnia (Pco2: 120 mm Hg). After heparinase, morphology showed a reduced thickness of the glycocalyx in cortical microvessels by 43% ( P < 0.05) compared with saline-treated controls. Under normocapnic conditions, a 15% ( P < 0.05) transient increase of cerebral flow velocity occurred 2.5 to 5 minutes after heparinase injection. Laser–Doppler flow and CBF returned to control values ten minutes after the injection. However, during severe hypercapnia, heparinase treatment resulted in a persisting increase in laser–Doppler flow (6%, P < 0.05) and CBF (30%, P < 0.05). These observations indicate the existence of a flow resistance in cerebral capillaries exerted by the glycocalyx. The transient nature of the CBF increase during normocapnia may be explained by a vascular compensation that is exhausted during severe hypercapnia.
Involvement of Oxidative Stress and Caspase-3 in Cortical Infarction after Photothrombotic Ischemia in Mice
Authors: Kim Gyung W. , Sugawara Taku , Chan Pak H.
Published: November 2003

Apoptosis-related cell death is linked to oxidative stress and caspases in experimental cerebral ischemia, However, the role of oxidative stress in caspase activation and subsequent apoptotic cell death after cerebral ischemia is unknown, The authors evaluated the role of oxidative stress in ischemic cerebral infarction after photothrombosis and the relation between oxidative stress and caspase-related cell death 6 and 24 hours after ischemia with and without U-74389G, a potent free radical scavenger (10 mg/kg, 30 minutes before and after ischemia induction). Reactive oxygen species, detected by hydroethidine oxidation, and cytosolic cytochrome c were detected in early ischemic lesions. Western blot analysis showed the cleaved form and the level of the proform of caspase-3 in the ischemic lesion 24 hours after ischemia. Decreased caspase-3 immunoreactivity was detected in the antioxidant-treated group after ischemia. Decreased DNA fragmentation and laddering were detected and the lesion was smaller in the treated group after ischemia compared with the untreated group. Oxidative stress and cytochrome c release occur in the ischemic lesion after photothrombotic ischemia. The free radical scavenger attenuated caspase-3 up-regulation, DNA fragmentation, and the final lesion. The authors concluded that oxidative stress may mediate caspase-related apoptotic cell death and subsequent cortical infarction after photothrombotic ischemia.
Prolonged but Delayed Postischemic Hypothermia: A Long-term Outcome Study in the Rat Middle Cerebral Artery Occlusion Model
Authors: Colbourne Frederick , Corbett Dale , Zhao Zonghang , Yang Jing , Buchan Alastair M.
Published: November 2003

Delayed but prolonged hypothermia persistently decreases cell death and functional deficits after global cerebral ischemia in rodents. Postischemic hypothermia also reduces infarction after middle cerebral artery occlusion (MCAO) in rat. Because initial neuroprotection is sometimes transient and may not subserve functional recovery, especially on demanding tasks, the authors examined whether postischemic cooling would persistently reduce infarction and forelimb reaching deficits after MCAO. Male spontaneously hypertensive rats were trained to retrieve food pellets in a staircase test that measures independent forelimb reaching ability. Later, rats underwent 90 minutes of normothermic MCAO, through a microclip, or sham operation. In some rats, prolonged cooling (33°C for 24 hours and then 35°C for 24 hours) began 2.5 hours after the onset of ischemia (60 minutes after the start of reperfusion; n = 17 with subsequently 1 death) or sham procedures (n = 4), whereas untreated sham (n = 4) and ischemic (n = 16 with subsequently 1 death) rats maintained normothermia. An indwelling abdominal probe continually measured core temperature, and an automated fan and water spray system was used to produce hypothermia. One month later rats were reassessed in the staircase test over five days and then killed. The contralateral limb impairment in food pellet retrieval was completely prevented by hypothermia ( P = 0.0001). Hypothermia reduced an infarct volume of 67.5 mm3 after untreated ischemia to 35.8 mm3 ( P < 0.0001). These findings of persistent benefit encourage the clinical assessment of hypothermia.
Redistribution of Neuroactive Amino Acids in Hippocampus and Striatum during Hypoglycemia: A Quantitative Immunogold Study
Authors: Gundersen Vidar , Fonnum Frode , Ottersen Ole Petter , Storm-Mathisen Jon
Published: November 2003

Postembedding immunocytochemistry was used to localize aspartate, glutamate, gamma-aminobutyric acid (GABA), and glutamine in hippocampus and striatum during normo- and hypoglycemia in rat. In both brain regions, hypoglycemia caused aspartatelike immunoreactivity to increase. In hippocampus, this increase was evident particularly in the terminals of known excitatory afferents—in GABA-ergic neurons and myelinated axons. Aspartate was enriched along with glutamate in nerve terminals forming asymmetric synapses on spines and with GABA in terminals forming symmetric synapses on granule and pyramidal cell bodies. In both types of terminal, aspartate was associated with clusters of synaptic vesicles. Glutamate and glutamine immunolabeling were markedly reduced in all tissue elements in both brain regions, but less in the terminals than in the dendrosomatic compartments of excitatory neurons. In glial cells, glutamine labeling showed only slight attenuation. The level of GABA immunolabeling did not change significantly during hypoglycemia. The results support the view that glutamate and glutamine are used as energy substrates in hypoglycemia. Under these conditions both excitatory and inhibitory terminals are enriched with aspartate, which may be released from these nerve endings and thus contribute to the pattern of neuronal death characteristic of hypoglycemia.
Experimental Stroke in the Female Diabetic, db/db, Mouse
Authors: Vannucci Susan J. , Willing Lisa B. , Goto Shozo , Alkayed Nabil J. , Brucklacher Robert M. , Wood Teresa L. , Towfighi Javad , Hurn Patricia D. , Simpson Ian A.
Published: November 2003

Diabetic hyperglycemia increases brain damage after cerebral ischemia in animals and humans, although the underlying mechanisms remain unclear. Gender-linked differences in ischemic tolerance have been described but have not been studied in the context of diabetes. In the current study, we used a model of unilateral common carotid artery ligation, combined with systemic hypoxia, to study the effects of diabetes and gender on hypoxic–ischemic (HI) brain damage in the genetic model of Type II diabetes, the db/db, mouse. Male and female, control and db/db, mice were subjected to right common carotid artery ligation followed by varying periods of hypoxia (8% oxygen/92% nitrogen) to assess mortality, infarct volume, and tissue damage by light microscopic techniques. End-ischemic regional cerebral blood flow (CBF) was determined using [14C] iodoantipyrine autoradiography. Glycolytic and high energy phosphate compounds were measured in blood and brain by enzymatic and fluorometric techniques. Gender and diabetes had significant effects on mortality from HI and extent of brain damage in the survivors. Female mice were more resistant than their male counterparts, such that the severity (mortality and infarction size) in the male diabetics > female diabetics ~ male controls > female controls. End-ischemic CBF and depletion of cerebral high energy reserves were comparable among all groups. Surprisingly, female diabetic mice were more hyperglycemic and demonstrated a greater prolonged lactacidosis than the males; however, they were more resistant to damage. The results suggest a unique pathophysiology of hypoxia–ischemia in the female diabetic brain.
Can Cerebral Blood Volume Be Measured Reproducibly with an Improved near Infrared Spectroscopy System?
Authors: Van de Ven Marjo J. T. , Colier Willy N. J. M. , van der Sluijs Marco C. , Walraven Diederik , Oeseburg Berend , Folgering Hans
Published: November 2003

In some circumstances, cerebral blood volume (CBV) can be used as a measure for cerebral blood flow. A new near infrared spectroscope was used for determining the reproducibility of CBV measurements assessed by the O2-method. Twenty-seven healthy subjects were investigated. An intrasubject coefficient of variation (CV) was calculated, based on four identical episodes of desaturation–resaturation (O2-method) procedures for CBV measurements. Two trials were performed, with (trial 1) and without (trial 2) disconnecting the equipment. A mean CV of 12.6% and 10.0% was found in trial 1 and 2, respectively. Cerebral blood volume values yield 3.60 ± 0.82 mL 100 g−1. Cerebral blood volume could be measured reproducible in adults using near infrared spectroscopy, if the arterial desaturation is limited to approximately 5% from baseline level.
17-β Estradiol Can Reduce Secondary Ischemic Damage and Mortality of Subarachnoid Hemorrhage
Authors: Yang Shao-Hua , He Zhen , Wu Samuel S. , He Yun-Ju , Cutright Jason , Millard William J. , Day Arthur L. , Simpkins James W.
Published: November 2003

Subarachnoid hemorrhage (SAH) is a unique disorder commonly occurring when an aneurysm ruptures, leading to bleeding and clot formation, with a higher incidence in females. To evaluate the influence of 17-β estradiol (E2) in the outcome of subarachnoid hemorrhage, SAH was induced by endovascular puncture of the intracranial segment of internal carotid artery in 15 intact females (INT), 19 ovariectomized females (OVX), and 13 ovariectomized female rats with E2 replacement (OVX + E2). Cerebral blood flow was recorded before and after SAH. All animals were decapitated immediately after death or 24 hours after SAH for clot area analysis. Brains were sliced and stained with 2,3,5-triphenyltetrazolium chloride (TTC) for secondary ischemic lesion analysis. The cortical cerebral blood flow (CBF), which was measured by a laser–Doppler flowmeter, decreased to 29.6% ± 17.7%, 22.8% ± 8.3%, and 43.5% ± 22.9% on the ipsilateral side ( P = 0.01), and decreased to 63.4% ± 14.1%, 57.4% ± 11.0%, and 66.6% ± 17.9% on the contralateral side ( P = 0.26) in INT, OVX, and OVX + E2, respectively. The subcortical CBF, which were measured by the H2 clearance method, were 7.77 ± 12.03, 7.80 ± 8.65, and 20.58 ± 8.96 mL 100 g−1 min−1 on the ipsilateral side ( P < 0.01), and 21.53 ± 2.94, 25.13 ± 3.01, and 25.30 ± 3.23 mL 100 g−1 min−1 on the contralateral side in INT, OVX, and OVX + E2, respectively. The mortality was 53.3%, 68.4%, and 15.4% in INT, OVX, and OVX + E2, respectively ( P = 0.01), whereas no significant difference in clot area was noted among the groups. The secondary ischemic lesion volume was 9.3% ± 8.4%, 24.3% ± 16.3%, and 7.0% ± 6.4% in INT, OVX, and OVX + E2, respectively ( P < 0.01). This study demonstrated that E2 can reduce the mortality and secondary ischemic damage in a SAH model without affecting the clot volume.
Dynamic Imaging of Cerebral Blood Flow Using Laser Speckle
Authors: Dunn Andrew K. , Bolay Hayrunnisa , Moskowitz Michael A. , Boas David A.
Published: November 2003

A method for dynamic, high-resolution cerebral blood flow (CBF) imaging is presented in this article. By illuminating the cortex with laser light and imaging the resulting speckle pattern, relative CBF images with tens of microns spatial and millisecond temporal resolution are obtained. The regional CBF changes measured with the speckle technique are validated through direct comparison with conventional laser-Doppler measurements. Using this method, dynamic images of the relative CBF changes during focal cerebral ischemia and cortical spreading depression were obtained along with electrophysiologic recordings. Upon middle cerebral artery (MCA) occlusion, the speckle technique yielded high-resolution images of the residual CBF gradient encompassing the ischemic core, penumbra, oligemic, and normally perfused tissues over a 6 × 4 mm cortical area. Successive speckle images demonstrated a further decrease in residual CBF indicating an expansion of the ischemic zone with finely delineated borders. Dynamic CBF images during cortical spreading depression revealed a 2 to 3 mm area of increased CBF (160% to 250%) that propagated with a velocity of 2 to 3 mm/min. This technique is easy to implement and can be used to monitor the spatial and temporal evolution of CBF changes with high resolution in studies of cerebral pathophysiology.
Gender and Strain Influence on Neurogenesis in Dentate Gyrus of Young Rats
Authors: Perfilieva Ekaterina , Risedal Anette , Nyberg Jenny , Johansson Barbro B. , Eriksson Peter S.
Published: November 2003

To investigate whether rat hippocampal neurogenesis varies with strain and gender, the authors examined proliferating progenitor cells and their progeny in young male and female Sprague-Dawley (SD) and spontaneously hypertensive rats (SHR) using the thymidine analog bromodeoxyuridine (BrdU) combined with immunohistochemistry for the neuronal marker Calbindin D28k and glial fibrillary acidic protein. Rats were given 7 consecutive daily BrdU injections and were killed 1 day or 4 weeks later to allow for discrimination between proliferation and cell survival. Stereologic analysis of the numbers of BrdU-immunoreactive cells in the dentate gyrus revealed both a strain difference with significantly higher cell proliferation and net neurogenesis in SHR than in SD and a gender difference with males from both strains producing significantly more cells than their female counterparts. Whereas the number of progenitors four weeks after BrdU injections was still significantly greater in male than in female SHRs, resulting in a greater net neurogenesis in the male, the number of BrdU-immunoreactive cells did not differ between male and female SD rats, suggesting a greater survival of newly generated cells in the dentate gyrus in female than in male SD rats. No sex or strain difference was observed in the relative ratio of neurogenesis and gliogenesis.
Cortical Spreading Depression Induces Proinflammatory Cytokine Gene Expression in the Rat Brain
Authors: Jander Sebastian , Schroeter Michael , Peters Oliver , Witte Otto W. , Stoll Guido
Published: November 2003

Cortical spreading depression (CSD) is characterized by reversible neuronal dysfunction in the absence of cell death. Preconditioning by CSD induces tolerance against subsequent lethal ischemia. In this study, we used quantitative reverse transcriptase-polymerase chain reaction and immunocytochemistry to analyze proinflammatory cytokine expression after CSD induced by topical application of potassium chloride (KCl) to the cortical surface of rat brains. Relative to control cortex, we found an increase of tumor necrosis factor-α (mean 62-fold, P < 0.001) and interleukin (IL)-1β (mean 24-fold, P < 0.001) mRNA levels within 4 hours ipsilateral to the site of KCl application. At 16 hours cytokine expression was decreasing toward baseline levels. Ipsilateral cytokine induction was abolished by pretreatment with the noncompetitive N-methyl-d-aspartate antagonist, MK-801. In contrast to focal cortical infarction, cytokine induction in CSD was not accompanied by the expression of inducible nitric oxide synthase mRNA. In immunocytochemical studies, expression of IL-1β protein was localized to ramified microglia in cortical layers I to III of the ipsilateral hemisphere. Our finding that NMDA receptor signaling without subsequent neuronal cell death is sufficient to induce inflammatory cytokine expression in the brain has basic implications for central nervous system immunoregulation. We postulate that cytokine expression in CSD forms part of a physiologic stress response that contributes to the development of ischemic tolerance in this and other preconditioning paradigms.
Cell Permeable Exogenous Ceramide Reduces Infarct Size in Spontaneously Hypertensive Rats Supporting In Vitro Studies That Have Implicated Ceramide in Induction of Tolerance to Ischemia
Authors: Furuya Kazuhide , Ginis Irene , Takeda Hidetaka , Chen Yong , Hallenbeck John M.
Published: November 2003

Previous work in primary cell culture has shown that TNF-α and ceramide are involved in the signaling that induces tolerance to brain ischemia ( Ginis et al., 1999 ; Liu et al., 2000 ). To validate the in vitro studies, the authors administered cell permeable analogs of ceramides intracisternally or intravenously to examine their effect on neuroprotection after focal cerebral ischemia. Permanent middle cerebral artery occlusion (MCAO) was performed in spontaneously hypertensive rats. Infarct volumes were assessed at 24 hours after surgery. D- erythro-N-acetylsphingosine (C2-ceramide) or its vehicle was infused intracisternally for 1 hour before MCAO. In a second set of studies, D- erythro-N-octanoylsphingosine (C8-ceramide) or its vehicle was injected intravenously 48 or 24 hours before MCAO to mimic preconditioning (PC) and was also injected 5 minutes after MCAO. C2-ceramide infusion significantly reduced infarct volumes by approximately 14% ( P < 0.05). C8-ceramide injection reduced infarct volumes approximately 17% compared with controls. This effect was constant and significant compared with controls over the time periods examined ( P < 0.01). This work supports findings in primary brain cell cultures that implicate ceramide as a downstream signal that is proximate to development of tolerance to brain ischemia. Because the degree of protection represents approximately 50% of the maximal infarct reduction observed in this model, there are probably additional signaling pathways that subserve tolerance.
The Metabotropic Glutamate Receptor System Protects against Ischemic Free Radical Programed Cell Death in Rat Brain Endothelial Cells
Authors: Lin Shi-Hua , Maiese Kenneth
Published: November 2003

As one of the key determinants of ischemic injury, cerebrovascular endothelial cell (EC) degeneration may be dependent upon the generation of the free radical nitric oxide (NO) and the subsequent induction of programed cell death (PCD). Although the mechanisms that can prevent EC injury are most likely multifactorial in origin, the metabotropic glutamate receptor (mGluR) system may represent a novel therapeutic approach for ECs given the ability of the mGluR system to reverse neuronal cell injury. This study examined the modulation of individual subtypes of mGluRs during anoxia and NO toxicity in primary rat cerebrovascular ECs. Cell injury was determined through trypan blue dye exclusion, intracellular lactate dehydrogenase release, DNA fragmentation, membrane phosphatidylserine (PS) exposure, and cysteine protease activity. Anoxia, through the generation of NO, and exposure to exogenous NO were directly toxic to ECs. Exposure to NO rapidly decreased EC viability from 98% ± 2% to 40% ± 9%, increased DNA fragmentation from 2% ± 2% to 61% ± 9%, and increased membrane PS exposure from 3% ± 3% to 66% ± 6% over a 24-hour period. Activation of the mGluR system significantly increased EC survival through the prevention of NO-induced DNA fragmentation and cellular membrane PS residue exposure. In contrast, antagonism of the mGluR system failed to prevent PCD. Cytoprotection by the mGluR system was dependent, at least in part, upon the direct inhibition of NO-generated caspase 1- and caspase 3-like activities. Further investigation into the ability of the mGluR system to prevent PCD in ECs may open new therapeutic avenues for the treatment of cerebrovascular injury.
Cerebrovascular Adaptation in Chronic Hydrocephalus
Authors: Luciano Mark G. , Skarupa David J. , Booth Amy M. , Wood Arcangela S. , Brant Christine L. , Gdowski Martha J.
Published: November 2003

This study characterizes the regional changes in vascularity, which accompanies chronic progressive hydrocephalus. Fifteen dogs underwent surgical induction of hydrocephalus and were used for histologic studies. Animals were divided into 4 groups: surgical control, short term (≤5 weeks), intermediate term ((8 weeks), and long term (10 to 12 weeks). Vessel diameter, density, and luminal area were calculated by imaging quantification after manual vessel identification in the cortical gray, white matter, and caudate nucleus. Capillary vessel diameter decreased 23.5% to 30.2% ( P < 0.01) in the caudate, but then returned to normal at 12 weeks. Capillary vessel density decreased 53.5% ( P < 0.05) in the cortical gray, but then increased to 234.8% ( P < 0.01) over surgical controls at 12 weeks. There was no initial decrease in capillary density in the caudate; however, the long-term group capillary density was significantly greater (172.8% to 210.5%, P < 0.01) than surgical controls. Overall, there was a short-term decrease in lumen area, with recovery in the longer term. Glial fibrillary acidic protein (GFAP) immunohistochemistry demonstrated the pattern of GFAP staining and reactive astrocytes differed in the caudate compared with the occipital cortex. This data suggest that an increase in capillary density and diameter may be an adaptive process allowing maintenance of adequate cerebral perfusion and metabolic support in the hypoxic environment of chronic hydrocephalus.
A Strategy for Removing the Bias in the Graphical Analysis Method
Authors: Logan Jean , Fowler Joanna S. , Volkow Nora D. , Ding Yu Shin , Wang Gene-Jack , Alexoff David L.
Published: November 2003

The graphical analysis method, which transforms multiple time measurements of plasma and tissue uptake data into a linear plot, is a useful tool for rapidly obtaining information about the binding of radioligands used in PET studies. The strength of the method is that it does not require a particular model structure. However, a bias is introduced in the case of noisy data resulting in the underestimation of the distribution volume (DV), the slope obtained from the graphical method. To remove the bias, a modification of the method developed by Feng et al. (1993) , the generalized linear least squares (GLLS) method, which provides unbiased estimates for compartment models was used. The one compartment GLLS method has a relatively simple form, which was used to estimate the DV directly and as a smoothing technique for more general classes of model structures. In the latter case, the GLLS method was applied to the data in two parts, that is, one set of parameters was determined for times 0 to T1 and a second set from T1 to the end time. The curve generated from these two sets of parameters then was used as input to the graphical method. This has been tested using simulations of data similar to that of the PET ligand [11C]- d-threo-methylphenidate (MP, DV = 35 mL/mL) and 11C raclopride (RAC, DV = 1.92 mL/mL) and compared with two examples from image data with the same tracers. The noise model was based on counting statistics through the half-life of the isotope and the scanning time. Five hundred data sets at each noise level were analyzed. Results (DV) for the graphical analysis (DVg), the nonlinear least squares (NLS) method (DVnls), the one-tissue compartment GLLS method (DVf), and the two part GLLS followed by graphical analysis (DVfg) were compared. DVFG was found to increase somewhat with increasing noise and in some data sets at high noise levels no estimate could be obtained. However, at intermediate levels it provided a good estimation of the true DV. This method was extended to use a reference tissue in place of the input function to generate the distribution volume ratio (DVR) to the reference region. A linearized form of the simplified reference tissue method of Lammertsma and Hume (1996) was used. The DVR generated directly from the model (DVRfl) was compared with DVRfg (determined from a “smoothed” uptake curve as for DVfg) using the graphical method.
Evidence for the Involvement of Par-4 in Ischemic Neuron Cell Death
Authors: Culmsee Carsten , Zhu Yuan , Krieglstein Josef , Mattson Mark P.
Published: November 2003

After a stroke many neurons in the ischemic brain tissue die by a process called apoptosis, a form of cell death that may be preventable. The specific molecular cascades that mediate ischemic neuronal death are not well understood. The authors recently identified prostate apoptosis response-4 (Par-4) as a protein that participates in the death of cultured hippocampal neurons induced by trophic factor withdrawal and exposure to glutamate. Here, the authors show that Par-4 levels increase in vulnerable populations of hippocampal and striatal neurons in rats after transient forebrain ischemia; Par-4 levels increased within 6 hours of reperfusion and remained elevated in neurons undergoing apoptosis 3 days later. After transient focal ischemia in mice, Par-4 levels were increased 6 to12 hours after reperfusion in the infarcted cortex and the striatum, and activation of caspase-8 occurred with a similar time course. Par-4 immunoreactivity was localized predominantly in cortical neurons at the border of the infarct area. A Par-4 antisense oligonucleotide protected cultured hippocampal neurons against apoptosis induced by chemical hypoxia and significantly reduced focal ischemic damage in mice. The current data suggest that early up-regulation of Par-4 plays a pivotal role in ischemic neuronal death in animal models of stroke and cardiac arrest.
Induction of gp91-phox, a Component of the Phagocyte NADPH Oxidase, in Microglial Cells during Central Nervous System Inflammation
Authors: Green Simon P. , Cairns Belinda , Rae Julie , Errett-Baroncini Carol , Hongo Jo-Anne S. , Erickson Richard W. , Curnutte John T.
Published: November 2003

Gp91- phox is an integral component of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex that generates reactive oxygen species (ROS) in activated circulating phagocytes. The authors previously demonstrated that gp91- phox knockout (KO) mice show significant protection from neuronal injury after cerebral ischemia–reperfusion injury, suggesting a pivotal role for this enzyme. Moreover, results from chimeric mice suggested that elimination of gp91- phox from both circulating phagocytes and a putative central nervous system (CNS) source were required to confer neuroprotection. In the current study, the authors demonstrated gp91- phox–specific immunostaining of perivascular cells in the CNS of control rats. However, after transient cerebral ischemia, gp91- phox–positive phagocytes were observed within the core ischemic region and activated microglial cells were positive in the penumbra. Such activated microglial cells were also gp91- phox–positive in the CNS of a chimpanzee with mild meningitis. Finally, in humans, both normal adult CNS tissues and isolated fetal microglial cells expressed gp91- phox mRNA. These microglia also expressed mRNA for the five other known components that comprise the NADPH oxidase complex. These data strongly suggest that microglial cells may contain a functionally active NADPH oxidase capable of generating ROS during CNS inflammation.
Local Injection of Antisense Oligonucleotides Targeted to the Glial Glutamate Transporter GLAST Decreases the Metabolic Response to Somatosensory Activation
Authors: Cholet Nathalie , Pellerin Luc , Welker Egbert , Lacombe Pierre , Seylaz Jacques , Magistretti Pierre , Bonvento Gilles
Published: November 2003

The mechanisms responsible for the local increase in brain glucose utilization during functional activation remain unknown. Recent in vitro studies have identified a new signaling pathway involving an activation of glial glutamate transporters and enhancement of neuron–astrocyte metabolic interactions that suggest a putative coupling mechanism. The aim of the present study was to determine whether one of the glutamate transporters exclusively expressed in astrocytes, GLAST, is involved in the neurometabolic coupling in vivo. For this purpose, rats were microinjected into the posteromedial barrel subfield (PMBSF) of the somatosensory cortex with GLAST antisense or random phosphorothioate oligonucleotides. The physiologic activation was performed by stimulating the whisker-to-barrel pathway in anesthetized rats while measuring local cerebral glucose utilization by quantitative autoradiography in the PMBSF. Twenty-four hours after injection of two different antisense GLAST oligonucleotide sequences, and despite the presence of normal whisker-related neuronal activity in the PMBSF, the metabolic response to whisker stimulation was decreased by more than 50%. Injection of the corresponding random sequences still allowed a significant increase in glucose utilization in the activated area. The present study highlights the contribution of astrocytes to neurometabolic coupling in vivo. It provides evidence that glial glutamate transporters are key molecular components of this coupling and that neuronal glutamatergic activity is an important determinant of energy utilization. Results indicate that astrocytes should also be considered as possible sources of altered brain metabolism that could explain the distinct imaging signals observed in some pathologic situations
Differentiation of Glucose Transport in Human Brain Gray and White Matter
Authors: de Graaf Robin A. , Pan Jullie W. , Telang Frank , Lee Jing-Huei , Brown Peter , Novotny Edward J. , Hetherington Hoby P. , Rothman Douglas L.
Published: November 2003

Localized 1H nuclear magnetic resonance spectroscopy has been applied to determine human brain gray matter and white matter glucose transport kinetics by measuring the steady-state glucose concentration under normoglycemia and two levels of hyperglycemia. Nuclear magnetic resonance spectroscopic measurements were simultaneously performed on three 12-mL volumes, containing predominantly gray or white matter. The exact volume compositions were determined from quantitative T1 relaxation magnetic resonance images. The absolute brain glucose concentration as a function of the plasma glucose level was fitted with two kinetic transport models, based on standard (irreversible) or reversible Michaelis-Menten kinetics. The steady-state brain glucose levels were similar for cerebral gray and white matter, although the white matter levels were consistently 15% to 20% higher. The ratio of the maximum glucose transport rate, Vmax, to the cerebral metabolic utilization rate of glucose, CMRGlc, was 3.2 ± 0.10 and 3.9 ± 0.15 for gray matter and white matter using the standard transport model and 1.8 ± 0.10 and 2.2 ± 0.12 for gray matter and white matter using the reversible transport model. The Michaelis-Menten constant Km was 6.2 ± 0.85 and 7.3 ± 1.1 mmol/L for gray matter and white matter in the standard model and 1.1 ± 0.66 and 1.7 ± 0.88 mmol/L in the reversible model. Taking into account the threefold lower rate of CMRGlc in white matter, this finding suggests that blood–brain barrier glucose transport activity is lower by a similar amount in white matter. The regulation of glucose transport activity at the blood–brain barrier may be an important mechanism for maintaining glucose homeostasis throughout the cerebral cortex.
Insulin-Like Growth Factor-1 Reduces Postischemic White Matter Injury in Fetal Sheep
Authors: Guan Jian , Bennet Laura , George Shirley , Wu David , Waldvogel Harry J. , Gluckman Peter D. , Faull Richard L. M. , Crosier Philip S. , Gunn Alistair J.
Published: November 2003

Insulin-like growth factor-1 (IGF-1) is known to be important for oligodendrocyte survival and myelination. In the current study, the authors examined the hypothesis that exogenous IGF-1 could reduce postischemic white matter injury. Bilateral brain injury was induced in near-term fetal sheep by 30 minutes of reversible carotid artery occlusion. Ninety minutes after ischemia, either vehicle (n = 8) or a single dose of 3 μg IGF-1 (n = 9) was infused intracerebroventricularly over 1 hour. White matter changes were assessed after 4 days recovery in the parasagittal intragyral white matter and underlying corona radiata. Proteolipid protein (PLP) mRNA staining was used to identify bioactive oligodendrocytes. Glial fibrillary acidic protein (GFAP) and isolectin B-4 immunoreactivity were used to label astrocytes and microglia, respectively. Myelin basic protein (MBP) density and the area of the intragyral white matter tracts were determined by image analysis. Insulin-like growth factor-1 treatment was associated with significantly reduced loss of oligodendrocytes in the intragyral white matter ( P < 0.05), with improved MBP density ( P < 0.05), reduced tissue swelling, and increased numbers of GFAP and isolectin B-4 positive cells compared with vehicle treatment. After ischemia there was a close association of PLP mRNA labeled cells with reactive astrocytes and macrophages/microglia. In conclusion, IGF-1 can prevent delayed, postischemic oligodendrocyte cell loss and associated demyelination.
Adenosine in Relation to Calcium Homeostasis: Comparison between Gray and White Matter Ischemia
Authors: Dohmen Christian , Kumura Eiji , Rosner Gerd , Heiss Wolf-Dieter , Graf Rudolf
Published: November 2003

In vitro studies suggest that adenosine may attenuate anoxic white matter damage as an intrinsic protective substance. The authors investigated ischemic alterations of purines in relation to tissue depolarization and extracellular calcium and amino acid concentrations in vivo using microdialysis and ion-selective electrodes in cortical gray and subcortical white matter of 10 cats during 120 minutes of global brain ischemia. Immediately on induction of ischemia, regional cerebral blood flow ceased in all cats in both gray and white matter. The direct current potential rapidly decreased, the decline being slower and shallower in white matter. Extracellular calcium levels decreased in gray matter. In contrast, they first increased in white matter and started to decrease below control levels only after approximately 30 minutes. Adenosine levels transiently increased in both tissue compartments; the peak was delayed by 30 minutes in white matter. Thereafter, levels declined faster in gray than in white matter and remained elevated in the latter tissue compartment. Inosine and hypoxanthine elevations were progressive in both regions but smaller in white matter. Levels of gamma-aminobutyric acid, another putatively protective agent, steadily increased, starting immediately in gray matter and delayed by almost 1 hour in white matter. The delayed and prolonged accumulation of adenosine correlates with a slower adenosine triphosphate breakdown in white matter ischemia and may result in protection of white matter by suspending cellular calcium influx.
Both Caspase-Dependent and Caspase-Independent Pathways May Be Involved in Hippocampal CA1 Neuronal Death Because of Loss of Cytochrome c from Mitochondria in a Rat Forebrain Ischemia Model
Authors: Zhan Ren-Zhi , Wu Chaoran , Fujihara Hideyoshi , Taga Kiichiro , Qi Sihua , Naito Makoto , Shimoji Koki
Published: November 2003

In a rat forebrain ischemia model, the authors examined whether loss of cytochrome c from mitochondria correlates with ischemic hippocampal CA1 neuronal death and how cytochrome c release may shape neuronal death. Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 10 minutes. After reperfusion, an early rapid depletion of mitochondrial cytochrome c and a late phase of diffuse redistribution of cytochrome c occurred in the hippocampal CA1 region, but not in the dentate gyrus and CA3 regions. Intracerebroventricular administration of Z-DEVD-FMK, a relatively selective caspase-3 inhibitor, provided limited but significant protection against ischemic neuronal damage on day 7 after reperfusion. Treatment with 3 minutes of ischemia (ischemic preconditioning) 48 hours before the 10-minute ischemia attenuated both the early and late phases of cytochrome c redistribution. In another subset of animals treated with cycloheximide, a general protein synthesis inhibitor, the late phase of cytochrome c redistribution was inhibited, whereas most hippocampal CA1 neurons never regained mitochondrial cytochrome c. Examination of neuronal survival revealed that ischemic preconditioning prevents, whereas cycloheximide only delays, ischemic hippocampal CA1 neuronal death. DNA fragmentation detected by terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) in situ was largely attenuated by ischemic preconditioning and moderately reduced by cycloheximide. These results indicate that the loss of cytochrome c from mitochondria correlates with hippocampal CA1 neuronal death after transient cerebral ischemia in relation to both caspase-dependent and -independent pathways. The amount of mitochondrial cytochrome c regained may determine whether ischemic hippocampal CA1 neurons survive or succumb to late-phase death.
Up-Regulation of Neuropilin-1 in Neovasculature after Focal Cerebral Ischemia in the Adult Rat
Authors: Zhang Zheng Gang , Tsang Wayne , Zhang Li , Powers Cecylia , Chopp Michael
Published: November 2003

During development, neuropilin-1 is a receptor for semaphorin 3a-mediated axonal guidance and for vascular endothelial growth factor (VEGF) promotion of angiogenesis. The authors measured neuropilin-1 expression in the adult ischemic brain using Northern blot, in situ hybridization, and immunohistochemistry. Neuropilin-1 mRNA was significantly up-regulated as early as 2 hours and persisted at least 28 days after focal cerebral ischemia. Acute up-regulation of neuropilin-1 mRNA primarily localized to the ischemic neurons. A marked increase in both mRNA and protein of neuropilin-1 was detected in endothelial cells of cerebral blood vessels at the border and in the core of the ischemic lesion 7 days after ischemia, and neuropilin-1 gene expression persisted on these vessels for at least 28 days after ischemia. In these areas, neovascularization was detected using three-dimensional reconstructed images obtained from laser scanning confocal microscopy. Activated astrocytes also exhibited neuropilin-1 immunoreactivity during 7 to 28 days of ischemia. Double immunofluorescent staining showed colocalization of neuropilin-1 and VEGF to cerebral blood vessels and activated astrocytes. These data suggest that in addition to its role in axonal growth, up-regulation of neuropilin-1, in concert with VEGF and its receptors, may contribute to neovascular formation in the adult ischemic brain.
Changes in Human Regional Cerebral Blood Flow and Cerebral Blood Volume during Visual Stimulation Measured by Positron Emission Tomography
Authors: Ito Hiroshi , Takahashi Kazuhiro , Hatazawa Jun , Kim Seong-Gi , Kanno Iwao
Published: November 2003

The hemodynamic mechanism of increase in cerebral blood flow (CBF) during neural activation has not been elucidated in humans. In the current study, changes in both regional CBF and cerebral blood volume (CBV) during visual stimulation in humans were investigated. Cerebral blood flow and CBV were measured by positron emission tomography using H215O and 11CO, respectively, at rest and during 2-Hz and 8-Hz photic flicker stimulation in each of 10 subjects. Changes in CBF in the primary visual cortex were 16% ± 16% and 68% ± 20% for the visual stimulation of 2 Hz and 8 Hz, respectively. The changes in CBV were 10% ± 13% and 21% ± 5% for 2-Hz and 8-Hz stimulation, respectively. Significant differences between changes in CBF and CBV were observed for visual stimulation of 8 Hz. The relation between CBF and CBV values during rest and visual stimulation was CBV = 0.88CBF0.30. This indicates that when the increase in CBF during neural activation is great, that increase is caused primarily by the increase in vascular blood velocity rather than by the increase in CBV. This observation is consistent with reported findings obtained during hypercapnia.
Tissue-Type Plasminogen Activator is Involved in the Process of Neuronal Death Induced by Oxygen–Glucose Deprivation in Culture
Authors: Nagai Nobuo , Yamamoto Seiji , Tsuboi Takashi , Ihara Hayato , Urano Tetsumei , Takada Yumiko , Terakawa Susumu , Takada Akikazu
Published: November 2003

Effect of tissue-type plasminogen activator (tPA) on oxygen–glucose deprivation (OGD) was studied in cultured cortical neurons prepared from tPA gene knockout (tPA-KO) and wild-type (Wt) mice. Three hours of OGD induced 45% and 23% of neuronal death in Wt and tPA-KO mice, respectively. Neuronal death in tPA-KO mice was increased to 42% by additional tPA. Six hours of OGD induced 80% and 40% of neuronal death in Wt and tPA-KO mice, respectively, whereas the addition of tPA increased to 62% in tPA-KO mice. These results suggest that tPA is directly involved in the process of neuronal death induced by ischemia-mimic stress without involving vascular or circulatory components.