Dizocilpine

A double-tracer autoradiographic method for simultaneous measurement of regional glucose utilization (rCMR^sub glc^) and regional protein synthesis (PS) in consecutive brain sections is described and applied to study the metabolism of the ischemic penumbra 2 h after occlusion of the middle cerebral artery (MCAO) in rats. In halothane anesthesia, the left middle cerebral artery was permanently occluded. Two hours after MCAO an kv. bolus injection of ^sup 14^ C-deoxyglucose and ^sup 3^H-leucine was given and circulated for 45 min. Two sets of brain sections were processed for quantitative autoradiography. Neighboring brain sections exposed an X-ray film (^sup 3^H-insensitive), and ^sup 3^H-sensitive for determination of rCMR^sub glc^ and PS, respectively. Sections for PS determination were washed in trichloroacetic acid (TCA) prior to film exposure in order to remove ^sub 14^ C-deoxyglucose and unincorporated ^sup 3^H-leucine. Regional rates of PS and glucose utilization were measured by densitometric image analysis. Normal rates of metabolism were defined as mean +/- 2 SD of values in the non-ischemic cortex. The volumes of ischemic cortex displaying normal rates of PS and glucose utilization, respectively, were measured. The cortical volume with normal PS was significantly less than that of normal rCMR^sub glc^: 142 (127-147) mm^sup 3^ vs. 203 (184-206) mm^sup 3^. Treatment with the glutamate antagonists MK-801 (I mg kg^sup -1^) and NBQX (30 mg kg^sup -1^ x 2) did not significantly change this, although MK-801 tended to reduce the size of the metabolic penumbra calculated as the difference between ischemic cortex with reduced PS and ischemic cortex with reduced rCMR^sub glc^. [Neurol Res 1991; 21: 687-6941

Keywords: Focal cerebral ischemia; ^sup 14^ C-deoxyglucose; ^sup 3^H-leucine; autoradiography; MK-80 1; NBQX; rat

INTRODUCTION

Experimental focal ischemia in rats induced by occlusion of the middle cerebral artery (MCAO) produces an acute lesion consisting of an ischemic core with severely reduced cerebral blood flow (CBF) surrounded by a oligemic borderzone or penumbra with less pronounced CBF reductions1-3. If arterial occlusion is maintained this initially viable penumbra will subsequently be recruited in the infarction process leading to a progressive growth of the infarct4,5. In order to elucidate the mechanisms underlying this recruitment, knowledge of the acute metabolic alterations in the penumbra is required. For this reason autoradiographic imaging of CBF, regional cerebral glucose utilization (rCMF^sup glc^) and protein synthesis (PS) in the penumbra has become a valuable tool in studies of focal cerebral ischemia.

Quantitative autoradiographic studies of regional cerebral glucose utilization in normoglycemic experimental focal ischemia have shown a consistent pattern of depressed glucose metabolism in the ischemic core bordered by zone of increased glucose utilization early after MCAO6-10. The increased rCMR^sub glc^ most likely represents enhanced anerobic glycolysis in hypoperfused tissue characterized by an uncoupling of flow and metabolism8,9.

Permanent MCAO in rats leads to a total inhibition of protein synthesis in the ischemic focus. Surrounding the cortical focus a zone with protein synthesis rates approximately half of normal is present as shown in autoradiographic studies of radioactive amino acid incorporation11,12.

The mechanisms underlying these metabolic disturbances seem to be related to pathological electrical phenomena in the penumbra. Both the depression of protein synthesis and the increased glucose utilization in the penumbra have been suggested to be the metabolic consequences of recurrent peri-infarct depolarizations13-17. The number of peri-infarct depolarizations has been shown to correlate linearly with the final infarct volume18.

Pharmacological blockade of ionotropic glutamate receptors of both the NMDA- and AMPA subtypes not only prevents peri-infarct depolarizatioins but also consistently reduces infarct volume when administered before or early after MCAO in rats, e.g., the noncompetitive NMDA-antagonist MK-801 (dizocilpine maleate) and the competitive AMPA-antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulphamoylbenzo(F)quinoxaline) both diminish infarct volume presumably by preventing the peri-infarct depolarizations19-21,22-25. Furthermore, autoradiographic studies have shown that MK-801 both normalizes the increased glucose metabolism and reduces the inhibition of protein synthesis in the penumbra early after MCAO11,26 . No data regarding the influence of NBQX on penumbral glucose metabolism is available whereas protein synthesis in the penumbra has been shown not to improve after NBQX treatment11.

The CBF threshold for inhibition of protein synthesis is markedly different from the flow threshold at which disturbances of glucose metabolism begin to occur. Mies et al.12 found that in rats subjected to permanent MCAO, protein synthesis is inhibited by 50% at CBF values of 55 ml 100 g^sup -1^ min^sup -1^ and is completely suppressed below 35ml 100 g^sup -1^ min^sup -1^. In contrast9,27, glucose utilization increases when CBF is lowered 35 ml 100g^sup-1^ min^sup -1^ before it sharply declines below 25ml 100g^sup -1^ min^sup -1^.

Since the flow thresholds for inhibition of protein synthesis and disturbance of glucose utilization are different, we hypothesized that the penumbral regions displaying reduced protein synthesis might not correspond completely to the penumbra with aberrant glucose metabolism and, if present, this regional incongruity might represent a metabolic penumbra which could be amenable to pharmacotherapy and could be salvaged by neuroprotective drugs.

The objective of the study was hence three-fold:

1. Design of a double-tracer autoradiographic procedure that allows simultaneous determination of regional protein synthesis and local glucose consumption in parallel brain sections from individual rats.

2. Comparison of cortical tissue with disturbed PS to that of disturbed 2-deoxyglucose uptake in rats acutely after occlusion of the middle cerebral artery.

3. To study the effect of the glutamate antagonists, MK-801 and NBQX, which as mentioned reduce cortical infarct volume in rat models of focal ischemia.

MATERIALS AND METHODS

The experiments were performed in accordance with The European Community Council directive of November 24 1986 (86/609/EEC) and the guidelines from the Danish Animal Experiment Inspectorate.

Double tracer autoradiography

At the end of the tracer circulation period the animals were killed with a halothane overdose. The brains were rapidly removed from the skull, snap frozen in isopentane and stored at -80 deg C until further processing. Frontal 20 (mu)m cryosections of the forebrain were cut on a Leitz: Kryostat 1720 (Ernst Leitz GmbH, Wetzlar, Germany). For every 300 (mu)m two neighboring sections were collected on coverslips for determination of ^sup 3^Hleucine incorporation and ^sup 14^C-deoxyglucose uptake, respectively. The sections for determination of 3 Hleucine incorporation were washed three times for 30 min in 10% TCA and left overnight in cold TCA in order to remove all ^sup 14^C-deoxyglucose from the tissue. A pilot study had shown that this washing procedure effectively extracted all the ^sup 14^C-deoxyglucose and at the same time removed any unincorporated ^sup 3^H-leucine from the sections. The coverslips were mounted on cardboard and the sections along with appropriate standards for ^sup 3^H and ^sup 14^C (Amersham, Buckinghamshire, UK). The washed sections for protein synthesis determination exposed a ^sup 3^H sensitive beta-max film (Amersham) and sections for 2-deoxyglucose uptake exposed a Kodak X-- ray film for approximately three weeks. In addition, two unwashed sections from each brain exposed each film in order to evaluate the efficiency of the washout.

Measurement and calculation of protein synthesis and glucose utilization

The regional optical densities of the autoradiographs were analyzed on a Macintosh computer equipped with a video camera using the public domain NIH Image program (developed at the US National Institutes of Health and available on the Internet at http://rsb.info. nih.gov/nih-image/).

Regional rates of protein synthesis were calculated using the operational equation derived by Smith et aj.29. Rates of regional glucose utilization were calculated using the operational equation and rate constants published by Sokoloff etal.30.

Volumetry of the ischemic cortex

Using the computerized image analysis system, the volumes of cortex ipsilateral to the MCAO displaying normal, decreased and increased rates of protein synthesis and glucose utilization were quantitated in the following way: On the autoradiographs of the coronal brain sections the non-ischemic cortex, i.e. contralateral to MCAO, was outlined and the mean optical density and standard deviation were measured. Normal metabolism was defined as being within this mean value +/- 2 standard deviations. Then the ischemic cortex was outlined and on each section the areas with normal (mean +/- 2 SM decreased (mean+ 2 SD) metabolism were measured using the software's density slice function (a thresholding tool). The corresponding volumes in each brain were calculated by summing the areas and multiplying with the distance between the sections according to Cavalieri's principle32,33.

RESULTS

Peroperative monitoring

Volumetric comparison of glucose metabolism and protein synthesis

The design of the measuring procedure allowed identification and calculation of the cortical volume ipsilateral to the MCAO with normal, reduced and increased glucose metabolism and protein synthesis in each brain. The results of these measurements are shown in Table 2. In both the control group and the treated groups, the cortical volume displaying normal protein synthesis rates was significantly less than the volume with normal glucose utilization. For example, in control rats 66% of the ipsilateral cortex displayed normal protein synthesis whereas 80% of the cortex had normal glucose utilization (Figure 3). In other words, within each ischemic cortex inhibition of protein synthesis was more widespread than disturbances in glucose metabolism. Treatment with the glutamate antagonists did not significantly change this difference although MK-801 tended to improve both protein synthesis and glucose metabolism. The cortical volume with disturbed glucose utilization was significantly less in MK-801 treated animals than in controls (Table 2).

This difference in distribution of deranged protein synthesis and glucose metabolism allowed definition of a metabolic penumbra in the focal ischemic cortex as the tissue volume with reduced protein synthesis minus the volume with reduced glucose utilization (Table 2). In controls the volume of this metabolic penumbra comprised 45.6 mm^sup 3^ or 22% of the ipsilateral cortex. In MK-801 treated group the metabolic penumbra was reduced to 27.8 mm^sup 3^ equivalent to 14% of the cortex, which, however, was not significantly different from control. Similarly, NBQX treatment had no statistically significant effect on the size of the metabolic penumbra.

DISCUSSION

Methodological considerations

The study demonstrates that double-tracer autoradiography can be employed to study glucose consumption and protein synthesis in neighboring sections from the same rat brain combining two previously well-described and validated methods29,30. We found that TCA washing as described above, satisfactorily removes ^sup 14^C-deoxyglucose from sections in which ^sup 3^H-leucine incorporation is to be determined. Several multiple-tracer autoradiographic procedures for simultaneous measurement of different parameters of brain metabolism, e.g. rCBF, rCMR^sub glc^ and pH have been published8,9,34-38. The different principles of autoradiographic differentiation between radioactive isotopes are described in these papers. Only a few previous studies have simultaneously assessed rCMR^sub glc^ and protein synthesis but none of these has investigated the events following a focal ischemic insult37,39,40. In a triple-tracer study by Mies et al.37 autoradiographic measurement of rCBF, regional protein synthesis, and rCMR^sub glc^ was done using the same approach for autoradiographic differentiation between ^sup 14^C-deoxyglucose and ^sup 3^H-leucine as in the present study, i.e. by use of a washout procedure and two film materials with different sensitivity to the energy emitted by the two radionuclides.

In contrast to us, Mies et al. reported considerable problems obtaining a complete washout of ^sup 14^C-deoxglucose and ^sup 14^C-deoxyglucosephosphate from the sections. The ^sup 14^C content was reduced to approximately 14% compared to less than 2% in the present study. Mies et al. had to correct for the ^sup 14^C contamination of the 3H autoradiographs by a digital subtraction procedure. In our study significant cross-contamination was only encountered in a few series of sections, which we therefore decided to exclude from the study.

Since the washout media in both studies contained 10% TCA and the sections were immersed in the medium for approximately the same period of time, it appears that it is pivotal that the sections are not allowed to dry before performing the wash procedure. In the present study the sections were washed immediately after they were cut, whereas the sections in the study of Mies et aL had exposed a 14C-sensitive film for two weeks prior to washout. The washout procedure employed in the present study was satisfactory and without the necessity for subtraction of contamination (Figure 1).

Pathophysiological significance of the findings

This study shows that the spatial distribution of disturbances in protein synthesis and glucose metabolism differs in the third hour after permanent MCAO in rats. The tissue volume with suppressed protein synthesis was found to be significantly larger than the tissue volume with abnormal glucose metabolism. The explanation for this should probably be sought in the topography of the CBF reduction and the different CBF thresholds for inhibition of protein synthesis and glucose dysmetabolism. Protein synthesis is half-maximally suppressed when CBF declines to 55 ml 100 g^sup -1^ min^sup -1^ equivalent to approximately 50% of normal12. In comparison, glucose consumption typically exhibits a bimodel response to reductions in CBF. When flow is lowered to 35-40 ml 100g^sup -1^ min^sup -1^ (38%) of control values increases in glucose consumption begin to occur9,27. Below 25 ml 100g^sup -1^ min^sup -1^ glucose consumption has ceased as it is seen in the core of the focal ischemic lesion8,27. Thus, suppression of protein synthesis occurs at higher CBF values than glucose dysmetabolism. In addition, the reduction of blood flow within the penumbra is not uniform. Lowest values are found closest to the ischemic core, whereas less severe reductions are found in the watershed zones between the middle cerebral artery and the anterior and posterior cerebral artery and in the upper cortical layers due to collateral pial supply1,2,8,41. It therefore seems plausible that a heterogeneous residual flow in the penumbra in combination with the aforementioned different CBF thresholds may be responsible for the difference in the volumes of cortex with reduced protein synthesis and glucose dysmetabolism observed in the present study.

The image analysis procedure permitted us to define and identify a metabolic penumbra as the difference between cortex with reduced protein synthesis and cortex with reduced glucose consumption. In control rats, the size of the metabolic penumbra amounted to 22% of the entire cortex or 32% of the cortex suffering from reduced protein synthesis (Table 2). Interestingly, only 16% of the metabolic penumbra had increased 2-- deoxyglucose uptake meaning that most of the penumbra had normal glucose consumption although protein synthesis was suppressed in the same areas. The perfusion threshold for ATP depletion 2 h after MCAO is 19 ml 100 g^sup -1^ min^sup -1^, which is close to threshold of membrane failure and irreversible cell damage42. Since the perfusion threshold for arrested glucose metabolism is higher than this, it follows that ATP was probably not depleted in the metabolic penumbra identified in the present study and therefore should constitute potentially viable tissue with the possibility for salvage by reperfusion or neuroprotective drugs. However, it should be emphasized that the penumbra is dynamic rather than static in its nature43. During recurrent peri-infarct depolarizations the penumbra may transiently suffer from ATP shortage. Regrettably, no reports on the correlation between the penumbral zone with glucose hypermetabolism and the ATP content have been published. Our data lead us to speculate that the part of the metabolic penumbra tissue with 2-deoxyglucose hyperphosphorylations represents tissue which is characterized by anerobic glycolysis, intermittent bioenergetic compromise and is close to the point of no return. Tissue with reduced PS but normal glucose metabolism on the other hand depicts tissue in which ATP is preserved although transient depolarizations may occur. Even though both parts of the metabolic penumbra are threatened, the therapeutic window is wider for the latter. Progressive deterioration of both parts of the metabolic penumbra will take place, if arterial occlusion is permanent, although the possibility may exist that the outer part of the penumbra with reduced ^sup 3^H-leucine incorporation may regain protein synthesizing activity despite ongoing arterial occlusion. Indirect support for this notion is given by Mies et al. who found that from 1 h to 12 h after permanent MCAO in rats the perfusion threshold for ATP depletion increased to become closer to but not identical to the higher perfusion threshold for inhibition of protein synthesis12.

To investigate the reversibility of the metabolic alterations in the ischemic penumbra, we administered the glutamate antagonists MK-801 and NBQX at time points and in doses previously shown to reduce infarct size in similar rat models of focal ischemia 24,25,44,45 . No statistically significant effects of these compounds on either the volume of cortex with normal metabolism or the volume of metabolic penumbra in the treatment groups were found although MK-801 tended to improve metabolism (Table 2). The lacking effect of MK-801 and NBQX on the size of the metabolic penumbra in the present study may be explained by the long lasting halothane anesthesia which could have influenced the occurrence of peri-infarct depolarizations.

Halothane has been shown to block cortical spreading depressions in the normal cat brain and peri-infarct depolarizations in the same species46,47. Since the neuroprotective effect of NBQX and MK-801 is thought also to be due to blockade of peri-infarct depolarizations19-21, the effect of halothane may have masked or interfered with the effect of the glutamate antagonists. In support of this is the finding that only half of the rats in the control group had autoradiographic signs of periinfarct depolarizations in terms of deoxyglucose hyperphosphorylations.

CONCLUSION

This study demonstrates that double-tracer autoradiography can be used to study glucose consumption and protein synthesis in parallel, fresh cryosections from the same rat brain. TCA satisfactorily washed out ^sup 14^C-- deoxyglucose from sections in which ^sup 3^H-leucine incorporation was to be determined.

The major finding was that in the acute phase following occlusion of the middle cerebral artery, cortical penumbral regions with disturbed protein synthesis are not completely congruent with that of disturbed glucose metabolism. We speculate that areas with reduced glucose metabolism represent tissue destined to infarction whereas part of the metabolic penumbra characterized by reduced protein synthesis but preserved glucose metabolism may recover provided proper therapy is initiated. This study, however, failed to demonstrate any statistically significant effect of the glutamate antagonists MK-801 and NBQX on the size of the metabolic penumbra.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the technical assistance and expertise of Ms Berit Jensen. The study was financially supported by the Michaelsen Foundation and the Danish Medical Research Council.

REFERENCES

1 Tyson GW, Teasdale GM, Graham DI, McCulloch J. Focal cerebral

ischemia in the rat: Topography of hemodynamic and histopathological changes. Ann Neurol, 1984; 15: 559-567

2 Bolander HG, Persson L, Hillered L, d'Argy R, Ponten U, Olsson Y. Regional cerebral blood flow and histopathologic changes after middle cerebral artery occlusion in rats. Stroke 1989; 20: 930-937

3 Memezawa H, Minamisawa H, Smith M, Siesjo BK. Ischemic penumbra in a model of reversible middle cerebral artery occlusion in the rat. Exp Brain Res 1992; 89: 67-78

4 Memezawa H, Smith M, Siesjo BK. Penumbral tissues salvaged by reperfusion following middle cerebral artery occlusion in rats. Stroke 1992; 23: 552-559

5 Kaplan 13, Brint S, Tanabe J, Jacewicz M, Wang X, Pulsinelli W. Temporal thresholds for neocortical infarction in rats subjected to reversible focal cerebral ischemia. Stroke 1991; 22: 1032-1039

6 Ginsberg MID, Reivich M, Giandomenico A, Greenberg JH. Local glucose utilization in acute focal cerebral ischemia: Local dysmetabolism and diaschisis. Neurology 1977; 27: 1042-1048

7 Welsh FA, Greenberg JH, Jones SC, Ginsberg MID, Reivich M. Correlation between glucose utilization and metabolite levels during focal ischemia in cat brain. Stroke 1980; 11: 79-84

8 Nedergaard M, Gjedde A, Diemer NH. Focal ischemia of the rat brain: Autoradiographic determination of cerebral glucose utilization, glucose content, and blood flow. J Cereb Blood Flow Metab 1986; 6: 414-424

9 Sako K, Kobatake K, Yamamoto YL, Diksic M. Correlation of local cerebral blood flow, glucose utilization, and tissue pH following a middle cerebral artery occlusion in the rat. Stroke 1985; 16: 828-834

10 Shiraishi K, Sharp FR, Simon RP. Sequential metabolic changes in rat brain following middle cerebral artery occlusion: A 2deoxyglucose study. I Cereb Blood Flow Metab 1989; 9: 765-773

11 Christensen T, Bruhn T, Frank L, Diemer NH. Differential effect of NMDA and AMPA receptor blockade on protein synthesis in the rat infarct borderzone. Acta Neurol Scand 1996; 93: 160-167

12 Mies G, Ishimaru S, Xie Y, Seo K, Hossmann K-A. Ischemic thresholds of cerebral protein synthesis and energy state following

middle cerebral artery occlusion in rat. J Cereb Blood Flow Metab 1991; 11: 753-761

13 Kocher A Metabolic and hemodynamic activation of postischemic rat brain by cortical spreading depression. J Cereb Blood Flow Metab 1990; 10: 564-571

14 Mies G. Inhibition of protein synthesis during repetitive cortical spreading depression. J Neurochem 1993; 60: 360-363

15 Mies G, Kohno K, Hossmann K-A. MK-8011, a glutamate antagonist, lowers flow threshold for inhibition of protein synthesis after middle cerebral artery occlusion of rat. Neurosci Lett 1993; 155: 65-68

16 Hossmann K-A. Periinfarct depolarizations. Cerebrovasc Brain Metab Rev 1996; 8: 195-208

17 Hossmann K-A. Viability thresholds and the penumbra of focal ischemia. Ann Neurol 1994; 36: 557-565

18 Mies G, lijima T, Hossmann K-A. Correlation between peri-infarct DC shifts and ischaemic neuronal damage in rat. Neuroreport 1993; 4: 709-711

19 Gill R, Andine P, Hillered L, Persson L, Hagberg H. The effect of MK-801 on cortical spreading depression in the penumbral zone following focal ischaemia in the rat. J Cereb Blood Flow Metab 1992; 12: 371-379

20 lijima T, Mies G, Hossmann KA. Repeated negative DC deflections in rat cortex following middle cerebral artery occlusion are abolished by MK-801: Effect on volume of ischemic injury. J Cereb Blood Flow Metab 1992; 12: 727-733

21 Mies G, Kohno K, Hossmann K-A. Prevention of periinfarct direct current shifts with glutamate antagonist NBQX following occlusion of the middle cerebral artery in the rat. J Cereb Blood Flow Metab 1994;14:802-807

22 Gill R, Brazell C, Woodruff GN, Kemp JA. The neuroprotective action of dizocilpine (MK-801) in the rat middle cerebral artery occlusion model of focal ischaemia. Br J Pharmacol 1991; 103: 2030-2036

23 Park CK, Nehls DG, Graham DI, Teasdale GM, McCulloch J. The glutamate antagonist MK-801 reduces focal ischemic brain damage in the rat. Ann Neurol 1988; 24: 543-551

24 Gill R, Nordholm L, Lodge D. The neuroprotective actions of 2,3dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) in a rat focal ischaemia model. Brain Res 1992; 580: 35-43

25 Buchan AM, Xue D, Huang ZG, Smith KH, Lesiuk H. Delayed AMPA receptor blockade reduces cerebral infarction induced by focal ischemia. Neuroreport 1991; 2: 473-476

26 Liu Y, Mituska S, Hashizume K, Hosaka T, Nukui H. The time course of glucose metabolism in rat cerebral ischemia with middle cerebral artery occlusion-reperfusion model and the effect of MK801. Neurol Res 1996; 18: 505-508

27 Paschen W, Mies G, Hossmann K-A. Threshold relationship between cerebral blood flow, glucose utilization, and energy metabolites during development of stroke in gerbils. Exp Neurol 1992;117:325-333

28 Tamura A, Graham DI, McCulloch J, Teasdale GM. Focal cerebral ischaemia in the rat: 1. Description of the technique and early neuropathological consequences following middle cerebral artery occlusion. J Cereb Blood Flow Metab 1981; 1: 53-60

29 Smith CB. The measurement of regional rates of cerebral protein synthesis in vivo. Neurochem Res 1991; 16: 1037-1046

30 Sokoloff L, Reivich M, Kennedy C, Des RM, Patlak CS, Pettigrew KD, Sakurada 0, Shinohara M. The [1 4C]deoxyglucose method for the measurement of local cerebral glucose utilization: Theory, procedure, and normal values in the conscious and anesthetized albino rat. j Neurochem 1977; 28: 897-916

31 Bruhn T, Cobo M, Berg M, Diemer NH. Limbic seizure-induced changes in extracellular amino acid levels in the hippocampal formation: A microdialysis study of freely moving rats. Acta Neurol Scand 1992. 86: 455-461

32 Cavalieri B. Geometria indivisibilibus continuorum nova quadam ratione promot, Bologna: Duci, 1635

33 Gundersen JH, Bendtsen TF, Korbo L, Marcussen N, Moller A, Nielsen K, Nyengaard JR, Pakkenberg B, Sorensen FB, Vesterby A, et aL Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 1988; 96: 379-394

34 Lear JL, Jones SC, Greenberg JH, Fedora T, Reivich A Use of 1231 and 14C in a double radionuclide autoradiographic technique for simultaneous measurement of LCBF and LCMRgl. Theory and method. Stroke 1981; 12: 589-597

35 Sako K, Kato A, Diksic M, Yamamoto LY. Use of short-lived 18F and long-lived 14C in double tracer autoradiography for simultaneous measurement of LCBF and LCGU. Stroke 1984; 15: 896-900

36 Mies G, Niebuhr 1, Hossmann K-A. Simultaneous measurement of blood flow and glucose metabolism by autoradiographic techniques. Stroke 1981; 12: 581-588

37 Mies G, Bodsch W, Paschen W, Hossmann K-A. Triple-tracer autoradiography of cerebral blood flow, glucose utilization, and protein synthesis in rat brain. J Cereb Blood Flow Metab 1986; 6: 59-70

38 Gjedde A, Diemer NH. Double-tracer study of the fine regional blood-brain glucose transfer in the rat by computer-assisted autoradiography. J Cereb Blood Flow Metab 1985; 5: 282-289

39 Paschen W, Mies G, Bodsch W, Yamori Y, Hossmann K-A. Regional cerebral blood flow, glucose metabolism, protein synthesis, serum protein extravasation, and content of biochemical substrates in stroke-prone spontaneously hypertensive rats. Stroke 1985; 16: 841-845

40 Kirikae M, Diksic M, Yamamoto YL. Quantitative measurements of regional glucose utilization and rate of valine incorporation into proteins by double-tracer autoradiography in the rat brain tumor model. J Cereb Blood Flow Metab 1989; 9: 87-95

41 Back T, Zhao W, Ginsberg MD. Three-dimensional image analysis of brain glucose metabolism-blood flow uncoupling and its electrophysiological correlates in the acute ischemic penumbra following middle cerebral artery occlusion. J Cereb Blood Flow Metab 1995; 15: 566-577

42 Kohno K, Hoehn-Berlage M, Mies G, Back T, Hossmann K-A. Relationship between diffusion-weighted MR images, cerebral blood flow, and energy state in experimental brain infarction. Magn Reson Imaging 1995; 13: 73-80

43 Obrenovitch TP. The ischaemic penumbra: Twenty years on. Cerebrovasc Brain Metab Rev 1997; 7: 297-323

44 Roussel S, Pinard E, Seylaz J. Effect of MK-801 on focal brain infarction in normotensive and hypertensive rats. Hypertension 1992;19:40-46

45 Bielenberg GW, Beck T. The effects of dizocilpine (MK-801), phencyclidine, and nimodipine on infarct size 48 h after middle cerebral artery occlusion in the rat. Brain Res 1991; 552: 338-342

46 Saito R, Graf R, Hubei K, Taguchi J, Rosner G, Fujita T, Heiss WD. Halothane, but not alpha-chloralose, blocks potassium-evoked cortical spreading depression in cats. Brain Res 1995; 699: 109-115

47 Saito R, Graf R, Hubei K, Fujita T, Rosner G, Heiss WID. Reduction of infarct volume by halothane: Effect on cerebral blood flow or perifocal spreading depression-like depolarizations. J Cereb Blood Flow Metab 1997; 17: 857-864

Thomas Christensen, Torbert Balchen, Torben Bruhn and Nils H. Diemer

Institute of Molecular Pathology, Laboratory for Neuropathology, University of Copenhagen, Copenhagen, Denmark

Correspondence and reprint requests to: Thomas Christensen, MD, institute of Molecular Pathology, Laboratory for Neuropathology, University of Copenhagen, Frederik V's vej 11, DK-2100 Copenhagen, Denmark. Accepted for publication March 1999.

Copyright Forefront Publishing Group Oct 1999
Provided by ProQuest Information and Learning Company. All rights Reserved