Actu Physiol Scand 1990, 138, 417-422
Extracellular levels of quinolinic acid are moderately increased in rat neostriatum following severe insulin- induced hypoglycaemia E. W E S T E R B E R G , K. MAGNUSSON, T. W I E L O C H , U. UNGE R ST E D T ", C. S P E C I A L E t and R. SCHWARCZ? Laboratory for Experimental Brain Research, Lund University Hospital, Sweden, Department of Pharmacology, Karolinska Institute, Stockholm, Sweden, and Maryland Psychiatric Research Center, Baltimore, MD, USA
" t
WESTERBERG, E., MAGNUSSON, K., WIELOCH, T., UNGERSTEDT, U., SPECIALE, C. & SCHWARU, R. 1990. Extracellular levels of quinolinic acid are moderately increased in rat neostriatum following severe insulin-induced hypoglycaemia. Actu Phjisiol Scund 138, 417-427. Received z October 1989, accepted 22 October 1989. ISSN 000143772. Laboratory for Experimental Brain Research, Lund University Hospital, Sweden, Department of Pharmacology, Karolinska Institute, Stockholm, Sweden, and Maryland Psychiatric Research Center, Baltimore, MD, USA. Extracellular concentrations of the brain metabolite quinolinic acid, an endogenous excitotoxin, were monitored by microdialysis in rat neostriatum and hippocampus/ cortex during and following a 30-min period of insulin-induced hypoglycaemia. During hypoglycaemia-induced isoelectricity, extracellular levels of quinolinic acid in the striatum (basal value, 1 . 1 k0.3 pmol per 30-pl fraction) were elevated 1.7 times as compared to the control period. Thirty to ninety minutes following hypoglycaemia a significant increase in extracellular quinolinic acid to 2.2 times basal level was noted. After 2 h recovery, the beginning of neuronal necrosis was observed in the dorsolateral striatum. Implantation of the dialysis probe did not influence the extent of neuronal damage. No changes in extracellular quinolinic acid levels were observed in the hippocampus/cortex. The data indicate that following a severe hypoglycaemic insult vulnerable striatal cells are exposed to hyperphysiological extracellular quinolinic acid concentrations over an extended period of time. Considering the pronounced susceptibility of rat striatal neurons to the toxin, the small but prolonged elevation in the extracellular levels of quinolinic acid could be of significance for the development of delayed neuronal death in hypoglycaemia.
Key words :excitotoxins, hypoglycaemia, microdialysis, neuronal death, quinolinic acid.
Severe hypoglycaemia, defined by electroencephalogram (EEG) isoelectricity, leads to neuronal damage in selectively vulnerable areas of the rat brain (Auer et al. 1984a). T h e neostriatum, the hippocampal region (dentate gyrus and CAI pyramidal cells) and the outer cortical layers are particularly susceptible to a hypoglycaemic insult. Neuronai necrosis in the Correspondence : Tadeusz Wieloch PhD, Laboratory for Experimental Brain Research, Lund University Hospital, S-ZZI 85 Lund, Sweden.
ctriatum is precipitated within 4-6 h of recovery following a 30-min hypoglycaemic insult (Kalimo et a/. 1985). I n contrast in the hippocampus and the cerebral cortex irreversible neuronal damage begins during the hypoglycaemia (Auer et al. 1984a, b). Recent evidence supports the idea that excitotoxins are causally involved in the pathogenesis of hypoglycaemic brain damage (Auer et al. 1985, Wieloch 1985a). T h e idea derives primarily from the ultrastructural ('axonsparing') appearance of this lesion (Auer et a[.
417
19x5, Wieloch 158ja) and from deafferentation \\-ere cannulated and repeated arterial samples were esperiments, which demonstrated that taken anaerobically from the catheter in glass glutamatergic denervation can protect the capillaries for determination of pH, Pco, and Po,, or datum against hj-poglycaemic damage collected directly for measurement of blood glucose (\Vieloch t't cil. 198j, Idinden ril. 1987). The with Glucostis reagent strips and a reflectometer mosr convincing evidence for ;i participation of s!stem (r\mes Co., Elkhart, IN, USA). Two burr holes were drilled, one above the caudate nucleus (coexcitatory amino acids in hypoglycaemic damage ordinates in mm : AP, o; MI,, 3. j ; Pasinos & Watson \\as the demonstration of selective ncuro1982) of one hemisphere, and a second above the protection b!- ,\-methyl-r>-aspartate ('iVD.4) dorsal hippocampus of the othcr hemisphere (coreceptor antagonists (Wieloch 1985 b, Simon ordinates: .\P. - 3 . j ; MI,, 2.5). A rectal thermometer r t i i l 1986, Westerberg rt u l . 1988, l l o n y e r et al. was placed, and a pair of needle electrodes were rg8q). Notably, eytracellular lei-els of glutamate inserted in the muscles lateral to the skull bone for LEG recording. The body temperature was kept at and aspartate, the two most abundant endogenous rxcitotoxins, increase se\ era]-fold in the 37 "C. .\t the end of the operative procedures, halothane striarum durinp hypoglycaemia, b u t are rapidl! administration was discontinued, D-tubocurarine normalized upon glucose administration ( 0 .j mg kg-I) was administered intravenously, and the (Sandberg LI/. 1986, [Vieloch r f [ I / . 1986, EEG monitored. In order to minimize bleeding, Butcher r t i l l . 1987, U'esterberp r t ( I / . 1988). heparin was not administered systemically, but was Since glutamate and aspartate arc marginally placed in the arterial catheter used for blood pressure neurotosic to the rat striatum even when recording onlJ-. Following 30 min of isoelectric EEG, administered in very large quantities ( l l a n g a n o normalization of the blood glucose concentration was CY- Scht\arcz r983), alternatii e ewitotoxic brain achieved by repeated intravenous injections of a 30 O0 constituents should be considered as potential (\v/v) glucose solution in saline. The animals were neurndegenerative agents in hypoglycaemic allowed z h of recovery with normal blood glucose damage. Quinolinic acid (QLXX), a selective levels prior to sacrifice by transcardial perfusion fisation with jo0phosphate-buffered formaldehyde. Sl411.4 receptor agonist (Stone & Perkins 1981) The brains were embedded in paraffin and serially and a potent neurotoxin in the rat striatum sectioned. The sections were stained with celestine (SchHarcz t't al. 1983), may serve this role. blue-acid fuchsin. Using microdialysis, we have therefore .tlic-roJicr!ysr's teshniqur. Dialysis probes (CR/fA/10 ; investigated the extracellular levels of QUIN in Carncgie Medicin ,4B, Stockholm, Sweden) with a the striatum and hippocarnpu\/cortes before, menibrme length of j nim were positioned stereotasically into the caudate nucleus and the during and after the induction of h!-poglycaemic hippocampus to a depth of 6.5 and 4.4 nim below the coma. brain surface respectively. The positions of the probes \\ere chosen to assure that sampling was performed from the h! poalycacmia-sensitive parts of the xi -4'1F: R I A I, .L\ N 1111E H I> srructures. Since the thickness of the dorsal hippocanipus is approximately z mm, positioning of a . 4 m m / s . H! pogl!caemia was induced as previously described (.\uer t./ ,//. IgX4h). \tale \Vistar rats (SPF dialysis probe with a 4 mm membrane length also strain, Ylollegaards .\vlslaborarorium, Copenhagen, sampled from the cerebral cortex. Dialysis probes Denmark) weighing zjj-420 g \Yere used. The nere continuousl) perfused with Krebs-bicarbonate ~: 1 2 2 , KCI 3 , animals were f
Extracellular levels of quinolinic acid are moderately increased in rat neostriatum following severe insulin- induced hypoglycaemia E. W E S T E R B E R G , K. MAGNUSSON, T. W I E L O C H , U. UNGE R ST E D T ", C. S P E C I A L E t and R. SCHWARCZ? Laboratory for Experimental Brain Research, Lund University Hospital, Sweden, Department of Pharmacology, Karolinska Institute, Stockholm, Sweden, and Maryland Psychiatric Research Center, Baltimore, MD, USA
" t
WESTERBERG, E., MAGNUSSON, K., WIELOCH, T., UNGERSTEDT, U., SPECIALE, C. & SCHWARU, R. 1990. Extracellular levels of quinolinic acid are moderately increased in rat neostriatum following severe insulin-induced hypoglycaemia. Actu Phjisiol Scund 138, 417-427. Received z October 1989, accepted 22 October 1989. ISSN 000143772. Laboratory for Experimental Brain Research, Lund University Hospital, Sweden, Department of Pharmacology, Karolinska Institute, Stockholm, Sweden, and Maryland Psychiatric Research Center, Baltimore, MD, USA. Extracellular concentrations of the brain metabolite quinolinic acid, an endogenous excitotoxin, were monitored by microdialysis in rat neostriatum and hippocampus/ cortex during and following a 30-min period of insulin-induced hypoglycaemia. During hypoglycaemia-induced isoelectricity, extracellular levels of quinolinic acid in the striatum (basal value, 1 . 1 k0.3 pmol per 30-pl fraction) were elevated 1.7 times as compared to the control period. Thirty to ninety minutes following hypoglycaemia a significant increase in extracellular quinolinic acid to 2.2 times basal level was noted. After 2 h recovery, the beginning of neuronal necrosis was observed in the dorsolateral striatum. Implantation of the dialysis probe did not influence the extent of neuronal damage. No changes in extracellular quinolinic acid levels were observed in the hippocampus/cortex. The data indicate that following a severe hypoglycaemic insult vulnerable striatal cells are exposed to hyperphysiological extracellular quinolinic acid concentrations over an extended period of time. Considering the pronounced susceptibility of rat striatal neurons to the toxin, the small but prolonged elevation in the extracellular levels of quinolinic acid could be of significance for the development of delayed neuronal death in hypoglycaemia.
Key words :excitotoxins, hypoglycaemia, microdialysis, neuronal death, quinolinic acid.
Severe hypoglycaemia, defined by electroencephalogram (EEG) isoelectricity, leads to neuronal damage in selectively vulnerable areas of the rat brain (Auer et al. 1984a). T h e neostriatum, the hippocampal region (dentate gyrus and CAI pyramidal cells) and the outer cortical layers are particularly susceptible to a hypoglycaemic insult. Neuronai necrosis in the Correspondence : Tadeusz Wieloch PhD, Laboratory for Experimental Brain Research, Lund University Hospital, S-ZZI 85 Lund, Sweden.
ctriatum is precipitated within 4-6 h of recovery following a 30-min hypoglycaemic insult (Kalimo et a/. 1985). I n contrast in the hippocampus and the cerebral cortex irreversible neuronal damage begins during the hypoglycaemia (Auer et al. 1984a, b). Recent evidence supports the idea that excitotoxins are causally involved in the pathogenesis of hypoglycaemic brain damage (Auer et al. 1985, Wieloch 1985a). T h e idea derives primarily from the ultrastructural ('axonsparing') appearance of this lesion (Auer et a[.
417
19x5, Wieloch 158ja) and from deafferentation \\-ere cannulated and repeated arterial samples were esperiments, which demonstrated that taken anaerobically from the catheter in glass glutamatergic denervation can protect the capillaries for determination of pH, Pco, and Po,, or datum against hj-poglycaemic damage collected directly for measurement of blood glucose (\Vieloch t't cil. 198j, Idinden ril. 1987). The with Glucostis reagent strips and a reflectometer mosr convincing evidence for ;i participation of s!stem (r\mes Co., Elkhart, IN, USA). Two burr holes were drilled, one above the caudate nucleus (coexcitatory amino acids in hypoglycaemic damage ordinates in mm : AP, o; MI,, 3. j ; Pasinos & Watson \\as the demonstration of selective ncuro1982) of one hemisphere, and a second above the protection b!- ,\-methyl-r>-aspartate ('iVD.4) dorsal hippocampus of the othcr hemisphere (coreceptor antagonists (Wieloch 1985 b, Simon ordinates: .\P. - 3 . j ; MI,, 2.5). A rectal thermometer r t i i l 1986, Westerberg rt u l . 1988, l l o n y e r et al. was placed, and a pair of needle electrodes were rg8q). Notably, eytracellular lei-els of glutamate inserted in the muscles lateral to the skull bone for LEG recording. The body temperature was kept at and aspartate, the two most abundant endogenous rxcitotoxins, increase se\ era]-fold in the 37 "C. .\t the end of the operative procedures, halothane striarum durinp hypoglycaemia, b u t are rapidl! administration was discontinued, D-tubocurarine normalized upon glucose administration ( 0 .j mg kg-I) was administered intravenously, and the (Sandberg LI/. 1986, [Vieloch r f [ I / . 1986, EEG monitored. In order to minimize bleeding, Butcher r t i l l . 1987, U'esterberp r t ( I / . 1988). heparin was not administered systemically, but was Since glutamate and aspartate arc marginally placed in the arterial catheter used for blood pressure neurotosic to the rat striatum even when recording onlJ-. Following 30 min of isoelectric EEG, administered in very large quantities ( l l a n g a n o normalization of the blood glucose concentration was CY- Scht\arcz r983), alternatii e ewitotoxic brain achieved by repeated intravenous injections of a 30 O0 constituents should be considered as potential (\v/v) glucose solution in saline. The animals were neurndegenerative agents in hypoglycaemic allowed z h of recovery with normal blood glucose damage. Quinolinic acid (QLXX), a selective levels prior to sacrifice by transcardial perfusion fisation with jo0phosphate-buffered formaldehyde. Sl411.4 receptor agonist (Stone & Perkins 1981) The brains were embedded in paraffin and serially and a potent neurotoxin in the rat striatum sectioned. The sections were stained with celestine (SchHarcz t't al. 1983), may serve this role. blue-acid fuchsin. Using microdialysis, we have therefore .tlic-roJicr!ysr's teshniqur. Dialysis probes (CR/fA/10 ; investigated the extracellular levels of QUIN in Carncgie Medicin ,4B, Stockholm, Sweden) with a the striatum and hippocarnpu\/cortes before, menibrme length of j nim were positioned stereotasically into the caudate nucleus and the during and after the induction of h!-poglycaemic hippocampus to a depth of 6.5 and 4.4 nim below the coma. brain surface respectively. The positions of the probes \\ere chosen to assure that sampling was performed from the h! poalycacmia-sensitive parts of the xi -4'1F: R I A I, .L\ N 1111E H I> srructures. Since the thickness of the dorsal hippocanipus is approximately z mm, positioning of a . 4 m m / s . H! pogl!caemia was induced as previously described (.\uer t./ ,//. IgX4h). \tale \Vistar rats (SPF dialysis probe with a 4 mm membrane length also strain, Ylollegaards .\vlslaborarorium, Copenhagen, sampled from the cerebral cortex. Dialysis probes Denmark) weighing zjj-420 g \Yere used. The nere continuousl) perfused with Krebs-bicarbonate ~: 1 2 2 , KCI 3 , animals were f
