Regional brain blood flow in mouse: quantitative measurement using a single-pass radio-tracer method and a mathematical algorithm.

We have developed a reliable experimental method for measuring local regional cerebral blood flows in anesthetized mice. This method is an extension of the well-established single-pass dual-label indicator method for simultaneously measuring blood flow and glucose influx in rat brains. C57BL6J mice (n = 10) were anesthetized and regional blood flows (ml/min/g) were measured using the radio-tracer method. To test the sensitivity of this method we used a mathematical algorithm to predict the blood flows and compared the two sets of results.Measured regional blood flows between 0.7 and 1.7 ml/min/g were similar to those we have previously reported in the rat. The predicted blood flows using an assumed linearly increasing arterial tracer concentration-versus-time profile (that is, a ramp) were similar to the values measured in the physiological experiments (R(2) 0.99; slope 0.91). Thus,measurements of local regional cerebral blood flow in anesthetized mice using a single-pass radio-tracer method appear to be reliable.

Distribution of NBCn2 (SLC4A10) splice variants in mouse brain.

The five known Na-coupled HCO(3)(-) transporters (NCBTs) of the solute carrier 4 (SLC4) family play important roles in pH regulation and transepithelial HCO(3)(-) transport. Nearly all of the NCBTs have multiple splice variants. One particular NCBT, the electroneutral Na/HCO(3)(-) cotransporter NBCn2 (SLC4A10), which is predominantly expressed in brain, has three known splice variants-NBCn2-A, -B, and -C-as well as a potential variant-D. It is important to know the tissue-specific expression of the splice variants for understanding the physiological roles of NBCn2 in central nervous system. In the present study, we developed three novel rabbit polyclonal antibodies against NBCn2: (1) anti-ABCD, which recognizes all four variants; (2) anti-BD, which recognizes NBCn2-B and -D; (3) anti-CD, which recognizes NBCn2-C and -D. By western blotting, we examined the expression and distribution of NBCn2 splice variants in five brain regions: cerebral cortex, subcortex, cerebellum, hippocampus, and medulla. The expression pattern revealed with anti-ABCD is distinct from those revealed with anti-BD and anti-CD. Moreover, by using immunoprecipitation in combination with western blotting, we demonstrate that NBCn2-D does indeed exist and that it is predominantly expressed in subcortex, to a lesser extent in medulla, but at very low levels in cortex, cerebellum, and hippocampus. NBCn2-A may be the dominant variant in mouse brain as a whole, and may also dominate in cerebral cortex, cerebellum, and hippocampus. Immunohistochemistry with anti-ABCD shows that NBCn2 is highly expressed in choroid plexus, cortex, molecular layer of cerebellum, hippocampus, and some specific regions of the brainstem.

Effect of alternate energy substrates on mammalian brain metabolism during ischemic events.

Regulation of brain metabolism and cerebral blood flow involves complex control systems with several interacting variables at both cellular and organ levels. Quantitative understanding of the spatially and temporally heterogeneous brain control mechanisms during internal and external stimuli requires the development and validation of a computational (mathematical) model of metabolic processes in brain. This paper describes a computational model of cellular metabolism in blood-perfused brain tissue, which considers the astrocyte-neuron lactate-shuttle (ANLS) hypothesis. The model structure consists of neurons, astrocytes, extra-cellular space, and a surrounding capillary network. Each cell is further compartmentalized into cytosol and mitochondria. Inter-compartment interaction is accounted in the form of passive and carrier-mediated transport. Our model was validated against experimental data reported by Crumrine and LaManna, who studied the effect of ischemia and its recovery on various intra-cellular tissue substrates under standard diet conditions. The effect of ketone bodies on brain metabolism was also examined under ischemic conditions following cardiac resuscitation through our model simulations. The influence of ketone bodies on lactate dynamics on mammalian brain following ischemia is studied incorporating experimental data.

Statistical analysis of metabolic pathways of brain metabolism at steady state.

The estimation of metabolic fluxes for brain metabolism is important, among other things, to test the validity of different hypotheses which have been proposed in the literature. The metabolic model that we propose considers, in addition to the blood compartment, the cytosol, and mitochondria of both astrocyte and neuron, including detailed metabolic pathways. In this work we use a recently developed methodology to perform a statistical Flux Balance Analysis (FBA) for this model. The methodology recasts the problem in the form of Bayesian statistical inference and therefore can take advantage of qualitative information about brain metabolism for the simultaneous estimation of all reaction fluxes and transport rates at steady state. By a Markov Chain Monte Carlo (MCMC) sampling method, we are able to provide for each reaction flux and transport rate a distribution of possible values. The analysis of the histograms of the reaction fluxes and transport rates provides a very useful tool for assessing the validity of different hypotheses about brain energetics proposed in the literature, and facilitates the design of the pathways network that is in accordance with what is understood of the functioning of the brain. In this work, we focus on the analysis of biochemical pathways within each cell type (astrocyte and neuron) at different levels of neural activity, and we demonstrate how statistical tools can help implement various bounds suggested by experimental data.

Ultrastructural analysis of a murine model of congenital hydrocephalus produced by overexpression of transforming growth factor-beta1 in the central nervous system.

The purpose of this study was to elucidate using transmission electron microscopy (TEM) the ultrastructural changes that occur within the cortical gray matter of a novel reproducible model of congenital hydrocephalus in mice created to overexpress the cytokine transforming growth factor-beta1 (TGF-beta1) in the central nervous system. Brain tissue was obtained from mice from a colony engineered to overexpress TGF-beta1 at two days postpartum and compared to a wild-type aged-matched control. This tissue was fixed using a solution containing 1.25% paraformaldehyde and 1.25% glutaraldehyde in phosphate buffer at least 3-4 h and then cut into 40-50 microm sections. Randomly selected thin sections were stained with uranyl acetate and lead citrate, and then analyzed using a JEOL-100CX or 1200EX transmission electron microscope at accelerating voltage 80 kV. Dramatic neuronal and glial pathology was observed throughout the cortical neuropil in TGF-beta1 mice. The most striking change in the hydrocephalic mice was severe edema with extracellular fluid, possibly due to cerebrospinal fluid (CSF) penetration into the cortex. In addition, severe disruption of the cytoplasmic matrix was seen throughout the cortex, with damage to cellular organelles and particularly severe damage to mitochondria. Our results suggest that congenital hydrocephalus may be associated with significant damage to cortical tissue.

Hypoxia tolerance in mammalian heterotherms.

Heterothermic mammals tolerate severe hypoxia, as well as a variety of central nervous system insults, better than homeothermic mammals. Tolerance to hypoxia may stem from adaptations associated with the ability to survive hibernation and periodic arousal thermogenesis. Here, we review evidence and mechanisms of hypoxia tolerance during hibernation, euthermy and arousal in heterothermic mammals and consider potential mechanisms for regenerative-like processes, such as synaptogenesis, observed within hours of hypoxic stress associated with arousal thermogenesis.

X-ray contrast media induce aortic endothelial damage, which can be prevented with prior heparin treatment.

X-ray contrast media induce damage to the endothelial layer of vessels and initiate the formation of thrombosis, which is a complication for clinical diagnostic procedures. The future determination of the mechanisms, which underlie the damaging effect of X-ray contrast medium on vascular wall cells, especially vascular endothelium and possible prevention of this damage by vasoprotector, will result in a larger application in diagnostic procedures. The aim of the present study is to analyze the effect of X-ray contrast media (Verographin, Iodamid and Iodolipol) on the arterial endothelium morphology by using ultrastructural techniques (scanning and transmission electron microscopy, SEM and TEM respectively). Experiments have been carried out on New Zealand white rabbits (6 month old) and Wistar rats (6-8 month old) after a single injection of X-ray contrast media with and without prior heparin treatment. Control groups of animals were exposed to the same procedure but without X-ray contrast media injection and only received isotonic saline solution. The following time points were selected: 1, 6, 24, 72 h and 7 days. At the end of the experiments, animals were anesthetized by pentobarbital and then perfused with a balanced buffer for 1 min and followed by perfusion fixation with Karnovsky's fixative containing buffered solution of 2.5% paraformaldehyde and 2.5% glutaraldehyde (pH 7.36) at least 30 min. The aortic tissue was removed and immediately placed into a fresh portion of the same fixative. Aortic samples were then prepared for scanning and transmission electron microscopy (SEM and TEM respectively). Immediately after the injection of X-ray contrast media, the number of microvilli and blebs on the luminal surface of the endothelial cells (EC) significantly increased. Very often, nuclear portions of the EC sharply protruded into the vessel lumen. Clusters of spindle-shaped EC were seen throughout the endothelial monolayer. These changes persist through the 72-h period after X-ray contrast media injection. Moreover, the desquamation and denudation of the EC from the monolayer is often observed and this is accompanied by the presence of a microthrombus on the vessel surface. Seven days after the post-injection period, endothelial monolayers still show severe damage, which often coexists with the presence of a different sized microthrombus on the vessel surface. However, the degree of lesion formation in most areas is substantially decreased as compared to the early period of post-injection (24 and 72 h). Heparin treated group shows intact morphology similar to the control experimental groups (saline injected group). Infrequently, minimal morphological changes of the endothelium, such as increased number of microblebs and microvilli, were seen with heparin treatment. We conclude that the negative side effects of the X-ray contrast media can be eliminated by a single injection of heparin or other vasoprotector prior to the diagnostic procedure.

Increased expression of NOS and ET-1 immunoreactivity in human colorectal metastatic liver tumours is associated with selective depression of constitutive NOS immunoreactivity in vessel endothelium.

The absence of perivascular nerves in tumour vessels suggests that endothelium derived vasoactive substances [nitric oxide (NO) and endothelin-1 (ET-1)] may be key factors in controlling tumour blood flow during tumour growth and metastasis. We have studied the ultrastructural distribution and immunoreactivity of different NO synthase (NOS) isoforms and ET-1 in human colorectal metastatic liver tumour tissues using pre-embedding peroxidase-anti-peroxidase and post-embedding immunoelectron microscopic triple gold labelling techniques. Dramatically lower NOS 1 immunoreactivity was observed in tumour vascular endothelium (1-3% and 15-20% in tumour and normal groups, respectively). As compared to control groups there were significantly less NOS3 immunopositive EC in metastatic tumour vessels (45-50% and 1-3% in normal and tumour groups, respectively). A striking rise in NOS2 was observed in tumour vessel endothelium (< 1% in normal and 65-70% in tumour vessel endothelium). ET-1 immunoreactivity levels were also significantly higher in tumour vessel endothelium (85-90% in tumour, 15-20% in normal group). This increased expression of NOS2 and ET-1 immunoreactivity was accompanied by the increased expression of three NOS isoforms and ET-1 immunoreactivity in liver parenchymal cells. These data suggest that metastatic tumour vessel endothelium is characterized by increased expression of NOS2 and ET-1 and by decreases in NOS1 and NOS3. These characteristics are associated with the overexpression of all three NOS isoforms and ET-1 immunoreactivity in non-vascular cells.

The effect of agonists and antagonists on the morphology of non-transformed human smooth muscle cell in vitro.

In the present study we used scanning electron microscopy (SEM) to investigate morphological changes of non-transformed line of human bronchial smooth muscle cells (bSMC) induced by different agonists. Explants of normal bronchi were dissected and subcultured between 2 and 6 passage. In addition, smooth muscle actin content was assessed by SDS-PAGE electrophoresis, and its isoelectric point by IPG followed by immunoblotting. SMC were fixed by 2.0% paraformaldehyde and 2% glutaraldehyde and then were post-fixed by OSO4 and followed by dehydration and gold coating. Cytosolic free calcium was measured using adherent cells incubated with 500 microM Fura-2 acetoxymethylester and monitored by single excitation fluorimetry. Cultured cells possess predominantly charged actin isoforms with pI at 4.95; they respond to acetylcholine (100 microM), bradykinin (5 microM) and sulfidopeptide leukotriens (0.3-1.0 microM) with contraction, marked morphological lesions, such as widespread monolayer disorganization, extension of cell contacts. The number of microvilli on the cell surfaces was correlated with the degree of the alterations of the cellular morphology. Receptor antagonists antagonized these changes: atropine (0.3 microM), HOE 140 (1 microM) and MK 571 (1 microM). Acetylcholine and bradykinin induced a biphasic elevation of cytosolic calcium, which was antagonized by their receptor antagonists. Calcium changes in response to agonists were maintained over repetitive passages. Therefore, morphological changes seen in human bronchial SMC in culture with physiological response to various, structurally unrelated agonists can be future concern for the study the possible testing of the different pharmacological substances.

Atherosclerotic lesions are associated with increased immunoreactivity for inducible nitric oxide synthase and endothelin-1 in thoracic aortic intimal cells of hyperlipidemic Watanabe rabbits.

The development and progression of atherosclerotic lesions in Watanabe heritable hyperlipidemic rabbits is associated with increases in inducible nitric oxide synthase (NOS2) and endothelin-1 (ET-1) immunoreactivity. In contrast, there is a reduction of immunoreactivity for neuronal NOS (NOS1) in aortic endothelial cells, but no change in endothelial NOS (NOS3) immunoreactivity. However, subendothelial macrophages and smooth muscle showed a different pattern of immunoreactivity of NADPH-diaphorase (NADPH-d), NOS2, ET-1, and NOS1. The lipid-rich macrophages in the intima were positively labeled for NADPH-d, NOS1, NOS2, NOS3, and ET-1. Smooth muscle cells in the subendothelium and the medial layers of the vascular wall were also positive for these markers. These results are consistent with the reduction of endothelium-dependent vasorelaxation that is known to occur during the development and progression of atherosclerosis in familial hypercholesterolemia. The data suggest a key role for vasoactive substances in the development of atherosclerosis.

Expression of hypoxia-inducible factor-1alpha in the brain of rats during chronic hypoxia.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates adaptive responses to the lack of oxygen in mammalian cells. HIF-1 consists of two proteins, HIF-1alpha and HIF-1beta. HIF-1alpha accumulates under hypoxic conditions, whereas HIF-1beta is constitutively expressed. HIF-1alpha and HIF-1beta expression were measured during adaptation to hypobaric hypoxia (0.5 atm) in rat cerebral cortex. Western blot analyses indicated that HIF-1alpha rapidly accumulated during the onset of hypoxia and did not fall for 14 days but fell to normal by 21 days despite the continuous low arterial oxygen tension. Immunostaining showed that neurons, astrocytes, ependymal cells, and possibly endothelial cells were the cell types expressing HIF-1alpha. Genes with hypoxia-responsive elements were activated under these conditions, as evidenced by elevated vascular endothelial growth factor and glucose transporter-1 mRNA levels. When 21-day-adapted rats were exposed to a more severe hypoxic challenge (8% oxygen), HIF-1alpha accumulated again. On the basis of these results, we speculate that the vascular remodeling and metabolic changes triggered during prolonged hypoxia are capable of restoring normal tissue oxygen levels.

Decreased constitutive nitric oxide synthase, but increased inducible nitric oxide synthase and endothelin-1 immunoreactivity in aortic endothelial cells of donryu rats on a cholesterol-enriched diet.

The Donryu rat is resistant to a high cholesterol diet in that typical atheromatous lesions do not develop. Using electron microscopic immunocytochemical techniques, the effects of a CCT diet (4% cholesterol with 1% cholic acid and 0.5% thiouracil) on the distributions of neuronal, macrophage, and endothelial specific nitric oxide synthase (NOS I, NOS II, and NOS III) and endothelin-1 (ET-1) immunoreactivity were examined in the thoracic aortic intima. Atheromatous lesions were absent, but immunocytochemistry showed 1. 4+/-0.52% and 4.0+/-0.9% endothelial cells (EC) with positive staining for NOS I and NOS III, respectively, compared with 16.3+/-2. 5% and 88.6+/-2.48% in control Donryu rats. The CCT-supplemented diet induced expression of NOS II immunoreactivity in thoracic aortic intimal cells. EC, subendothelial macrophages, and smooth muscle cells (SMC) also showed high NOS II-positive staining. The percentage of NOS II-immunoreactive EC was 43+/-1.8%. In control groups, no NOS II immunoreactive cells were observed. The percentage of ET-1 immunopositive cells was also significantly increased by 9. 2+/-0.66% and 64.2+/-1.4% in control and CCT-fed groups, respectively. It is concluded that the administration of a high cholesterol diet in Donryu rats produces endothelial dysfunction associated with changes in the balance of the different isoforms of NOS and ET-1. Therefore, the increase in inducible NOS and ET-1 immunoreactivity seen during the cholesterol-enriched diet appears to be a compensatory reaction of aortic wall cells to the high cholesterol supplementation.

The role of mitochondria in the regulation of hypoxia-inducible factor 1 expression during hypoxia.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that regulates transcriptional activation of several genes responsive to the lack of oxygen, including erythropoietin, vascular endothelial growth factor, glycolytic enzymes, and glucose transporters. Because the involvement of mitochondria in the regulation of HIF-1 has been postulated, we tested the effects of mitochondrial electron transport chain deficiency on HIF-1 protein expression and DNA binding in hypoxic cells. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) inhibits electron transport chain at the level of complex I. MPTP is first converted to a pharmacologically active metabolite 1-methyl-4-phenylpyridinum (MPP+). MPP+ effectively inhibited both complex I activity and hypoxic accumulation of HIF-1alpha protein in dopaminergic cell lines PC12 and CATH.a. In C57BL/6 mice, a single dose of MPTP (15 mg/kg, intraperitoneal) inhibited complex I activity and HIF-1alpha protein accumulation in the striatum in response to a subsequent hypoxic challenge (8% O(2), 4 h). In a genetic model system, 40% complex I-inhibited human-ape xenomitochondrial cybrids, hypoxic induction of HIF-1alpha was severely reduced, and HIF-1 DNA binding was diminished. However, succinate, the mitochondrial complex II substrate, restored the hypoxic response in cybrid cells, suggesting that electron transport chain activity is required for activation of HIF-1. A partial complex I deficiency and a mild reduction in intact cell oxygen consumption effectively prevented hypoxic induction of HIF-1alpha protein.

Prospects for noninvasive imaging of brain amyloid beta in Alzheimer's disease.

The brain in patients with Alzheimer's disease (AD) contains large amounts of fibrillary amyloid beta protein. Studies attempting to use levels of amyloid beta protein in plasma, cerebrospinal fluid or skin as diagnostic tests for the disease have not been fruitful. A method for the noninvasive detection of cerebral amyloid beta would be valuable for dementia differential diagnosis, pathophysiology and monitoring of anti-amyloid therapies. Anti-amyloid monoclonal antibody 10H3 has been evaluated as an amyloid-imaging ligand, without success. Important considerations in the development of amyloid-imaging ligands include choice of radiolabel and physical and biological half-lives, route of administration, protein binding, use of control molecules, and imaging techniques. It is important that imaging studies be designed to reflect the slow nature of the process of amyloid deposition. We used a transgenic mouse model overexpressing beta protein precursor (beta PP) to assess the binding of basic fibroblast growth factor (bFGF) and serum amyloid P component (SAP) to amyloid beta (A beta) plaques in mouse brain. Although the binding of these ligands is similar to AD, neither is found endogenously associated with A beta deposits. Because SAP is a component of mouse serum, these findings suggest the blood-brain barrier in transgenic mice is not affected as it is in AD. These findings suggest that the transgenic mouse may be used as a model for evaluation of A beta imaging methods.

Perfusion-limited recovery of evoked potential function after spinal cord injury.

STUDY DESIGN: The current study was designed to determine whether progressive spinal cord damage during residual compression is caused by low blood flow and ischemia. OBJECTIVES: The purpose of this experiment was to determine the effects of sustained spinal cord compression on regional blood flow and evoked potential recovery after time-dependent decompression. SUMMARY OF BACKGROUND DATA: Spinal cord injury after trauma is commonly associated with residual cord compression. Although decreased blood flow has been reported after spinal cord contusion, the effect of residual spinal cord displacement on reperfusion of blood flow or recovery of neurologic function remains unclear. METHODS: Eighteen beagles were anesthetized, and the spinal cord at T13 was loaded dorsally under precision loading conditions until evoked potential amplitudes were reduced by 50%. At this function endpoint, spinal cord displacement was maintained for 90 minutes. Somatosensory-evoked potentials were measured at regular intervals until 3 hours after decompression. Regional spinal cord blood flow was measured with a fluorescent microsphere technique at regular time points during and after spinal cord decompression. RESULTS: Within 5 minutes after dynamic cord compression was discontinued, evoked potential signals were absent in all dogs. Evoked potential recovery was observed after decompression in 7 of 18 dogs. Regional spinal cord blood flow at baseline, 21.8 +/- 1.9 mL/100 g. min (mean +/- SE), decreased to 3.9 +/- 0.9 mL/100 g. min after dynamic compression was discontinued. Although spinal cord-piston interface pressure dissipated by 87% of maximum interface pressure during sustained compression, mean blood flow recovered to only 34% of baseline flow. In the 7 dogs that recovered evoked potential function, blood flow increased to 11.3 +/- 2.7 mL/100g. min immediately before decompression (P < or = 0.05). In the 11 dogs that did not recover evoked potential function after decompression, regional blood flow did not improve during sustained compression. CONCLUSIONS: Recovery of evoked potential function after decompression corresponded with a greater return of blood flow during sustained displacement and greater reperfusion of blood flow associated with decompression.

Methyl isobutyl amiloride alters regional brain reperfusion after resuscitation from cardiac arrest in rats.

In a rat model of cardiac arrest and resuscitation, [(14)C]-iodoantipyrene (IAP) autoradiography was used to measure the regional variations in cerebral blood flow 15 and 60 min after reperfusion. The purpose of this study was to investigate the hypothesis that the inhibition of the Na+/H+ antiporter with methyl isobutyl amiloride (MIA) would decrease postischemic pericapillary cytotoxic edema and, therefore, improve vascular perfusion dynamics. Vehicle-treated rats responded to cardiac arrest and resuscitation as expected with initial hyperemia after 15 min of reperfusion, except for thalamic and midbrain structures which were hypoperfused. All brain structures were perfused at half the baseline blood flow at 60 min after resuscitation, and the residual blood flow in each region was proportional to the baseline flow of each region. MIA treatment was associated with decreased blood flow in every region examined at both 15 min and 60 min of reperfusion. No hyperemia was observed at 15 min in any region after MIA treatment. Sixty minutes after resuscitation in MIA-treated rats, all structures were hypoperfused (to 25+/-7% of baseline, 48+/-8% of vehicle-treated rats). These effects are unlikely to be due to prevention of cytotoxic edema, but may be due to MIA protection of capillary endothelium by prevention of neutrophil activation.

Rapid and slow swelling during hypoxia in the CA1 region of rat hippocampal slices.

The role of swelling in hypoxic/ischemic neuronal injury is incompletely understood. We investigated the extent and time course of cell swelling during hypoxia, and recovery of cell volume during reoxygenation, in the CA1 region of rat hippocampal slices in vitro. Cell swelling was measured optically and compared with simultaneous measurements of the extracellular DC potential, extracellular [K+], and synaptic transmission in the presence and absence of hypoxic depolarization. Hypoxia-induced swelling consisted of rapid and/or slow components. Rapid swelling was observed frequently and always occurred simultaneously with hypoxic depolarization. Additionally, rapid swelling was followed by a prolonged phase of swelling that was approximately 15 times slower. Less frequently, slow swelling occurred independently, without either hypoxic depolarization or a preceding rapid swelling. For slices initially swelling rapidly, recovery of both cell volume and the slope of field excitatory postsynaptic potentials were best correlated with the duration of hypoxia (r = 0.77 and 0.87, respectively). This was also the case for slices initially swelling slowly (r = 0.70 and 0.58, respectively). In contrast, the degree of recovery of cell volume was the same at 30 or 60 min of reoxygenation, indicating that prolonging the duration of reoxygenation within these limits was ineffective in improving recovery. Spectral measurements indicated that the hypoxia-induced changes in light transmittance were related to changes in cell volume and not changes in the oxidation state of mitochondrial cytochromes. The persistent impairment of synaptic transmission in slices swelling slowly (i.e., without hypoxic depolarization) indicates that swelling may play a role in this injury and that hypoxic depolarization is not required. Additionally, the correlation between the degree of recovery of cell volume and the degree of recovery of synaptic transmission during reoxygenation supports a role for swelling in hypoxic neuronal injury.

Effects of oxygen deprivation on parapyramidal neurons of the ventrolateral medulla in the rat.

We characterized the electrophysiological properties and responses of neurons located in the parapyramidal region of the ventral aspect of the rat medulla oblongata (parapyramidal neurons, PP neurons) to oxygen deprivation, in order to understand the mechanisms involved in hypoxia induced respiratory depression. The responses of PP neurons to oxygen deprivation were compared to those of the functionally dissimilar neurons of the dentate gyrus (DG). Neurons from the PP region were found to fire spontaneously with a frequency of 3-3.5 spikes/sec in both adults and neonates and responded to an anoxic insult with a complete loss of spontaneous firing. Discrete metabolite analysis showed a small (about 17%) decrease in tissue adenosine triphosphate (ATP) levels of the PP neurons during an anoxic insult and the decrease was significantly smaller than in the DG cell region (28%). In contrast to the DG neurons, the PP neurons recovered from an anoxic insult lasting more than 30 min, indicating a greater survival capacity of the PP neurons during oxygen deprivation. The PP neurons were also capable of withstanding successive anoxic insults better than the DG cells as demonstrated by their complete recovery following reoxygenation. It is suggested that the PP neurons may depress their electrical activity as an energy conservation mechanism, and thereby survive anoxic insults longer than the dentate neurons, whereas the loss of cellular activity in the DG neurons may be a result of energy depletion.

Endothelial activation following prolonged hypobaric hypoxia.

Prolonged exposure to low oxygen may induce adaptive changes which can be either beneficial or deleterious to cell survival. We examined the effect of prolonged moderate hypobaric hypoxia on CNS endothelial cell (EC) function. Exposure to hypoxia resulted in expression of EC activation markers, the cell surface adhesion proteins intracellular adhesion molecule-1 and E-selectin. Induction of the major histocompatibility complex (MHC) class II molecule as well as increased constitutive expression of the transferrin receptor and the glucose transporter-1 protein was also detected within 24 h of exposure to hypobaric hypoxia. Constitutive expression of the MHC class I molecule increased by 48 h. Expression of most EC activation markers increased with time from 0 to 2 weeks. By 3 weeks of exposure to hypobaric hypoxia, ECs returned to their quiescent state with the exception of sustained expression of E-selectin and elevated glut-1. Little to no significant increase in expression of vascular cell adhesion molecule-1 was seen at any time period.

Cerebral metabolic profile, selective neuron loss, and survival of acute and chronic hyperglycemic rats following cardiac arrest and resuscitation.

Cortical metabolites and regional cerebral intracellular pH (pHi) were measured in normoglycemic (NM), acute hyperglycemic (AH), and chronic hyperglycemic (CH, 2 week duration, streptozotocin-induced) Wistar rat brains during cardiac arrest and resuscitation. During total ischemia in AH and CH rats (plasma glucose approximately 30 mM), cortical ATP, PCr, glucose, and glycogen all fell significantly as expected. Lactate levels increased dramatically in association with a concomitant intracellular acidosis. Although lactate reached higher concentrations in AH and CH than NM, pHi was significantly lower only in the AH group. With 5 min of reperfusion, all groups recovered to near baseline in all variables, though lactate remained elevated. In a separate aspect of the study, animals from each experimental group were allowed to recover for 4 days following resuscitation, with outcome being gauged by mortality rate and hippocampal CA1 neuron counts. NM survival rate was significantly better than AH and CH. In particular, no CH rats survived for 4 days despite rapid initial recovery. After 4 days, the AH group had suffered significantly greater CA1 neuron loss than the NM rats. In summary, our research identified differences in intra-ischemic acid-base status in the two hyperglycemic groups, suggesting that chronic hyperglycemia may alter the brain's buffering capacity. These observations may account for differences between acutely and chronically hyperglycemic subjects regarding outcome, and they suggest that factors other than hydrogen ion production during ischemia are responsible for modulating outcome.