Johann Valtysson

Interstitial glycerol as a marker for membrane phospholipid degradation in the acutely injured human brain

OBJECTIVE Brain interstitial glycerol was studied as a potential marker for membrane phospholipid degradation in acute human brain injury. METHODS Glycerol was measured in microdialysis samples from the frontal lobe cortex in four patients in the neurointensive care unit, during the acute phase after severe aneurysmal subarachnoid haemorrhage. Microdialysis probes were inserted in conjunction with a ventriculostomy used for routine intracranial pressure monitoring. Clinical events involving hypoxia/ischaemia were diagnosed by neurological signs, neuroimaging (CT and PET), and neurochemical changes of the dialysate—for example, lactate/pyruvate ratios and hypoxanthine concentrations. RESULTS Altogether 1554 chemical analyses on 518 microdialysis samples were performed. Clinical events involving secondary hypoxia/ischaemia were generally associated with pronounced increases (up to 15-fold) of the dialysate glycerol concentration. In a patient with a stable condition and no signs of secondary hypoxia/ischaemia the glycerol concentration remained low. Simultaneous determination of glycerol in arterial plasma samples showed that the changes in brain interstitial glycerol could not be attributed to systemic changes and an injured blood brain barrier. CONCLUSIONS This study suggests that membrane phospholipid degradation occurs in human cerebral ischaemia. Interstitial glycerol harvested by microdialysis seems to be a promising tool for monitoring of membrane lipolysis in acute brain injury. The marker may be useful for studies on cell membrane injury mechanisms mediated by for example, Ca2+ disturbances, excitatory amino acids, and reactive oxygen species; and in the evaluation of new neuroprotective therapeutic strategies.

Simultaneous Intracerebral Microdialysis and Positron Emission Tomography in the Detection of Ischemia in Patients with Subarachnoid Hemorrhage

Intracerebral microdialysis (MD) was applied in patients with subarachnoid hemorrhage. The regional CBF, the CMRO2, and oxygen extraction ratio (OER) were measured with simultaneous positron emission tomography (PET). The aim was to directly correlate alterations in dialysate levels of energy-related metabolites (lactate, lactate/pyruvate ratio, hypoxanthine) and excitatory amino acids (EAAs) (glutamate and aspartate) to the energy state in the MD probe region as determined by PET. Regional ischemia was defined according to Heiss et al. and Lassen (Heiss et al., 1992; Lassen, 1966). Whole-brain ischemia was considered present when the OER for the whole brain exceeded the mean whole-brain OER + 2 SD of six reference patients. In general, the presence of whole-brain ischemia and/or regional ischemia within the region of the MD probe was associated with increased levels of energy-related metabolites and EAAs retrieved by MD. Increased levels of energy-related metabolites and EAAs were only occasionally seen when PET did not show any signs of ischemia or when signs of regional ischemia were found remote from the MD probe region. Thus, the energy-related metabolites and EAAs may be used as extracellular “markers” of ischemia. PET may be of use in defining critical ischemic regions (tissue at risk) where the MD probe can be inserted for- chemical monitoring.

Neuropathological endpoints in experimental stroke pharmacotherapy: The importance of both early and late evaluation

SummaryThis study adresses the issue of endpoint selection in the evaluation of neuroprotective drugs in experimental focal ischaemia. Previous work with the permanent middle cerebral artery (MCA) occlusion model in the rat has demonstrated that the ischaemic lesion does not acquire its final appearance until at least 28 days after the ictus. Therefore, the effect of the NMDA receptor blocker MK-801 (dizocilpine maleate) was evaluated both early (3 days) and late (28 days) after MCA occlusion to determine if the previously reported protective effect of a single post-ischaemic dose of MK801 found in acute experiments remained after 28 days.Mk-801 (0.5mg/kg, i.v.) or isotonic saline was randomly given to rats 30 min after MCA occlusion. Infarct volume and volume of ipsilateral and contralateral hemispheres were estimated from camera lucida drawings of 8 defined coronal histological sections of the brain. As expected, a 40% (p<0.05) reduction of infarct size was found in MK-801 treated rats after 3 days. In animals evaluated 28 days after MCA occlusion, no significant difference in infarct size, total tissue loss (infarct volume + ipsilateral hemisphere atrophy) or remaining non-infarcted tissue (contralateral hemisphere — total tissue loss) was seen between the MK-801 and placebo treated rats.The results suggest that the single dose treatment with MK-801 postponed the evolution of the infarct, which at 3 days after MCA occlusion is still in progress, possibly by ameliorating oedema formation. It remains to be shown if a multiple dose treatment with NMDA receptor antagonists improves the final neuropathological outcome after experimental stroke. The study illustrates the importance of including a late endpoint when evaluating the efficacy of neuroprotective stroke therapy.

Central nervous system effects of subdissociative doses of (S)‐ketamine are related to plasma and brain concentrations measured with positron emission tomography in healthy volunteers

Plasma concentrations, maximum regional brain concentrations, and specific regional binding in the brain after administration of 0, 0.1, and 0.2 mg/kg doses of (S)‐ketamine were measured in a randomized, double‐blind, crossover study in five volunteers and were related to induced effects such as analgesia, amnesia, and mood changes. Specific binding in the brain was assessed by simultaneous administration of (S)‐[N‐methyl‐11C]ketamine quantified by positron emission tomography. High radioactivities in the brain corresponded to regional distribution of N‐methyl‐D‐aspartate receptor complexes. A significant and dose‐dependent reduction of binding was measured as a result of displacement of (S)‐[N‐methyl‐11C]ketamine. Memory impairment and psychotomimetic effects were related to dose, plasma concentration 4 minutes after administration, and decreased regional binding of (S)‐ketamine in the brain and were consistently seen at plasma and maximum regional brain (S)‐ketamine concentrations higher than 70 and 500 ng/ml, respectively. The magnitude of specific binding of (S)‐ketamine, measured with positron emission tomography, can be related directly to drug effects.

Increase of interstitial glycerol reflects the degree of ischaemic brain damage: a PET and microdialysis study in a middle cerebral artery occlusion-reperfusion primate model.

OBJECTIVE To evaluate interstitial glycerol as a marker of ischaemia by studying the changes in glycerol in direct relation to changes in regional cerebral metabolic rate of oxygen (CMRO2), the lactate/pyruvate ratio (LP ratio), and glutamate. METHODS Transorbital 2 hour middle cerebral artery occlusion (MCAO) was performed in eight monkeys, which were studied with continuous microdialysis for 24 hours. Interstitial fluids were collected by microdialysis and analysed for glycerol, lactate, pyruvate, and glutamate with an enzymatic assay and high performance liquid chromatography. Sequential PET studies of cerebral blood flow (CBF), CMRO2, oxygen extraction ratio (OER), and cerebral blood volume (CBV) were performed. The microdialysis probe regions were classified as severe ischaemia or penumbra, depending on whether the mean CMRO2 side to side ratio was below or above 60%, respectively. RESULTS A nine-fold, sustained increase in glycerol was registered after MCAO in severe ischaemia regions. In penumbra regions, the increase in glycerol was five-fold, but the glycerol concentration returned to baseline within 8 hours of clip removal. The difference between severe ischaemia and penumbra glycerol values was statistically significant. As expected from previous studies, the interstitial LP ratio and glutamate increased markedly in severe ischaemia, with a less pronounced change in penumbra regions. There was a time lag between the biochemical changes in severe ischaemia regions, with the LP ratio preceding glutamate, followed by glycerol. CONCLUSIONS A marked, sustained increase in interstitial glycerol is indicative of severe ischaemia in this stroke model. A transient, diminutive increase in interstitial glycerol may reflect a penumbra situation. Interstitial glycerol in combination with other biochemical markers such as the LP ratio and glutamate may be useful for clinical monitoring of the ischaemic brain, reflecting a sequence of secondary pathophysiological events.

Middle Cerebral Artery Occlusion and Reperfusion in Primates Monitored by Microdialysis and Sequential Positron Emission Tomography

Background and Purpose— In a previous investigation concerning the hemodynamic and metabolic changes over time displayed by sequential positron emission tomography (PET) in a middle cerebral artery (MCA) occlusion/reperfusion primate model, a metabolic threshold for irreversible ischemia could be identified (reduction of metabolic rate of oxygen[CMRO2] to ≈60% of the contralateral hemisphere). To evaluate the potential of microdialysis (MD) as an instrument for chemical brain monitoring, the aim of this subsequent study was to relate the chemical changes in MD levels directly to the regional metabolic status (CMRO2 above or below the metabolic threshold) and the occurrence of reperfusion, as assessed by PET. Methods— Continuous MD (2 probes in each brain) and sequential PET measurements were performed during MCA occlusion (2 hours) and 18 hours (mean) of reperfusion in 8 monkeys (Macaca mulatta). Energy-related metabolites (lactate, pyruvate, and hypoxanthine) and glutamate were analyzed. The MD probe regions were divided into 3 categories on the basis of whether CMRO2 was below or above 60% of the contralateral region (metabolic threshold level) during MCA occlusion and whether reperfusion was obtained: severe ischemia with reperfusion (n=4), severe ischemia without reperfusion (n=4), and penumbra with reperfusion (n=5). Results— The lactate/pyruvate ratio, hypoxanthine, and glutamate showed similar patterns. MD probe regions with severe ischemia and reperfusion and probe regions with severe ischemia and no reperfusion displayed high and broad peaks, respectively, during MCA occlusion, and the levels almost never decreased to baseline. Penumbra MD probe regions displayed only slight transient increases during MCA occlusion and returned to baseline. Conclusions— This experimental study of focal ischemia showed that the extracellular changes of energy-related metabolites and glutamate differed depending on the ischemic state of the brain during MCA occlusion and depending on whether reperfusion occurred. If MD proves to be beneficial in clinical practice, it appears important to observe relative changes over time.

A metabolic threshold of irreversible ischemia demonstrated by PET in a middle cerebral artery occlusion–reperfusion primate model*

Objective– to evaluate the predictive value of measurements of regional cerebral blood flow (CBF), oxygen metabolism (CMRO2) and oxygen extraction ratio (OER) for assessment of the fate of ischemic brain tissue. Materials and methods– Sequential PET measurements were performed during middle cerebral artery occlusion (MCAO; 2 h) and 12–24 h (mean 18 h) of reperfusion in a primate model (Macaca mulatta, n=8). A penumbra region was delineated on the MCAO PET image (OER >125% and CMRO2≥45% of the values observed in the contralateral hemisphere, respectively) and an infarction region was delineated on the last PET image (CMRO2<45% of the values observed in the contralateral hemisphere). The penumbra regions delineated during MCAO and the infarction regions delineated at the final PET, were copied on to the images from all other PET sessions for measurements of CBF, CMRO2 and OER. Ratios were calculated by dividing the mean values obtained by the values of the corresponding contralateral region. Results– Histopathology verified the adequacy of the criteria applied in the last PET for delineation of the infarction region. The penumbra region and infarction region were separated in all cases, except in two cases where a minimal overlap was seen. CBF and OER showed considerable variation over time and there was no consistent difference between the penumbra and infarction regions. CMRO2 showed a more stable pattern and the difference between penumbra and infarction regions was maintained from the time of MCAO throughout the entire reperfusion phase. With CMRO2 as predictor, all 50 observations could be correctly predicted as penumbra or infarction when using an optimal threshold ratio value estimated to be in the interval of 61% to 69% of the corresponding contralateral region. CBF and OER proved to have low power as predictors. Conclusions– The results indicate that CMRO2 is the best predictor of reversible or irreversible brain damage and the critical metabolic threshold level appears to be a reduction of oxygen metabolism to between 61% and 69% of the corresponding contralateral region.

Extracellular Ischaemia Markers in Repeated Global Ischaemia and Secondary Hypoxaemia Monitored by Microdialysis in Rat Brain

Summary The aim of this study was to explore the usefulness of extracellular markers of cerebral ischaemia. Cortical microdialysate concentrations of the energy related metabolites lactate, pyruvate, glucose, adenosine, inosine, hypoxanthine and xanthine, were measured in rats subjected to repeated transient ischaemia. The animals were subjected to one or two 10-min periods of global ischaemia produced by induced intracranial hypertension, with a 2 h period of reperfusion after each insult. In addition, the effect of superimposed secondary hypoxaemia and hypotension was studied. In general, there was a good agreement between the extracellular markers and known intracellular energy disturbances under similar conditions, including marked transient increases of lactate, lactate/pyruvate ratio, inosine and hypoxanthine. Several new observations were made: (1) glucose appeared to be a useful marker of severe ischaemia and recirculation, (2) a marked post-ischaemic xanthine formation was observed implicating a substantial loss of salvageable hypoxanthine as well as increased production of superoxide radicals, (3) a blunted purine response was noted after the second insult, reflecting a reduced intracellular adenine nucleotide pool, and (4) a different pattern of ischaemia markers was observed during secondary hypoxia as compared to hypoxia with hypotension.In conclusion, extracellular lactate, pyruvate, glucose, adenosine, inosine hypoxanthine and xanthine all seem valuable as ischaemia markers. The results support the usefulness of intracerebral microdialysis for monitoring of energy metabolic disturbances caused by cerebral ischaemia/hypoxia. The pattern of extracellular ischaemia markers may help differentiate between various causes of energy perturbations, such as different degrees of ischaemia and hypoxia.

Colonic ischaemia and intra-abdominal hypertension following open repair of ruptured abdominal aortic aneurysm

The aim was to investigate the association between colonic ischaemia and intra‐abdominal pressure (IAP) after surgery for ruptured abdominal aortic aneurysm (rAAA).

Effect of tirilazad mesylate given after permanent middle cerebral artery occlusion in rat

SummaryThe effect of the antioxidant drug tirilazad mesylate (U-74006F) on histopathological and neurological outcome 3 days after permanent middle cerebral artery (MCA) occlusion was evaluated in rats. Several previous studies have demonstrated the efficacy of tirilazad in reducing infarct size when administered before and during MCA occlusion, whereas post-treatment may be less effective in permanent focal ischaemia. We sought to determine if a protective effect of tirilazad could be demonstrated when administered after the insult only.U-74006F (3 mg/kg, i.v.) or sterile vehicle, was randomly given to rats 10 minutes and 3 hours after permanent MCA occlusion produced by transcranial proximal electrocauterization. Infarct volume and hemisphere volumes were estimated blindly from histological sections of defined levels of the brain after 72 h of ischaemia. Neurological score was determined blindly 1, 2, and 3 days after insult. There was no significant difference in infarct volume, volume of non-infarcted tissue, or neurological score between the tirilazad and placebo-treated rats.In conclusion, our results support the conception that post-treatment with tirilazad mesylate is not efficacious in reducing infarct size in permanent focal ischaemia, while pre-treatment, as reported by other groups, appears to be effective in both permanent and temporary focal ischaemia models. In temporary focal ischaemia, the limited data available suggest that also post-treatment with tirilazad may prove to be neuroprotective.