Brain Research, 569 (1992) 275-280 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$03.50
275
BRES 17331
Brain glutamine synthetase increases following cerebral ischemia in the rat Carol K. Petito 1, Marilda C. Chung 1, Lena M. Verkhovsky 2 and Arthur J.L. Cooper 2 Department of 1pathology (Neuropathology) and the Departments of 2Biochemistry and Neurology, Cornell University Medical College, New York, NY 10021 (U.S.A.) (Accepted 27 August 1991)
Key words: Glutamine synthetase; Cerebral ischemia; Astrocyte; Hippocampus; Cortex; Striatum
Changes in astrocyte glutamine synthetase (GS) in postischemic rat brain were evaluated and correlated with regional neuronal vulnerability or resistance to ischemia. Rats subjected to 20 or 30 min of cerebral ischemia were allowed to survive for 3 or 24 h after ischemia; normal animals served as controls. Resultant neuronal necrosis was severe in the striatum by 24 h and in the CAI region of the hippocampus at 72 h; neurons in paramedian cortex and CA3 region of the hippocampus were not permanently damaged. Glutamine synthetase (GS) immunocytochemistry was performed on vibratome sections of paraformaldehyde-fixed brains and enzyme activity was assayed in frozen samples of cerebral cortex, striatum and hippocampus. At 3 and 24 h after ischemia, GS immunoreactivity increased and was secondary to enlargement of GS-positive cell bodies and processes as well as to increased numbers of GS-positive astrocytes. Enzyme activity also increased in cortex, striatum and hippocampus at 3 and 24 h (P ~< 0.03). This study shows that increase in astrocyte GS occurs rapidly after ischemia, and prior studies indicate that this increase occurs in parallel with proliferative changes in astrocyte organelles. The results also suggest that astrocyte metabolism of glutamate increases after ischemia. The increased capacity for glutamine synthetase may be important in normalizing extracellular glutamate following ischemia and protecting brain from the neurotoxic effects of this excitatory amino acid. INTRODUCTION
The excitotoxic action of glutamate may be important in initiating brain injury following cerebral ischemia 32. As originally described by Benveniste et al. 1 and Simon et al. 36, extracellular concentrations of glutamate rise during ischemia to potentially neurotoxic levels and blockade of glutamate receptors reduce ischemic brain damage. Extracellular glutamate is normally taken up by astrocytes s'H't2 where it is metabolized to a-ketoglutarate 41 and glutamine 2°. The ischemia-related increase in glutamate may enhance astrocyte uptake or metabolism of this amino acid which could potentially protect brain from its excitotoxicity. Accordingly, we studied the a m o u n t and the activity of astrocyte GS in postischemic rat brain. This enzyme is preferentially localized in astrocytes in the CNS 18'2°, where it catalyzes the conversion of glutamate to glutamine. Transient cerebral ischemia was produced in rats by temporary occlusion of the carotid and vertebral arteries followed by reperfusion for 3 and 24 h. This insult results in severe neuronal necrosis in the dorsolateral striatum by 24 h and in the CA1 region of the hippocampus by 3 days, but leaves neurons in the paramedian
cerebral cortex and CA3 region of the hippocampus without p e r m a n e n t injury29. Preliminary accounts of the present study have been published previously26'28. MATERIALS AND METHODS
Animal preparation Cerebral ischemia was produced in male Wistar rats weighing 225-250 g using the modified 4-vessel occlusion method of Pulsinelli and Brierley3°. Under Fluothane anesthesia, the vertebral arteries were occluded by electrocautery and the common carotid arteries were isolated and surrounded by loose clasps. The animals were fasted overnight and, on the following day, the common carotid artery clasps were tightened to occlude carotid flow. This produces a reduction in the blood flow to forebrain structures to less than 3% of control, provided the unanesthetized animals remain unconscious and demonstrate bilateral loss of their normal righting reflex throughout the ischemic period 31. After 20 or 30 min, the carotid clasps were released and the animals were allowed to survive for 3 or 24 h. Normal, unoperated animals served as controls. For immunohistochemistry, animals were deeply anesthetized with intraperitoneal urethane and sequentially perfused-fixed through the ascending aorta with: (1) 0.1 M sodium phosphate (NaPO4) buffer, pH 7.4, for 30 s; (2) 4% (w/v) paraformaldehyde in 0,1 M NaPO4 buffer, pH 7.4, for 15 min; and (3) 0.1 M NaPO 4 buffer, pH 7.4, for 5 min. Brains were removed and placed into 0.1 M NaPO4 buffer overnight. Coronal sections at the level of the anterior commissure and at the level of the mid-dorsal hippocampus were cut 40 /~m thick with a vibratome. The adjacent coronal sections were dehydrated, embedded in paraffin, cut 7/~m thick, and stained with hematoxylin-eosin or Luxol-Fast blue-Cresyl vi-
Correspondence: C.K. Petito, Department of Pathology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, U.S.A.
276 olet. Animals used for biochemical studies were anesthetized with Fluothane, decapitated and the brains were rapidly removed. The paramedian cerebral cortex, anterior striatum and mid-dorsal hippocampus were dissected and quickly frozen at -70°C.
total protein content was determined by the method of Lowry et al? 7, using bovine serum albumin as a standard. Glutamine synthetase activity was expressed as ~mol/h/mg of protein.
lmmunohistochemistry
RESULTS
Glutamine synthetase immunochemistry was performed with a polyclonal rabbit anti-glutamine synthetase antibody (gift of Dr. Michael D. Norenberg) using the indirect antibody method, originally employed by Norenberg et al.2°. as well as the avidin-biotin complex (ABC) technique of Hsu et a1.13. The indirect antibody technique employed 40-/~m-thickvibratome pieces of cortex and striatum from animals subjected to 30 min ischemia and sacrificed at 3 and 24 h after ischemia (n = 4 in controls and in the 2 experimental groups). The sections were incubated for 1 h at 27°C with rabbit anti-GS antiserum (1:100 dilution), washed x3 with phosphate-buffered saline (PBS), and incubated for 60 min with peroxidase-conjugated goat anti-rabbit IgG (Boehringer-Mannheim Biochemicals). Horseradish peroxidase reaction product (HRP) was developed by incubation of the sections with HzO2 and 3,3"-diaminobenzidine according to the directions of the manufacturer. Substitution of primary antiserum with PBS served as a negative control. The sections were then postfixed in 1% (w/v) osmium tetroxide, dehydrated through graded alcohol, placed in epon and fiat-embedded between 2 plastic sheets, and lightly stained with 1% (w/v) Toluidine blue. Ultrathin sections were prepared and examined (either unstained or stained with uranyl acetate and lead citrate) under a JOEL 100S electron microscope. The numbers of GS-positive astrocytes and of capillaries surrounded by GS-positive astrocytes were counted in 3-5 1-~m plasticembedded sections of control animals, and at 3 to 24 h after ischemia. The results were expressed as number of GS-positive astrocytes or capillaries per 0.1 mm2. A digitizing pad (Summagraphics. Inc.) interfaced with a Macintosh IIcx computer was used to determine the area of the unstained one/~m thick section. The specimens were evaluated without knowledge of the experimental conditions. With the ABC technique, 40/.tm thick coronal sections of hippocampus, striatum and cortex were cut from controls (n = 4) and from animals subjected to 20 rain of ischemia followed by reperfusion intervals of 3 h (n = 4), 24 h (n = 5) and 48 h (n = 7). The sections were sequentially incubated with normal horse serum, 1:10 for 1 h; rabbit anti-GS IgG, 1:3200 overnight; and horse anti-rabbit IgG, I:100 for 1 h. The avidin-biotin complex, and subsequent incubation with H202 and 3,3"-diaminobenzidine was performed according to the directions of the manufacturer (Vector Labs, Burlingame, CA).
Glutarnine synthetase activity Dissected frozen brain samples were obtained from controls (n = 10) and animals subjected to 20 min of ischemia followed by reperfusion intervals of 3 h (n = 8), and 24 h (n = 6). The specimens were homogenized in 10 vols (w/v) of ice-cold 0.15 M KC1 using a glass-to-glass homogenizer. The homogenates were frozen, thawed the next day and assayed. It should be noted that subsequent freeze-thawing resulted in some loss of glutamine synthetase activity (personal observations). The glutamine synthetase assay was modified from that of Pamiljans et al.12; 100/~1 of homogenate was incubated for 15 rain at 37°C in 0.2 ml of a reaction mixture containing 10 mM MgCI2, 50 mM L-glutamate, 100 mM imidazoleHCI buffer (pH 7.4), 10 mM 2-mercaptoethanol, 50 mM hydroxylamine-HCl (previously adjusted to pH 7.4 with 3 M sodium hydroxide) and 10 mM ATE The reaction was terminated by addition of 0.8 ml of a solution containing 0.37 M ferric chloride, 0.67 M HC1 and 0.2 M trichloroacetic acid. The precipitated protein was removed by centrifugation. After incubation for 15 min at room temperature, the absorbence of the supernate was read at 530 nm and compared to the absorbenee generated by standard quantities of v-glutamylhydroxamate (Sigma) treated with the ferric chloride reagent. The controls contained homogenate but lacked ATP. The
lmmunochemistry The indirect antibody technique and the A B C technique both d e m o n s t r a t e d glutamine synthetase i m m u n o reactivity that was confined to astrocyte cell bodies and processes. Successful immunohistochemical demonstration of GS was d e p e n d e n t not only upon length of time in fixative2°, but also on the interval between sacrifice and the immunohistochemical procedure. In normal rat brain, astrocytes had thin rims of GSpositive cytoplasm surrounding the nuclei and fine delicately branching GS-positive cell processes and perivascular processes (Fig. la). The GS-positive astrocytes were most frequent in the subpial cortex and hippocampus, and infrequent in the white matter structures, including the c e n t r u m semi-ovale a n d white matter bundles of the corpus striatum. Astrocytes in the s t n a t u m contained more reaction product than those in the cerebral cortex using the indirect method, but stained poorly or not at all in the 40-/zm thick sections prepared with the ABC technique. Electron microscopy of stained and unstained ultrathin sections revealed small amounts of electron-dense H R P reaction product in astrocyte cell bodies and processes (Fig. 2a). The n u m b e r , size and staining intensity of GS-positive astrocytes and astrocytic processes increased at 3 h after 20 or 30 min of cerebral ischemia (Fig. tb). These changes occurred both in cortex and in striatum, and were observed in the plastic-embedded sections by light and electron microscopy, as well as in the 40-ktm sections prepared with the A B C technique. Electron microscopy demonstrated enlarged astrocyte cell bodies and processes which were filled with a b u n d a n t amounts of electron dense reaction product (Fig. 2b). Focal regions of striatum contained shrunken neurons whose cytoplasm and nucleus were diffusely stained with H R P reaction product which represented non-specific staining of necrotic cells. E n l a r g e m e n t of the GS-positive cell bodies and processes was less p r o m i n e n t at 24 h than at 3 h postischemia (Fig. lc), but still persisted in comparison with controls, especially in the perivascular astrocyte foot processes. Electron microscopy again showed an astrocytic localization of the H R P reaction product in both regions. In the cerebral cortex, the average n u m b e r of capillaries surrounded by GS-positive processes and of astrocytes per/~m 2 was 7.4 and 10.9 in controls. 45.8 and 25.3 per p m 2 at 3 h and 25.3 and 19.0 per ,um2 at 24 h In the
277 striatum, the number of GS-positive capillaries and astrocytes per ~m 2 was 11.5 and 4.3 in controls, 27.8 and 21.7 at 3 h after ischemia and 21.7 and 20.7 at 24 h after ischemia. While repeated measure analyses of variance failed to show significant differences according to astrocytes vs capillaries or time in either site, small or moderate differences would likely go undetected since the sample sizes were small and the standard deviations were high. The number of GS-positive astrocytes and their staining intensity was also estimated blindly in the 40-~m sections, prepared with the ABC technique. In controls, mild or no staining was present in the striatum and hippocampus in all 5 animals; and in the cortex in 4 of 5 animals. One had moderate staining in cortical astrocytes. At 3 h after ischemia, moderate to marked staining was present in the cortex in 3 of 4 animals; in the striatum in 2 of 4; and in the hippocampus in 1 of 4. At 24 and 48 h after ischemia, moderate to marked staining was observed in 6 of 12 animals in the cortex, 5 of 12 in the striatum and 5 of 12 in the hippocampus. While the results among groups are variable, they do show increases in GS immunoreactivity during the postischemic period.
Glutamine synthetase activity In control animals, GS activity (~mol/h/mg protein) was 0.45 + 0.13 in the hippocampus, 0.45 -+ 0.07 in the paramedian cerebral cortex and 0.29 -+ 0.04 in the striatum, values similar to that reported in previous studies of GS activity in normal rat brain TM. Glutamine synthetase activity significantly increased at 3 and at 24 h following cerebral ischemia in all 3 brain regions examined (Table I). Repeated measure of analyses of variance, using brain regions as the within factor and time as the between factor, showed no significant differences in glutamine synthetase activity across brain regions. However, a significant difference across time (P < 0.03) was noted. Multiple comparisons using the Student/NewmanKeuls method, showed that glutamine synthetase activity in controls differed from that of each of the postischemic intervals of 3 and 24 h, but that the two postischemic time points did not differ from each other.
Fig. 1. Glutamine synthetase (GS) immunohistochemistry in 1 micron-thick plastic-embedded sections of striatum. Perivascular astrocyte processes (short arrow) and astrocyte cell bodies (long arrow) contain no or small amounts of GS immunoreactivity in controls (a) but increased amounts at 3 h (b) and at 24 h (c) after 30 min ischemia. Toluidine blue; bar = 25 ~m.
278
Fig. 2. Glutamine synthetase immunoreactivity in astrocyte cell body is sparse in controls (a) but increased in the cell bodies and pericapillary foot processes at 3 h after ischemia (b). Bar = 5 #m.
DISCUSSION TABLE I Glutamine synthetase activity (l~mol/h/mg protein) in different regions of the brain 3 and 24 h after an ischemia insult
Cortex Striatum Hippoeampus
Control (n)
3 h (n)*
24 h (n)*
0.45_+0.07(10) 0.29_+0.04( 9 ) 0.45+_0.13 (10)
0.72_+0.14 (8) 0.53_+0.13 (8) 0.48+_0.06 (8)
0.60+0.10 (6) 0.46+0.07 (6) 0.70_+0.16(6)
* As a group the GS activity at both 3 and 24 h is different from the controls with P ~< 0.03; Student/Newraan-Keuls method. Numbers in parentheses refer to total number of animals in each group.
G l u t a m i n e synthetase in brain is largely confined to astrocytes 2° where it plays a k e y rote in m e t a b o l i s m of extracellular glutamate and N H 4 - ions 5. The distribution of astrocyte G S in vivo 14'2° and its expression following exposure to h o r m o n e s or second messengers in vitro 14"23 is h e t e r o g e n e o u s , and m a y correlate with glutamatergic neuronal activity. M o d u l a t i o n of glutamine synthetase in vivo has b e e n described in a few e x p e r i m e n t a l models. G l u t a m i n e synthetase m R N A increases with acute hepatic e n c e p h a l o p a t h y 38, although GS enzyme activity and immunoreactivity is u n a l t e r e d 3'~'19 or decreased 2 with
279 chronic liver disease. Brain trauma causes a rapid, transient increase in GS immunoreactivity 19 whereas GS activity declines following hyperoxygenation 33. The postischemic increases in GS found in the present study are at variance with a previous report that examined the effects of ischemia on GS activity in vivo. In that study, cortical GS activity was decreased at 3 h following 10 min of bilateral common carotid artery occlusion in the gerbil and returned to normal levels at 24 h 2a. Several explanations may account for this apparent contradiction. Cortical damage is mild or moderate following 20-30 min ischemia in the rat model 29, but is absent or minimal following 10 min ischemia in the gerbil model 1°. The magnitude and duration of elevated extracellular glutamate 9'35 as well as the degree of reactive astrocytosis t°'25'27 are proportional to the severity and duration of the ischemia and the extent of neuronal damage. Although these differences might account for enhanced GS activity following 30 min but not 10 min of cerebral ischemia, they fail to explain the reduction in enzyme activity found in the gerbil study. Furthermore, if free radical oxidative inactivation of GS occurs after ischemia 21'33, it is not clear why GS reduction did not occur with our model in which free radicals are thought to be produced during the early postischemic period 4°. The possibility that the different assay methods used to measure GS activity caused the apparent discrepancy in postischemic GS activity cannot be ruled out. The v-glutamyltransferase reaction used in the gerbil study 21 is much more sensitive than the synthetase reaction used in the rat study. However, there may be pitfalls associated with its u s e 4 which may result in an artifactual decrease in enzyme activity. The mechanisms underlying the rapid increase in astrocyte glutamine synthetase are unknown but include several possibilities. Firstly, an actual increase in GS protein content could occur by: (1) increased translation or stabilization of its mRNA; (2) increased transcription; and (3) impaired protein degradation. Although total protein synthesis is decreased in postischemic brain 7'15, selective synthesis of certain proteins such as ornithine decarboxylase 7'16 can occur within minutes. Early proliferative changes within astrocytes, including increases in rough endoplasmic reticulum and mitochondria 25, indicate a potential for enhanced protein synthesis. Secondly, posttranslational modification of glutamine synthetase protein may take place during reperfusion. This could expose more antigenic sites to the applied antisera and induce a higher level of enzyme activity. Increases in GS may be due to its induction within glial cells that, in normal brain, possess little GS activity. Such candidates include oligodendroglia and perineuronal glia. Oligodendrocytes recently have been shown
to contain GS although not in as high a quantity as astrocytes 6'37. Perineuronal glia, which have the morphological characteristics of medium-light oligodendrocytes by electron microscopy, are transformed into reactive astrocytes within 1 to 3 h after cerebral ischemia 24. Studies are in progress to determine whether they acquire GS immunoreactivity after ischemia, and at what time point the acquisition occurs. The time course of increased GS correlates well with the onset of reactive astrocytosis in the 4VO model of cerebral ischemia. Although elevations in glial fibrillary acidic protein are delayed for 1-3 days after ischemia in this 27 as well as in other models 1°, increases in astrocyte mitochondria and rough endoplasmic reticulum occur within 3 h 25. The present study does not determine whether or not the increased GS is a specific astrocytic response to ischemia or is part of the early proliferative response of postischemic astrocytes. The postischemic increases in astrocyte glutamine synthetase suggest that the capacity to convert glutamate to glutamine increases following ischemia and that this enhancement may be due to a rise in extracellular glutamate. GS activity of astrocytes in vitro increases with increasing elevations of extracellular glutamate 39. Increases in GS also may indicate that astrocyte uptake of glutamate increases after ischemia, a finding recently shown to occur when astrocytes are exposed to hypoxia 34. Increased astrocyte GS may represent an important mechanism whereby neurons are protected from the deleterious effects of excess glutamate in extracellular fluid during the postischemic period 26. Recently, Sher and H u 34 demonstrated that increased GS activity and glutamate uptake in neuronal-astrocytic cultures exposed to chronic hypoxia directly correlated with improved neuronal viability. Astrocytes are closely associated with neurons and neuronal processes and thus are in a prime location to quickly respond to changes in the neuronal environment. Previous studies from our laboratory 25 have shown proliferation of intracellular organelles within 1-3 h after ischemia and it was suggested that metabolic activity of these cells is enhanced. The present finding of increases in GS immunoreactivity and GS enzyme activity supports this previous hypothesis.
Acknowledgements. The authors thank Mrs. Kathy Pike, Mrs. Helen Hanes and Miss Rachel Tucker for expert technical assistance, and Mrs. Geraldine Winfrey for secretarial help. The manuscript review by James C.K. Lai, Ph.D. and William A. Pulsinelli, M.D., Ph.D., and statistical analyses performed by Martin L. Lesser, Ph.D. are gratefully appreciated. This work was supported by grants from the Stroke Council of the American Heart Association, Student Scholarship in Cerebrovascular Disease (M.C.C.) and The National Institute of Health NS003346 (C.K.P.) and DK 16739 (A.J.L.C.).
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275
BRES 17331
Brain glutamine synthetase increases following cerebral ischemia in the rat Carol K. Petito 1, Marilda C. Chung 1, Lena M. Verkhovsky 2 and Arthur J.L. Cooper 2 Department of 1pathology (Neuropathology) and the Departments of 2Biochemistry and Neurology, Cornell University Medical College, New York, NY 10021 (U.S.A.) (Accepted 27 August 1991)
Key words: Glutamine synthetase; Cerebral ischemia; Astrocyte; Hippocampus; Cortex; Striatum
Changes in astrocyte glutamine synthetase (GS) in postischemic rat brain were evaluated and correlated with regional neuronal vulnerability or resistance to ischemia. Rats subjected to 20 or 30 min of cerebral ischemia were allowed to survive for 3 or 24 h after ischemia; normal animals served as controls. Resultant neuronal necrosis was severe in the striatum by 24 h and in the CAI region of the hippocampus at 72 h; neurons in paramedian cortex and CA3 region of the hippocampus were not permanently damaged. Glutamine synthetase (GS) immunocytochemistry was performed on vibratome sections of paraformaldehyde-fixed brains and enzyme activity was assayed in frozen samples of cerebral cortex, striatum and hippocampus. At 3 and 24 h after ischemia, GS immunoreactivity increased and was secondary to enlargement of GS-positive cell bodies and processes as well as to increased numbers of GS-positive astrocytes. Enzyme activity also increased in cortex, striatum and hippocampus at 3 and 24 h (P ~< 0.03). This study shows that increase in astrocyte GS occurs rapidly after ischemia, and prior studies indicate that this increase occurs in parallel with proliferative changes in astrocyte organelles. The results also suggest that astrocyte metabolism of glutamate increases after ischemia. The increased capacity for glutamine synthetase may be important in normalizing extracellular glutamate following ischemia and protecting brain from the neurotoxic effects of this excitatory amino acid. INTRODUCTION
The excitotoxic action of glutamate may be important in initiating brain injury following cerebral ischemia 32. As originally described by Benveniste et al. 1 and Simon et al. 36, extracellular concentrations of glutamate rise during ischemia to potentially neurotoxic levels and blockade of glutamate receptors reduce ischemic brain damage. Extracellular glutamate is normally taken up by astrocytes s'H't2 where it is metabolized to a-ketoglutarate 41 and glutamine 2°. The ischemia-related increase in glutamate may enhance astrocyte uptake or metabolism of this amino acid which could potentially protect brain from its excitotoxicity. Accordingly, we studied the a m o u n t and the activity of astrocyte GS in postischemic rat brain. This enzyme is preferentially localized in astrocytes in the CNS 18'2°, where it catalyzes the conversion of glutamate to glutamine. Transient cerebral ischemia was produced in rats by temporary occlusion of the carotid and vertebral arteries followed by reperfusion for 3 and 24 h. This insult results in severe neuronal necrosis in the dorsolateral striatum by 24 h and in the CA1 region of the hippocampus by 3 days, but leaves neurons in the paramedian
cerebral cortex and CA3 region of the hippocampus without p e r m a n e n t injury29. Preliminary accounts of the present study have been published previously26'28. MATERIALS AND METHODS
Animal preparation Cerebral ischemia was produced in male Wistar rats weighing 225-250 g using the modified 4-vessel occlusion method of Pulsinelli and Brierley3°. Under Fluothane anesthesia, the vertebral arteries were occluded by electrocautery and the common carotid arteries were isolated and surrounded by loose clasps. The animals were fasted overnight and, on the following day, the common carotid artery clasps were tightened to occlude carotid flow. This produces a reduction in the blood flow to forebrain structures to less than 3% of control, provided the unanesthetized animals remain unconscious and demonstrate bilateral loss of their normal righting reflex throughout the ischemic period 31. After 20 or 30 min, the carotid clasps were released and the animals were allowed to survive for 3 or 24 h. Normal, unoperated animals served as controls. For immunohistochemistry, animals were deeply anesthetized with intraperitoneal urethane and sequentially perfused-fixed through the ascending aorta with: (1) 0.1 M sodium phosphate (NaPO4) buffer, pH 7.4, for 30 s; (2) 4% (w/v) paraformaldehyde in 0,1 M NaPO4 buffer, pH 7.4, for 15 min; and (3) 0.1 M NaPO 4 buffer, pH 7.4, for 5 min. Brains were removed and placed into 0.1 M NaPO4 buffer overnight. Coronal sections at the level of the anterior commissure and at the level of the mid-dorsal hippocampus were cut 40 /~m thick with a vibratome. The adjacent coronal sections were dehydrated, embedded in paraffin, cut 7/~m thick, and stained with hematoxylin-eosin or Luxol-Fast blue-Cresyl vi-
Correspondence: C.K. Petito, Department of Pathology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, U.S.A.
276 olet. Animals used for biochemical studies were anesthetized with Fluothane, decapitated and the brains were rapidly removed. The paramedian cerebral cortex, anterior striatum and mid-dorsal hippocampus were dissected and quickly frozen at -70°C.
total protein content was determined by the method of Lowry et al? 7, using bovine serum albumin as a standard. Glutamine synthetase activity was expressed as ~mol/h/mg of protein.
lmmunohistochemistry
RESULTS
Glutamine synthetase immunochemistry was performed with a polyclonal rabbit anti-glutamine synthetase antibody (gift of Dr. Michael D. Norenberg) using the indirect antibody method, originally employed by Norenberg et al.2°. as well as the avidin-biotin complex (ABC) technique of Hsu et a1.13. The indirect antibody technique employed 40-/~m-thickvibratome pieces of cortex and striatum from animals subjected to 30 min ischemia and sacrificed at 3 and 24 h after ischemia (n = 4 in controls and in the 2 experimental groups). The sections were incubated for 1 h at 27°C with rabbit anti-GS antiserum (1:100 dilution), washed x3 with phosphate-buffered saline (PBS), and incubated for 60 min with peroxidase-conjugated goat anti-rabbit IgG (Boehringer-Mannheim Biochemicals). Horseradish peroxidase reaction product (HRP) was developed by incubation of the sections with HzO2 and 3,3"-diaminobenzidine according to the directions of the manufacturer. Substitution of primary antiserum with PBS served as a negative control. The sections were then postfixed in 1% (w/v) osmium tetroxide, dehydrated through graded alcohol, placed in epon and fiat-embedded between 2 plastic sheets, and lightly stained with 1% (w/v) Toluidine blue. Ultrathin sections were prepared and examined (either unstained or stained with uranyl acetate and lead citrate) under a JOEL 100S electron microscope. The numbers of GS-positive astrocytes and of capillaries surrounded by GS-positive astrocytes were counted in 3-5 1-~m plasticembedded sections of control animals, and at 3 to 24 h after ischemia. The results were expressed as number of GS-positive astrocytes or capillaries per 0.1 mm2. A digitizing pad (Summagraphics. Inc.) interfaced with a Macintosh IIcx computer was used to determine the area of the unstained one/~m thick section. The specimens were evaluated without knowledge of the experimental conditions. With the ABC technique, 40/.tm thick coronal sections of hippocampus, striatum and cortex were cut from controls (n = 4) and from animals subjected to 20 rain of ischemia followed by reperfusion intervals of 3 h (n = 4), 24 h (n = 5) and 48 h (n = 7). The sections were sequentially incubated with normal horse serum, 1:10 for 1 h; rabbit anti-GS IgG, 1:3200 overnight; and horse anti-rabbit IgG, I:100 for 1 h. The avidin-biotin complex, and subsequent incubation with H202 and 3,3"-diaminobenzidine was performed according to the directions of the manufacturer (Vector Labs, Burlingame, CA).
Glutarnine synthetase activity Dissected frozen brain samples were obtained from controls (n = 10) and animals subjected to 20 min of ischemia followed by reperfusion intervals of 3 h (n = 8), and 24 h (n = 6). The specimens were homogenized in 10 vols (w/v) of ice-cold 0.15 M KC1 using a glass-to-glass homogenizer. The homogenates were frozen, thawed the next day and assayed. It should be noted that subsequent freeze-thawing resulted in some loss of glutamine synthetase activity (personal observations). The glutamine synthetase assay was modified from that of Pamiljans et al.12; 100/~1 of homogenate was incubated for 15 rain at 37°C in 0.2 ml of a reaction mixture containing 10 mM MgCI2, 50 mM L-glutamate, 100 mM imidazoleHCI buffer (pH 7.4), 10 mM 2-mercaptoethanol, 50 mM hydroxylamine-HCl (previously adjusted to pH 7.4 with 3 M sodium hydroxide) and 10 mM ATE The reaction was terminated by addition of 0.8 ml of a solution containing 0.37 M ferric chloride, 0.67 M HC1 and 0.2 M trichloroacetic acid. The precipitated protein was removed by centrifugation. After incubation for 15 min at room temperature, the absorbence of the supernate was read at 530 nm and compared to the absorbenee generated by standard quantities of v-glutamylhydroxamate (Sigma) treated with the ferric chloride reagent. The controls contained homogenate but lacked ATP. The
lmmunochemistry The indirect antibody technique and the A B C technique both d e m o n s t r a t e d glutamine synthetase i m m u n o reactivity that was confined to astrocyte cell bodies and processes. Successful immunohistochemical demonstration of GS was d e p e n d e n t not only upon length of time in fixative2°, but also on the interval between sacrifice and the immunohistochemical procedure. In normal rat brain, astrocytes had thin rims of GSpositive cytoplasm surrounding the nuclei and fine delicately branching GS-positive cell processes and perivascular processes (Fig. la). The GS-positive astrocytes were most frequent in the subpial cortex and hippocampus, and infrequent in the white matter structures, including the c e n t r u m semi-ovale a n d white matter bundles of the corpus striatum. Astrocytes in the s t n a t u m contained more reaction product than those in the cerebral cortex using the indirect method, but stained poorly or not at all in the 40-/zm thick sections prepared with the ABC technique. Electron microscopy of stained and unstained ultrathin sections revealed small amounts of electron-dense H R P reaction product in astrocyte cell bodies and processes (Fig. 2a). The n u m b e r , size and staining intensity of GS-positive astrocytes and astrocytic processes increased at 3 h after 20 or 30 min of cerebral ischemia (Fig. tb). These changes occurred both in cortex and in striatum, and were observed in the plastic-embedded sections by light and electron microscopy, as well as in the 40-ktm sections prepared with the A B C technique. Electron microscopy demonstrated enlarged astrocyte cell bodies and processes which were filled with a b u n d a n t amounts of electron dense reaction product (Fig. 2b). Focal regions of striatum contained shrunken neurons whose cytoplasm and nucleus were diffusely stained with H R P reaction product which represented non-specific staining of necrotic cells. E n l a r g e m e n t of the GS-positive cell bodies and processes was less p r o m i n e n t at 24 h than at 3 h postischemia (Fig. lc), but still persisted in comparison with controls, especially in the perivascular astrocyte foot processes. Electron microscopy again showed an astrocytic localization of the H R P reaction product in both regions. In the cerebral cortex, the average n u m b e r of capillaries surrounded by GS-positive processes and of astrocytes per/~m 2 was 7.4 and 10.9 in controls. 45.8 and 25.3 per p m 2 at 3 h and 25.3 and 19.0 per ,um2 at 24 h In the
277 striatum, the number of GS-positive capillaries and astrocytes per ~m 2 was 11.5 and 4.3 in controls, 27.8 and 21.7 at 3 h after ischemia and 21.7 and 20.7 at 24 h after ischemia. While repeated measure analyses of variance failed to show significant differences according to astrocytes vs capillaries or time in either site, small or moderate differences would likely go undetected since the sample sizes were small and the standard deviations were high. The number of GS-positive astrocytes and their staining intensity was also estimated blindly in the 40-~m sections, prepared with the ABC technique. In controls, mild or no staining was present in the striatum and hippocampus in all 5 animals; and in the cortex in 4 of 5 animals. One had moderate staining in cortical astrocytes. At 3 h after ischemia, moderate to marked staining was present in the cortex in 3 of 4 animals; in the striatum in 2 of 4; and in the hippocampus in 1 of 4. At 24 and 48 h after ischemia, moderate to marked staining was observed in 6 of 12 animals in the cortex, 5 of 12 in the striatum and 5 of 12 in the hippocampus. While the results among groups are variable, they do show increases in GS immunoreactivity during the postischemic period.
Glutamine synthetase activity In control animals, GS activity (~mol/h/mg protein) was 0.45 + 0.13 in the hippocampus, 0.45 -+ 0.07 in the paramedian cerebral cortex and 0.29 -+ 0.04 in the striatum, values similar to that reported in previous studies of GS activity in normal rat brain TM. Glutamine synthetase activity significantly increased at 3 and at 24 h following cerebral ischemia in all 3 brain regions examined (Table I). Repeated measure of analyses of variance, using brain regions as the within factor and time as the between factor, showed no significant differences in glutamine synthetase activity across brain regions. However, a significant difference across time (P < 0.03) was noted. Multiple comparisons using the Student/NewmanKeuls method, showed that glutamine synthetase activity in controls differed from that of each of the postischemic intervals of 3 and 24 h, but that the two postischemic time points did not differ from each other.
Fig. 1. Glutamine synthetase (GS) immunohistochemistry in 1 micron-thick plastic-embedded sections of striatum. Perivascular astrocyte processes (short arrow) and astrocyte cell bodies (long arrow) contain no or small amounts of GS immunoreactivity in controls (a) but increased amounts at 3 h (b) and at 24 h (c) after 30 min ischemia. Toluidine blue; bar = 25 ~m.
278
Fig. 2. Glutamine synthetase immunoreactivity in astrocyte cell body is sparse in controls (a) but increased in the cell bodies and pericapillary foot processes at 3 h after ischemia (b). Bar = 5 #m.
DISCUSSION TABLE I Glutamine synthetase activity (l~mol/h/mg protein) in different regions of the brain 3 and 24 h after an ischemia insult
Cortex Striatum Hippoeampus
Control (n)
3 h (n)*
24 h (n)*
0.45_+0.07(10) 0.29_+0.04( 9 ) 0.45+_0.13 (10)
0.72_+0.14 (8) 0.53_+0.13 (8) 0.48+_0.06 (8)
0.60+0.10 (6) 0.46+0.07 (6) 0.70_+0.16(6)
* As a group the GS activity at both 3 and 24 h is different from the controls with P ~< 0.03; Student/Newraan-Keuls method. Numbers in parentheses refer to total number of animals in each group.
G l u t a m i n e synthetase in brain is largely confined to astrocytes 2° where it plays a k e y rote in m e t a b o l i s m of extracellular glutamate and N H 4 - ions 5. The distribution of astrocyte G S in vivo 14'2° and its expression following exposure to h o r m o n e s or second messengers in vitro 14"23 is h e t e r o g e n e o u s , and m a y correlate with glutamatergic neuronal activity. M o d u l a t i o n of glutamine synthetase in vivo has b e e n described in a few e x p e r i m e n t a l models. G l u t a m i n e synthetase m R N A increases with acute hepatic e n c e p h a l o p a t h y 38, although GS enzyme activity and immunoreactivity is u n a l t e r e d 3'~'19 or decreased 2 with
279 chronic liver disease. Brain trauma causes a rapid, transient increase in GS immunoreactivity 19 whereas GS activity declines following hyperoxygenation 33. The postischemic increases in GS found in the present study are at variance with a previous report that examined the effects of ischemia on GS activity in vivo. In that study, cortical GS activity was decreased at 3 h following 10 min of bilateral common carotid artery occlusion in the gerbil and returned to normal levels at 24 h 2a. Several explanations may account for this apparent contradiction. Cortical damage is mild or moderate following 20-30 min ischemia in the rat model 29, but is absent or minimal following 10 min ischemia in the gerbil model 1°. The magnitude and duration of elevated extracellular glutamate 9'35 as well as the degree of reactive astrocytosis t°'25'27 are proportional to the severity and duration of the ischemia and the extent of neuronal damage. Although these differences might account for enhanced GS activity following 30 min but not 10 min of cerebral ischemia, they fail to explain the reduction in enzyme activity found in the gerbil study. Furthermore, if free radical oxidative inactivation of GS occurs after ischemia 21'33, it is not clear why GS reduction did not occur with our model in which free radicals are thought to be produced during the early postischemic period 4°. The possibility that the different assay methods used to measure GS activity caused the apparent discrepancy in postischemic GS activity cannot be ruled out. The v-glutamyltransferase reaction used in the gerbil study 21 is much more sensitive than the synthetase reaction used in the rat study. However, there may be pitfalls associated with its u s e 4 which may result in an artifactual decrease in enzyme activity. The mechanisms underlying the rapid increase in astrocyte glutamine synthetase are unknown but include several possibilities. Firstly, an actual increase in GS protein content could occur by: (1) increased translation or stabilization of its mRNA; (2) increased transcription; and (3) impaired protein degradation. Although total protein synthesis is decreased in postischemic brain 7'15, selective synthesis of certain proteins such as ornithine decarboxylase 7'16 can occur within minutes. Early proliferative changes within astrocytes, including increases in rough endoplasmic reticulum and mitochondria 25, indicate a potential for enhanced protein synthesis. Secondly, posttranslational modification of glutamine synthetase protein may take place during reperfusion. This could expose more antigenic sites to the applied antisera and induce a higher level of enzyme activity. Increases in GS may be due to its induction within glial cells that, in normal brain, possess little GS activity. Such candidates include oligodendroglia and perineuronal glia. Oligodendrocytes recently have been shown
to contain GS although not in as high a quantity as astrocytes 6'37. Perineuronal glia, which have the morphological characteristics of medium-light oligodendrocytes by electron microscopy, are transformed into reactive astrocytes within 1 to 3 h after cerebral ischemia 24. Studies are in progress to determine whether they acquire GS immunoreactivity after ischemia, and at what time point the acquisition occurs. The time course of increased GS correlates well with the onset of reactive astrocytosis in the 4VO model of cerebral ischemia. Although elevations in glial fibrillary acidic protein are delayed for 1-3 days after ischemia in this 27 as well as in other models 1°, increases in astrocyte mitochondria and rough endoplasmic reticulum occur within 3 h 25. The present study does not determine whether or not the increased GS is a specific astrocytic response to ischemia or is part of the early proliferative response of postischemic astrocytes. The postischemic increases in astrocyte glutamine synthetase suggest that the capacity to convert glutamate to glutamine increases following ischemia and that this enhancement may be due to a rise in extracellular glutamate. GS activity of astrocytes in vitro increases with increasing elevations of extracellular glutamate 39. Increases in GS also may indicate that astrocyte uptake of glutamate increases after ischemia, a finding recently shown to occur when astrocytes are exposed to hypoxia 34. Increased astrocyte GS may represent an important mechanism whereby neurons are protected from the deleterious effects of excess glutamate in extracellular fluid during the postischemic period 26. Recently, Sher and H u 34 demonstrated that increased GS activity and glutamate uptake in neuronal-astrocytic cultures exposed to chronic hypoxia directly correlated with improved neuronal viability. Astrocytes are closely associated with neurons and neuronal processes and thus are in a prime location to quickly respond to changes in the neuronal environment. Previous studies from our laboratory 25 have shown proliferation of intracellular organelles within 1-3 h after ischemia and it was suggested that metabolic activity of these cells is enhanced. The present finding of increases in GS immunoreactivity and GS enzyme activity supports this previous hypothesis.
Acknowledgements. The authors thank Mrs. Kathy Pike, Mrs. Helen Hanes and Miss Rachel Tucker for expert technical assistance, and Mrs. Geraldine Winfrey for secretarial help. The manuscript review by James C.K. Lai, Ph.D. and William A. Pulsinelli, M.D., Ph.D., and statistical analyses performed by Martin L. Lesser, Ph.D. are gratefully appreciated. This work was supported by grants from the Stroke Council of the American Heart Association, Student Scholarship in Cerebrovascular Disease (M.C.C.) and The National Institute of Health NS003346 (C.K.P.) and DK 16739 (A.J.L.C.).
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