Altered PPARgamma expression and activation after transient focal ischemia in rats.

Stroke is a devastating disease with limited treatment options. Recently, we found that the peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists troglitazone and pioglitazone reduce injury and inflammation in a rat model of transient cerebral ischemia. The mechanism of this protection is unclear, as these agents can act through PPAR-gamma activation or through PPAR-gamma-independent mechanisms. Therefore, we examined PPAR-gamma expression, DNA binding and transcriptional activity following stroke. In addition, we used a PPAR-gamma antagonist, T0070907, to determine the role of PPAR-gamma during ischemia. Using immunohistochemical techniques and real-time PCR, we found low levels of PPAR-gamma mRNA and PPAR-gamma immunoreactivity in nonischemic brain; however, PPAR-gamma expression dramatically increased in ischemic neurons, peaking 24 h following middle cerebral artery occlusion. Interestingly, we found that in both vehicle- and agonist-treated brains, DNA binding was reduced in the ischemic hemisphere relative to the contralateral hemisphere. Expression of a PPAR-gamma target gene, lipoprotein lipase, was also reduced in ischemic relative to nonischemic brain. Both DNA binding and lipoprotein lipase expression were increased by the addition of the PPAR-gamma agonist rosiglitazone. Finally, we found that rosiglitazone-mediated protection after stroke was reversed by the PPAR-gamma antagonist T0070907. Interestingly, infarction size was also increased by T0070907 in the absence of PPAR-gamma agonist, suggesting that endogenous PPAR-gamma ligands may mitigate the effects of cerebral ischemia.

Peroxisome proliferator-activated receptor-gamma ligands reduce inflammation and infarction size in transient focal ischemia.

Newly developed insulin-sensitizing agents, which target the nuclear receptor peroxisome proliferator-activated receptor-gamma have recently been appreciated to exhibit potent anti-inflammatory actions. Since stroke is associated with an intense inflammatory response, we reasoned that these agents may ameliorate injury from stroke. We report that administration of troglitazone or pioglitazone 24 h before and at the time of cerebral infarction dramatically reduced infarction volume and improved neurological function following middle cerebral artery occlusion in rats. Furthermore, we find that delayed therapy also significantly reduced infarct volume. The brains of the drug-treated animals displayed reduced inflammation as evidenced by decreased immunoreactivity for microglial/macrophage markers and reduced protein and mRNA for interleukin-1beta, cyclooxygenase-2 and inducible nitric oxide synthase. We argue that the beneficial effects of these drugs are likely due to reduced expression of these inflammatory mediators, which are known to exacerbate ischemic injury following stroke. These results are of particular relevance to diabetic patients chronically treated with these agents who may benefit from the neuroprotective actions of these drugs.

Retina accumulates more glucose than does the embryologically similar cerebral cortex in diabetic rats.

AIMS/HYPOTHESIS: The retina is embryologically similar to cerebral cortex and the tissues of both are exposed to similar blood glucose concentrations. Nevertheless, in diabetes the retina develops metabolic abnormalities and microvascular lesions from which cerebrum seems relatively protected. We directly compared glucose concentrations and expression of GLUT-1 (the major carrier transporting glucose from blood into the neural retina and cerebrum) in the two tissues from normal and diabetic rats. METHODS: Tissue and intracellular glucose were measured using two methods: direct assay of glucose and assay of Amadori products on intracellular proteins. The expression of GLUT-1 was measured using western blots in tissue and in the isolated endothelial luminal membrane of the two vascular beds. RESULTS: Both methods assessing intracellular glucose indicate that intracellular concentrations of glucose in diabetes increased significantly in the retina but not in cerebral cortex. Concentrations of free glucose and Amadori product in retinas of diabetic animals were increased above normal by 334% and 122%, respectively, whereas there was no statistically significant increase in either parameter in the cerebral cortex of diabetic animals. In contrast to the observed increase in glucose in the retina in diabetes, expression of GLUT-1 on the luminal plasmalemma of the retinal vascular endothelium and in homogenates of whole retina decreased to a statistically significant extent (55% and 36%, respectively compared to normal). In the luminal cell membrane of the cerebral vasculature, diabetes did not decrease expression of GLUT-1 but tended to increase it slightly. CONCLUSIONS/INTERPRETATION: Even among tissues that do not require insulin for glucose uptake, tissue glucose concentration varies in diabetes. The greater increase in glucose concentration in retina than in cerebrum in diabetes probably contributes to the tissue differences in biochemical and histopathologic sequelae of the disease. The expression of GLUT-1 in the microvasculature is unlikely to account for the differences in tissue glucose between retina and cerebrum.

Characterization of a model of hydrocephalus in transgenic mice.

OBJECT: The purpose of this study was to elucidate the pathophysiological characteristics of hydrocephalus in a new transgenic model of mice created to overproduce the cytokine transforming growth factor-beta1 (TGFbeta1) in the central nervous system (CNS). METHODS: Galbreath and colleagues generated transgenic mice that overexpressed TGFbeta1 in the CNS in an effort to examine the role of this cytokine in the response of astrocytes to injury. Unexpectedly, the animals developed severe hydrocephalus and died. The authors have perpetuated this transgenic colony to serve as a model of congenital hydrocephalus, breeding asymptomatic carrier males that are heterozygous for the transgene with wild-type females. One hundred twelve (49.6%) of 226 mice developed clinical manifestations of hydrocephalus, characterized by dorsal doming of the calvaria, spasticity, limb tremors, ataxia, and, ultimately, death. The presence of the TGFbeta1 transgene was determined by performing polymerase chain reaction (PCR) analysis of sample tail slices. Animals with the hydrocephalic phenotype consistently carried the transgene, although some animals with the transgene did not develop hydrocephalus. Animals without the transgene did not develop hydrocephalus. Alterations in brain structure were characterized using magnetic resonance (MR) imaging, gross and light microscopic analysis, and immunocytochemical studies. Magnetic resonance imaging readily distinguished hydrocephalic animals from nonhydrocephalic controls and demonstrated an obstruction at the outlets of the fourth ventricle. Gross and light microscopic examination confirmed the MR findings. The results of immunofluorescent staining of brain tissue slices revealed the presence of the TGFbeta1 cytokine and its receptor preferentially in the meninges and subarachnoid space in both hydrocephalic and control mice. Reverse transcriptase-PCR analysis demonstrated tissue-specific expression of the TGFbeta1, gene in the brains of transgenic mice, and enzyme-linked immunosorbent assay confirmed overexpression of the TGFbeta1 cytokine in brain, cerebrospinal fluid, and plasma. CONCLUSIONS: The transgenic murine model provides a reproducible representation of congenital hydrocephalus. The authors hypothesize that overexpression of TGFbeta1 in the CNS causes hydrocephalus by altering the environment of the extracellular matrix and interfering with the circulation of cerebrospinal fluid. A model of hydrocephalus in which the genetic basis is known should be useful for evaluating hypotheses regarding the pathogenesis of this disorder and should also help in the search for new treatment strategies.

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.

Vascular endothelial growth factor in Alzheimer's disease and experimental cerebral ischemia.

Several growth factors have been implicated in the pathogenesis of Alzheimer's disease (AD). We considered whether the vascular endothelial growth factor (VEGF) is involved in the vascular pathology associated with most cases of AD. We observed enhanced VEGF immunoreactivity in clusters of reactive astrocytes in the neocortex of subjects with AD compared to elderly controls. VEGF reactivity was also noted in walls of many large intraparenchymal vessels and diffuse perivascular deposits. In addition, we established that astrocytic and perivascular VEGF reactivity was enhanced in cerebral cortex of rats subjected to cerebral ischemia and to chronic hypoxia; experimental conditions known to be associated with astrogliosis and angiogenesis. We suggest the increased VEGF reactivity, also observed in infarcted human brain tissue, implicates compensatory mechanisms to counter insufficient vascularity or reduced perfusion (oligemia) apparent in AD.

Substrates of energy metabolism attenuate methamphetamine-induced neurotoxicity in striatum.

High doses of methamphetamine (METH) produce a long-term depletion in striatal tissue dopamine content. The mechanism mediating this toxicity has been associated with increased concentrations of dopamine and glutamate and altered energy metabolism. In vivo microdialysis was used to assess and alter the metabolic environment of the brain during high doses of METH. METH significantly increased extracellular concentrations of lactate in striatum and prefrontal cortex. This increase was significantly greater in striatum and coincided with the greater vulnerability of this brain region to the toxic effects of METH. To examine the effect of supplementing energy metabolism on METH-induced dopamine content depletions, the striatum was perfused directly with decylubiquinone or nicotinamide to enhance the energetic capacity of the tissue during or after a neurotoxic dosing regimen of METH. When decylubiquinone or nicotinamide was perfused into striatum during the administration of METH, there was no significant effect on METH-induced striatal dopamine efflux, glutamate efflux, or the long-term dopamine depletions measured 7 days later. However, a delayed perfusion with decylubiquinone or nicotinamide for 6 h beginning immediately after the last METH injection attenuated the METH-induced striatal dopamine depletions measured 1 week later. These results support the hypothesis that the compromised metabolic state produced by METH administration predisposes dopamine terminals to the neurotoxic effects of glutamate, dopamine, and/or free radicals.

Nutrient consumption and metabolic perturbations.

The brain is entirely dependent on a continual supply of nutrients for the production of energy and the maintenance of function. Brain attack is a good example of a condition where the loss of blood flow and the consequent deprivation of nutrients can be devastating in terms of both structure and function. The unique metabolic characteristics of the brain make it more susceptible than most other organs of the body to nutrient restriction and starvation. In addition, changes occur during ischemia that compromise nutrient consumption even with reperfusion. Clearly, improving the outcome following brain attack will require a restoration of nutrient consumption to meet the relatively high energetic demands of the brain, and novel interventions to offset any metabolic lesions induced by the insult or reperfusion injury.

Decreased energy metabolism in brain stem during central respiratory depression in response to hypoxia.

Metabolic changes in the brain stem were measured at the time when oxygen deprivation-induced respiratory depression occurred. Eucapnic ventilation with 8% oxygen in vagotomized urethan-anesthetized rats resulted in cessation of respiratory drive, monitored by recording diaphragm electromyographic activity, on average within 11 min (range 5-27 min), presumably via central depressant mechanisms. At that time, the brain stems were frozen in situ for metabolic analyses. By using 20-microns lyophilized sections from frozen-fixed brain stem, microregional analyses of ATP, phosphocreatine, lactate, and intracellular pH were made from 1) the ventral portion of the nucleus gigantocellularis and the parapyramidal nucleus; 2) the compact and ventral portions of the nucleus ambiguus; 3) midline neurons; 4) nucleus tractus solitarii; and 5) the spinal trigeminal nucleus. At the time of respiratory depression, lactate was elevated threefold in all regions. Both ATP and phosphocreatine were decreased to 50 and 25% of control, respectively. Intracellular pH was more acidic by 0.2-0.4 unit in these regions but was relatively preserved in the chemosensitive regions near the ventral and dorsal medullary surfaces. These results show that hypoxia-induced respiratory depression was accompanied by metabolic changes within brain stem regions involved in respiratory and cardiovascular control. Thus it appears that there was significant energy deficiency in the brain stem after hypoxia-induce respiratory depression had occurred.

ATP catabolism and adenosine generation during ischemia in the aging heart.

Myocardial injury following ischemia and reperfusion is increased in the aging heart. The mechanisms underlying the increased susceptibility of the aging heart to ischemic injury remain unknown. We investigated whether decreased glycogen utilization with a more rapid depletion of ATP occurred during ischemia in the aging heart. Isolated buffer-perfused hearts from adult (6 months old) and aging (24 months old) Fischer 344 rats were subjected to 0, 2, 5, 10, 15 or 25 min of global stop-flow ischemia following a 15 min equilibration period (n = 5-6 for each ischemic time at each age). ATP level were decreased at preischemic baseline in aging hearts. ATP levels remained lower in the aging heart throughout ischemia (P < 0.001) with a similar pattern of decrease in both age groups. The decrease in tissue glycogen and increase in lactate contents was similar during ischemia in both age groups, suggesting that comparable glycogen utilization occurred during ischemia in adult and aging hearts. ATP catabolism leads to ADP, AMP and then adenosine. Tissue levels of adenosine, an important cardioprotective metabolite, were measured during ischemia. Tissue adenosine levels were decreased by 50% in the aging heart at 5 and 10 min, and remained depressed at 15 min and 25 min of ischemia compared to adult controls. Thus, increased ischemic injury in the aging heart is not related to differences in glycogen consumption. Lower tissue ATP levels and decreased adenosine levels were observed during ischemia. The differences in ATP content between adult and aging hearts occurred only during early ischemia and are unlikely to provide a mechanism for the increased damage observed following more prolonged periods of ischemia in the aging heart. The potential contribution of these decreases in tissue adenosine content to the increased injury observed in the aging heart will require further study.

Diet-induced ketosis does not cause cerebral acidosis.

Ketosis is beneficial for seizure control, possibly through induction of cerebral acidosis. However, cerebral intracellular pH has not previously been measured in ketotic humans and the animal data are sparse. We describe a high-fat diet, avidly consumed by rats, that induced consistent and moderate ketosis. Adult male rats were fed either the high-fat ketogenic diet, a high-carbohydrate diet with the same protein content as the ketogenic diet, or regular laboratory chow. Five to 6 weeks later, the rats were anesthetized, paralyzed, and injected with neutral red; their brains were frozen in situ. Intracellular pH of the cerebral cortex and cerebral glucose, lactate, ATP, phosphocreatine, and gama-aminobutyric acid (GABA) levels were measured. Rats fed the ketogenic diet had > 10-fold increase in their plasma ketones, but we noted no significant differences in cerebral pH or in cerebral metabolites and GABA levels among the three groups. Therefore, the antiepileptic effect of the ketogenic diet probably is not mediated by cerebral acidosis or changes in total cerebral GABA levels.

Effects of unbalanced diets on cerebral glucose metabolism in the adult rat.

We measured regional cerebral metabolic rates for glucose and selected cerebral metabolites in rats fed one of the following diets for 6 to 7 weeks: (1) regular laboratory chow; (2) high-fat, carbohydrate-free ketogenic diet deriving 10% of its caloric value from proteins and 90% from fat; and (3) high-carbohydrate diet deriving 10% of its caloric value from proteins, 78% from carbohydrates, and 12% from fat. In preliminary experiments, we found that moderate ketosis could not be achieved by diets deriving less than about 90% of their caloric value from fat. Rats maintained on the ketogenic diet had moderately elevated blood beta-hydroxybutyrate (O.4 mM) and acetoacetate (0.2 mM), and a five- to 10-fold increase in their cerebral beta-hydroxybutyrate level. Cerebral levels of glucose, glycogen, lactate, and citrate were similar in all groups. 2-Deoxyglucose studies showed that the ketogenic diet did not significantly alter regional brain glucose utilization. However, rats maintained on the high-carbohydrate diet had a marked decrease in their brain glucose utilization and increased cerebral concentrations of glucose 6-phosphate. These findings indicate that long-term moderate ketonemia does not significantly alter brain glucose phosphorylation. However, even marginal protein dietary deficiency, when coupled with a carbohydrate-rich diet, depresses cerebral glucose utilization to a degree often seen in metabolic encephalopathies. Our results support the clinical contention that protein dietary deficiency coupled with increased carbohydrate intake can lead to CNS dysfunction.

Brain glucose metabolism in hypobaric hypoxia.

Hypobaric hypoxia at one-half atmospheric pressure for 3 wk was reported to increase the brain capillary density and glucose transport at the blood-brain barrier in the adult rat. We examined the metabolic concomitants of these alterations in rats subjected to the same hypoxic insult. Hypoxic rats increased brain glucose and lactate concentrations and decreased brain glycogen. However, hypoxia had no significant effects on regional brain levels of ATP and phosphocreatine or on intracellular pH, indicating successful adaptation to the hypoxic insult. 2-Deoxyglucose studies showed that hypoxia increased the regional metabolic rate for glucose by 10-40%. These results indicate increased glycolysis in the hypoxic rat brain, which probably underlies the increased density of glucose transporters in brain microvessels and the increased blood-to-brain glucose influx in hypoxia.

Rapid recovery of rat brain intracellular pH after cardiac arrest and resuscitation.

We studied the intracellular pH in rat cerebral cortex of rats subjected to reversible total cerebral ischemia by cardiac arrest and resuscitation. Brain acidoses was more pronounced during ischemia in hyperglycemic rats (6.21 +/- 0.14) than in normoglycemic rats (6.56 +/- 0.07). Brain tissue lactate accumulated proportionally. Nevertheless, within 5 min of reperfusion, pHi in both normoglycemic and hyperglycemic groups had recovered to baseline levels, i.e. near 7.1-7.2, despite the fact that lactate concentrations were still elevated. These results demonstrate a rapid reversal of ischemic acidosis during recovery from 10 min of cardiac arrest, and suggest that acidosis, per se, may not be responsible for neuronal damage following cardiac arrest and resuscitation, even in hyperglycemic conditions.

Aging increases ischemia-reperfusion injury in the isolated, buffer-perfused heart.

Survival after acute myocardial infarction is decreased in elderly patients as compared with the overall adult population. Although several cardiac and noncardiac causes could contribute to the increased mortality rate, little is known regarding the relative susceptibility of aging myocardium to injury during ischemia and reperfusion. We hypothesized that the elderly heart is intrinsically more susceptible to damage than the adult heart. The recovery of isolated, buffer-perfused rat hearts from elderly animals (Fischer 344 rats, 24 months of age) was compared with that of adult hearts (6 months of age) obtained from the same strain. Hearts underwent 25 minutes of ischemia followed by 30 minutes of reperfusion. Hemodynamic recovery was decreased in elderly (n = 5) as compared with adult (n = 5) hearts, including developed pressure (% of preischemic baseline: elderly 31% +/- 4% vs adult 57% +/- 4%, p < 0.01). Elderly hearts also sustained greater tissue damage, with a markedly increased release of creatine kinase (elderly 2950 +/- 500 U vs adult 860 +/- 345 U, p < 0.01) during the 30-minute reperfusion period. The release of total protein and lactate dehydrogenase, other markers of myocyte injury, was also increased. Thus the elderly rat heart is more susceptible than the adult rat heart to ischemia-reperfusion injury. Greater injury during ischemia and reperfusion in an experimental model of aged myocardium raises the possibility of a more rapid progression of ischemic damage in elderly patients suffering acute myocardial infarction.

Early reversal of acidosis and metabolic recovery following ischemia.

Tissue acidosis is believed to be a key element in ischemic injury of neural tissue. The goal of this study was to determine whether persisting postischemic acidosis or the extent of acidosis would affect metabolic recovery following an ischemic event. Intracellular pH (pHi), adenosine triphosphate, phosphocreatine, and lactate levels were measured in the cerebral cortex during the early stages of reperfusion, following either 5 or 10 minutes of global ischemia in both normo- and hyperglycemic gerbils. A total of 130 gerbils were injected with a solution containing 1.5 ml Neutral Red (1%) (+/- 2.5 gm/kg glucose); 30 minutes later, the gerbils were placed under halothane anesthesia, and the carotid arteries were occluded for either 5 or 10 minutes. The brains were frozen in liquid nitrogen at 0, 15, 30, 60, and 120 seconds after reperfusion; they were sectioned and the block face was photographed to determine the pHi by using Neutral Red histophotometry. At the conclusion of the ischemia, the pHi in all groups had decreased significantly from a control value of 7.05 +/- 0.03) (mean +/- standard error of the mean). In normoglycemic brains, the pHi values fell to 6.71 +/- 0.04 and 6.68 +/- 0.11 after 5 and 10 minutes of ischemia, respectively. Hyperglycemic brains were more acidotic; values fell to 6.57 +/- 0.10 and 6.52 +/- 0.24 after 5 and 10 minutes of ischemia, respectively. Lactate levels were approximately fivefold greater than those of control tissue in normoglycemic brains, while lactate levels in hyperglycemic brains were increased eightfold. The adenosine triphosphate and phosphocreatine levels were depleted at the end of ischemia in all groups. After 2 minutes of reflow activity, the pHi levels in both normo- and hyperglycemic brains were restored to those of control values in the '5-minute ischemic group, while the pHi levels remained significantly depressed in the 10-minute ischemic group. Restoration of high-energy phosphates was similar in normoglycemic brains regardless of ischemic duration, recovering to only 20% of the restoration obtained in control tissue at 2 minutes. In hyperglycemic brains, however, there was complete recovery of high-energy phosphates by 2 minutes of reflow activity following 5 minutes of ischemia. Extending the ischemic period to 10 minutes in hyperglycemic brains slowed the rate of metabolic recovery to that observed in normoglycemic brains. The results indicate that the reflow period permits the rapid restoration of pHi levels substantially before the normalization of primary energetic compounds.(ABSTRACT TRUNCATED AT 400 WORDS)

Temporary vessel occlusion in spontaneously hypertensive and normotensive rats. Effect of single and multiple episodes on tissue metabolism and volume of infarction.

Temporary occlusion of an intracranial artery is frequently necessary in the surgical management of intracranial aneurysms, arteriovenous malformations, and tumors. While the risks of vessel damage associated with clip application have been lessened by improved design, the threat of ischemic damage remains. It is unclear whether multiple, brief periods of clip application are more or less safe than a single period of occlusion, and whether the underlying cerebrovascular status influences the outcome from either method. The effect of each of these paradigms (single: 1-hour occlusion; multiple: three 20-minute episodes separated by 10 minutes of reperfusion) on histopathological outcome was assessed in a middle cerebral artery (MCA) occlusion model using both normotensive and spontaneously hypertensive rats. The mean volume of infarction (+/- standard error of the mean) was not different between the single-ischemic (49.4 +/- 17.3 cu mm) and the multiple-ischemic (42.9 +/- 12.9 cu mm) episode groups of normotensive rats, whereas in the spontaneously hypertensive rats a significant difference existed between the volume of infarction for the single-occlusion group (126.7 +/- 18.7 cu mm) and the multiple-occlusion group (162.4 +/- 15.5 cu mm) (p < 0.05). The metabolic data obtained from spontaneously hypertensive animals did not provide an explanation for the larger infarction in that there were no significant differences between the single- and multiple-occlusion groups with respect to tissue glucose, adenosine triphosphate, or lactate levels. The results suggest that intermittent reperfusion may have different effects depending not only on the degree and duration of ischemia and reperfusion, but also on the underlying cerebrovascular status.