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(Stroke. 1995;26:290-297.)
© 1995 American Heart Association, Inc.

Articles

Neuron-Specific Enolase in Gerbil Brain and Serum After Transient Cerebral Ischemia

Markus Horn, MD; Florian Seger, MD Wolfgang Schlote, MD

From the Institute of Neurology (Edinger-Institut), University of Frankfurt/Main (M.H., W.S.), and the Department of Neurosurgery, University of Würzburg (F.S.) (Germany).

Correspondence to M. Horn, MD, Department of Neurology, University of Heidelberg, Im Neuenheimer Feld 400, D-69120 Heidelberg, FRG.

	Abstract

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Background and Purpose The sensitivity and validity of serum neuron-specific enolase as a marker of brain injury were tested after global cerebral ischemia.

Methods Sixty-nine Mongolian gerbils were perfusion fixed after variable reperfusion after 5-minute (group 1) or 15-minute (group 2) bilateral carotid occlusion. Neuron-specific enolase was analyzed by an enzyme immunoassay in serum of control, sham-operated, and ischemic animals before euthanasia and in nonischemic gerbil brains. Brains were processed for histology, immunohistochemistry, and morphometric evaluation of ischemic neuronal damage.

Results After cerebral ischemia, loss of neuronal immunoreactivity was closely associated with increased neuron-specific enolase serum levels, which were significantly elevated by 24 hours (group 1) or by 4 hours (group 2) of reperfusion (P<.001). Response of serum levels depended on the duration of preceding ischemia, and maximum concentrations were approximately 3-fold (group 1) or 20-fold (group 2) those of nonischemic control. Morphological damage became apparent 48 hours (group 1) or 12 hours (group 2) after ischemia, as indicated by histological and morphometric data.

Conclusions Significantly elevated neuron-specific enolase serum levels could be demonstrated as a consequence of ischemia-induced cytoplasmic loss of neuron-specific enolase in central nervous system neurons, corresponded quantitatively to the severity of cerebral ischemia, and were detectable before irreversible neuronal injury. Therefore, analysis of serum neuron-specific enolase is suggested to be both a valuable diagnostic tool in clinical management of the initial stages of global cerebral ischemia and a prognostic parameter during the postischemic course.

Key Words: cerebral ischemia • neuronal death • neuron-specific enolase • gerbils

	Introduction

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Brief forebrain ischemia in rats and gerbils results in delayed neuronal death in the hippocampal CA1 subfield if postischemic reperfusion exceeds 48 hours.^{1 2 3 4} Nevertheless, CA1 neurons show temporary recovery of their metabolic and electrophysiological activity and appear morphologically well preserved during the first days of reperfusion until necrosis is completed 72 to 96 hours after ischemia.^{5 6} Recent experimental studies on cerebral ischemia suggest that the ultimate fate of selectively vulnerable CA1 pyramidal cells is closely related to initial postischemic reperfusion.^{7 8} Meanwhile, there is also increasing evidence for the occurrence of delayed neuronal death in the human brain.^{9 10} The most important clinical implication of these findings is the possibility of a therapeutic window for postischemic treatment of the brain if early diagnosis can be achieved.^{7 8 11} For this reason, early detectors of ischemic neuronal damage are urgently needed in the clinical field. Concerning focal brain ischemia, computed tomographic and newer magnetic resonance imaging techniques are capable of obtaining positive findings in up to 95% of patients with middle cerebral artery occlusion within the first 1 to 6 hours after onset of neurological symptoms.^{12 13 14} In contrast, in global ischemia neuroradiological diagnostics usually fail to demonstrate brain damage within that time period.

Neuron-specific enolase (NSE) represents the ,-dimer of the protein enolase (2-phospho-D-glycerate hydrolase), which is a soluble enzyme of the glycolytic pathway with a total molecular weight of approximately 80 000 D.¹⁵ Physiologically, NSE is specifically present in neuronal cytoplasm and dendrites and in cells of the amine precursor uptake and decarboxylation (APUD) cell system.¹⁶ In addition, tumor cells in APUDomas, neuroblastomas, seminomas, and small-cell carcinoma of the lung are capable of producing NSE, usually accompanied by elevated NSE serum titers.^{17 18} For this reason, NSE has been established as a diagnostic and prognostic serum marker in clinical management of such neoplasms. Furthermore, early clinical studies are available demonstrating elevated serum NSE titers in stroke or cardiac arrest patients.^{19 20 21 22} Accordingly, variations in neuronal anti-NSE immunostaining in both rats and gerbils after focal cerebral ischemia^{23 24} and in humans resuscitated from cardiac arrest¹⁰ have been reported. Previously, studies on rats subjected to four-vessel occlusion have shown intracellular compounds, especially NSE, to accumulate in the cerebrospinal fluid (CSF) during recirculation.²⁵ However, controlled experimental studies on elevated NSE serum titers as a result of global brain ischemia are not available at this time.

The objective of this study was to elucidate the temporal profile of hypothesized changes in NSE serum levels and of variations in NSE immunoreactivity of hippocampal and neocortical neurons and their relation to the manifestation of ischemic neuronal death in the gerbil after transient forebrain ischemia.

	Materials and Methods

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Surgical Procedures
Sixty-nine adult Mongolian gerbils (weight, 50 to 70 g; source, Max Planck Institute for Brain Research, Frankfurt/Main, FRG) were used to study the effects of mild and severe forebrain ischemia. Before the experiments animals had free access to food and water. After premedication with atropine 1 µg SC, anesthesia was induced with 3% halothane inhalation and then maintained with 1.5% halothane in a gas mixture of 70% nitrogen and 30% oxygen. Rectal temperature was monitored continuously throughout the experiment and was kept close to physiological values (37.5°C) with a feedback-controlled heating system. Both common carotid arteries were exposed by a midline cervical incision and occluded with Biemer clips (FD 562, Aesculap). Anesthesia was discontinued as soon as the clips were placed. After 5 or 15 minutes, brains were recirculated spontaneously by removal of the clips. An additional 12 gerbils were sham operated, ie, their common carotid arteries were not occluded, and they served as histological and serological controls. The skin incision was sutured, and the animals were observed continuously concerning their postoperative neurological behavior during the first hour of recirculation. Afterward they were replaced in their cages and remained there until they were killed.

Histological Preparation, Morphometric Evaluation, and NSE Immunohistochemistry
After 4-, 8-, 12-, 24-, 48-, 72-, or 96-hour survival, gerbils were reanesthetized with pentobarbital (100 mg/kg IP) and fixed by transcardiac perfusion with 2% paraformaldehyde/0.2% picric acid in 0.1 mol/L acetate buffer (pH 6.0) at a pressure of 120 mm Hg.²⁴ Each gerbil was coded at random and afterward investigated only by its code number. Subsequent to removal and postfixation of the brains, material was paraffin embedded. Coronal sections (7 µm) were prepared at a level 1.4 to 1.7 mm posterior to the bregma and stained with hematoxylin and eosin or cresyl violet. At high-power light microscopy, the number of unaffected hippocampal neurons in the central portion of subfield CA1 (CA1b) and in the medial portion of CA3 (CA3c) was evaluated by the aid of morphometric equipment (VIDEOPLAN, Zeiss-Kontron). Only neurons showing normal nuclear morphology and intact nucleoli were included,²⁶ and neuronal density was expressed as cells per millimeter linear length of stratum pyramidale. Histopathologic analysis also included the overlying cerebral cortex. Immunohistochemistry was performed by means of a monoclonal antibody raised against ,-NSE (clone BBS/NC/VI-H14; Camon) and an avidin-biotin complex technique (Camon). Slices were counterstained with hemalum.

Serum NSE Detection
During general anesthesia as described above, blood samples were taken by transdiaphragmatic puncture of the right cardiac ventricle in normal controls (n=20) of identical age and body weight and derived from the same strain of gerbils as used for experimental cerebral ischemia, and in both sham-operated and postischemic gerbils before perfusion fixation. Serum NSE was measured by means of an enzyme immunoassay (EIA) based on the sandwich technique including a solid-phase monoclonal antibody raised against ,-NSE (clone 18E5; Cobas Core NSE EIA, Roche Diagnostica). NSE detection by EIA is a highly reliable method and has been shown to be superior to radioimmunoassay in regard to practicability, time spent, and disposal.¹⁸

Authenticity of Applied Antibodies
The specificity and sensitivity of monoclonal antibodies used for immunohistochemical and serological detection of NSE were tested by Western blot analysis according to the following protocol. Normal gerbil brain was taken from a control animal, and immediately after removal it was frozen in liquid nitrogen. After homogenization in sucrose buffer (6x800 rpm potter), the brain homogenate was centrifuged repeatedly (10 minutes at 1000g; 20 minutes at 10 000g) to remove nonsolubilized material. The resulting supernate, representing the cytosolic fraction of gerbil brain, and normal gerbil serum were applied to a 10% polyacrylamide gel. Proteins were separated by electrophoresis and transferred to a nitrocellulose membrane. After blocking of nonspecific binding sites, blots were incubated in primary anti-NSE antibodies (BBS/NC/VI-H14 or 18E5) and afterward in horseradish peroxidase–labeled conjugate. Immunoreaction was detected by enhanced chemiluminescence (ECL) and exposure to blue light–sensitive autoradiography film (ECL, Amersham Life Science). Results in brain and serum demonstrate specific binding of both applied primary antibodies to a protein located near 40 kD representing the monomeric -subunit of enolase¹⁵ (Fig 1).

View larger version (88K): [in this window] [in a new window]   Figure 1. Polyacrylamide gel electrophoresis performed on cytosolic fraction of normal gerbil brain (lane 1) and normal gerbil serum (lane 2) and Western blots using monoclonal anti–neuron-specific enolase antibodies 18E5 (lane 3, brain supernate; lane 4, serum) and BBS/NC/VI-H14 (lane 5, brain supernate; lane 6, serum). The gel was stained with Coomassie blue; Western blots were detected by enhanced chemiluminescence and exposure to blue light–sensitive autoradiography film. Results in brain and serum samples demonstrate specific binding of both antibodies to a protein located near 40 kD representing the monomeric -subunit of enolase. Note that in the analyzed serum sample neuron-specific enolase was present only in a concentration of 6.07 ng/mL. LMW indicates low-molecular-weight marker; lanes 1, 3, 5, supernate of centrifuged normal gerbil brain homogenate; and lanes 2, 4, 6, normal gerbil serum.

Statistical Analysis
Statistical analysis of NSE serum measurements and of morphometric quantification of ischemic neuronal damage in hippocampal CA1 and CA3 was performed by means of the two-sample Wilcoxon test. In addition, two-sided tests with Bonferroni correction were performed. Values are expressed as mean±SD.

	Results

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General Postoperative Outcome
One gerbil subjected to 5 minutes and two gerbils subjected to 15 minutes of bilateral carotid occlusion (BCO) became asystolic during removal of the clips and died of irreversible cardiocirculatory arrest. Another gerbil of the 15-minute ischemia group recovered initially but then died 2 days after recirculation, after showing signs of acute pneumonia. Results from these animals were excluded from the analysis. The remaining 65 gerbils survived without showing gross neurological deficits except for transient motor hyperactivity during the initial postischemic period, and they were killed at predetermined postischemic intervals. All 12 sham-operated controls recovered from anesthesia within a few minutes and afterward survived until they were fixed by transcardiac perfusion.

Histopathologic Findings During Postischemic Reperfusion
Brains of sham-operated gerbils revealed absence of ischemic cell changes and of so-called dark neurons known to be artificially produced as a result of removal of the incompletely fixed brain from the skull. Capillary lumina appeared devoid of blood cells and of regular caliber, thereby indicating adequate perfusion fixation.

After 5-minute BCO during postischemic reperfusion for up to 96 hours, neuronal damage was never visualized outside the hippocampus. Changes in the morphology of hippocampal neurons became evident only in subfield CA1 by 48-hour reperfusion. Afterward, progressive neuronal cell death took place, involving almost all CA1 pyramidal cells. In contrast, neurons of the remaining hippocampus were morphologically well preserved throughout the 4-day reperfusion interval.

After 15-minute BCO, extended and severe changes in neuronal cell morphology were detected in supragranular cortical layers (3 and 4) and in layers 5 and 6 by 8 hours and by 12 hours of reperfusion. At 24 hours after ischemia, major parts of cortical layers 3, 5, and 6 showed irreversible cell necrosis, but only slight additional changes were noted by 4 days of reperfusion. Within the dorsal hippocampus, ischemic cell change involving the major portion of subfield CA1 was frequent after 24 hours of reperfusion, while in subfield CA2 and the border zone between CA3 and CA4, definite necrosis was detectable at the same time. By 48 hours of reperfusion, subfield CA2 exhibited extended ischemic cell necrosis with few only slightly changed neurons left, accompanied by prominent microglial reaction. Between 48 and 96 hours after ischemia, delayed neuronal death extending to the whole CA1 pyramidal stratum occurred. In subfield CA3 and in the dentate gyrus, the ischemic lesion that finally resulted consisted of a small number of necrotic neurons distributed in a patchy pattern.

Morphometric Evaluation of Ischemic Neuronal Damage in Hippocampal Subfields CA1 and CA3
In brains of sham-operated gerbils, neuronal cell density was 223.4±3.8/mm in CA1 and 278.4±4.3/mm in CA3 (Table 1). After 5-minute BCO, the number of unaffected CA1 neurons diminished slightly during the first and second postischemic days and thereafter was reduced rapidly between day 2 and day 4 after ischemia (Fig 2, top panel). A significant reduction was observed by 48 hours of reperfusion (P<.001) in CA1, whereas CA3 cell density was nearly unchanged by 96 hours after ischemia. Forebrain ischemia lasting for 15 minutes resulted in more progressive but also delayed neuronal death of CA1 cells with subtotal destruction of the CA1 cell layer, and neuronal density was significantly reduced after 24 hours of reperfusion (P<.001) (Fig 2, bottom panel). In subfield CA3, cell density rapidly diminished between 12 and 48 hours after ischemia (P<.001).

View this table: [in this window] [in a new window]   Table 1. Number of Morphologically Affected Neurons per 1 mm Linear Length of Stratum Pyramidale of the Hippocampal Subfields CA1 and CA3 in Sham-Operated Gerbils and Gerbils Subjected to Mild (5-Minute) or Severe (15-Minute) Forebrain Ischemia

View larger version (91K): [in this window] [in a new window]   Figure 2. Schematic diagrams show changes in neuronal cell density in hippocampal subfields CA1 and CA3 after 5-minute (top) or 15-minute (bottom) ischemia as defined by morphometric quantification.

NSE Immunohistochemistry
In hippocampal specimens derived from sham-operated gerbils, neuronal cytoplasm of hippocampal cells in the stratum pyramidale and their cell processes within the stratum radiatum showed strong immunostaining for NSE, whereas immunoreaction was weak with surrounding neuropil (Fig 3). Similar results were obtained in the cerebral cortex.

View larger version (0K): [in this window] [in a new window]   Figure 3. Photomicrographs show anti–neuron-specific enolase (NSE) immunostaining of hippocampal subfield CA1 taken from a sham-operated control (A) and from gerbils subjected to 15-minute forebrain ischemia and killed after reperfusion intervals of 8 (B), 24 (C), 48 (D), or 96 (E) hours. Anti-NSE immunostaining detects progressive loss for NSE in CA1 neurons from 8 to 48 hours of postischemic survival. Note absence of immunoreactivity even in morphologically preserved neurons at 24 or 48 hours after ischemia (arrows). Photomicrograph taken at 96-hour survival shows final manifestation of the ischemic lesion consisting of total destruction of CA1 pyramidal stratum and provides evidence for strong microglial reaction. Avidin-biotin complex technique, counterstaining with hemalum; bar=50 µm.

After 5-minute BCO, regular NSE immunostaining was observed at 8 and 12 hours after ischemia. By 24 hours of reperfusion, a considerable number of faintly staining neurons were visualized, diffusively distributed through all layers of the cerebral cortex and in all hippocampal subfields except for the dentate gyrus. Subsequently, progressive diminution of cytoplasmic NSE immunoreactivity became evident in CA1 neurons, and after 48 hours of reperfusion the entire CA1 region remained negative. Recovery of regular immunolabeling was noted between 48 and 96 hours after ischemia in subfields CA2, CA3, and CA4 and in the neocortex.

After 15-minute BCO, immunohistochemistry indicated considerable loss of cytoplasmic NSE immunoreactivity in single, morphologically well-preserved neocortical neurons and in both CA1 and CA4 pyramidal cells by 8 hours of reperfusion (Fig 3). With advancing survival, the number of affected and unaffected neurons staining weak and finally negative for NSE increased in all brain areas under consideration, and by 48-hour survival the whole CA1 subfield revealed complete loss of immunoreactivity, including residual neurons, which displayed normal morphology. Because of the manifestation of delayed neuronal death between 48 and 96 hours after ischemia, the final stage of the ischemic lesion consisted of selective neuronal necrosis of CA1. The remaining cell debris either showed an unspecific faint positive or a negative immunoreaction to NSE antibodies. In contrast to findings in the 5-minute BCO group, restoration of regular immunolabeling was incomplete in the neocortex and in the remaining parts of the hippocampus at 96 hours after ischemia.

Serum NSE Detection
In normal controls and sham-operated gerbils, NSE serum levels detected by EIA ranged from 2.03 to 9.30 ng/mL (mean±SD, 6.16±2.49 ng/mL) (Table 2). In gerbils subjected to 5-minute BCO, levels were significantly elevated by 24-hour postischemic survival (P<.001), and maximum values were approximately threefold those of control. In contrast, after 15-minute BCO levels were significantly different from control at 4 hours after ischemia (P<.001), and a 20-fold increase of NSE serum titers was observed by 48 hours of reperfusion. Both groups showed considerably elevated NSE serum levels even at 96 hours after the ischemic insult (Table 2 and Fig 4).

View this table: [in this window] [in a new window]   Table 2. Neuron-Specific Enolase Levels in Normal Controls, Sham-Operated Gerbils, and Gerbils Subjected to Mild (5-Minute) or Severe (15-Minute) Forebrain Ischemia

View larger version (24K): [in this window] [in a new window]   Figure 4. Schematic diagram shows comparison of changes in neuron-specific enolase (NSE) serum levels during reperfusion in gerbils subjected to 5- or 15-minute forebrain ischemia. After severe ischemia, NSE serum levels were significantly elevated by as early as 4 hours of reperfusion, whereas in mild ischemia NSE serum titers were significantly different from control by 24 hours after the insult.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
NSE is known as a neuronal marker of cell cytoplasm and dendrites in both the rodent and the human brain and physiologically is present only in negligible amounts in the peripheral blood.¹⁵ Average brain levels of NSE are 12 500 ng/mg protein in rats and 16 500 ng/mg protein in humans.¹⁶ Previous immunohistochemical studies have shown early release of NSE by damaged neurons, thereby indicating functional disturbances or structural defects of the plasma membrane due to cerebral ischemia in rats,²³ gerbils,²⁴ and humans.¹⁰ In addition, NSE has been identified as a neurotrophic factor supporting survival of cultured neocortical rat neurons in a dose-dependent manner. Therefore, it was designated as a "neuronal survival factor" in the central nervous system, stressing the particular importance of this protein to the neuronal metabolism.²⁷

Earlier research on experimental cerebral ischemia in gerbils demonstrated that 5-minute BCO damaged selectively vulnerable neurons in the hippocampal CA1 subfield.^{1 2 28} With this fact in mind, in the present study brief forebrain ischemia was thought to be suitable for studying the sensitivity of serum NSE measurement in reflecting ischemic neuronal damage. A second group of gerbils was subjected to 15-minute BCO to investigate whether the extent of serum NSE rise shows any correlation with the magnitude of preceding ischemia. The methodological approach applied included the comparison of data obtained by NSE immunohistochemistry, serum NSE detection by EIA, histopathologic evaluation of ischemic neuronal damage in neocortex and hippocampus, and morphometric measurement of neuronal cell density in the hippocampal subfields CA1 and CA3 at various postischemic survival times. Results indicated statistically significant elevations of NSE serum titers by 24 hours of reperfusion after 5-minute BCO or by 4 hours of reperfusion after 15-minute BCO (P<.001). Gerbils of both experimental groups showed considerable reduction of neuronal NSE immunoreactivity during reperfusion. Cytoplasmic exhaustion for NSE was completed 48 hours after ischemia and therefore distinctly preceded final morphological damage in selectively vulnerable hippocampal CA1 neurons in both experimental groups. Histopathologic findings during reperfusion after brief or severe forebrain ischemia were consistent with previously reported data obtained in similar models of complete cerebral ischemia in the rodent.^{1 2 4 29 30} Morphometric quantification showed a significant reduction of hippocampal CA1 cell density during reperfusion from 48 to 96 hours after ischemia (5-minute BCO) or from 12 to 96 hours after ischemia (15-minute BCO) (P<.001). The different progression of neuronal damage in the two experimental groups obviously was related to a maturation phenomenon of cell injury in CA1, which is known to depend on the severity of preceding ischemia, and to additional damage to CA3 neurons in the 15-minute BCO group.^{4 28}

Impairment of the blood-brain barrier and neuronal membrane dysfunction were previously demonstrated as early consequences of 5- and 15-minute forebrain ischemia and as prerequisites for extravasation and neuronal uptake of serum proteins in the gerbil hippocampus.^{31 32} Conversely, defects of the neuronal plasma membrane due to ischemia cause early release of cytoplasmic NSE into the neuropil, explaining the observation of a marked decrease in neuronal NSE immunostaining during the first postischemic day.²⁴ Accumulation of NSE in the CSF²⁵ and ischemia-induced disruption of the blood-brain barrier⁶ are suggested to result in an increase of NSE serum levels.

Previous evidence also indicates that even brief ischemia is capable of producing severe impairment of protein synthesis in the selectively vulnerable hippocampal subfield CA1.³³ Until 18 hours after ischemia, NSE synthesis also is dramatically reduced in ischemically affected but viable neurons.³⁴ On the other hand, persistent inhibition of protein synthesis precedes irreversible damage in neurons destined to die.^{35 36} The present findings of both a transient postischemic reduction and recovery of NSE immunoreactivity in ischemia-resistant neocortical and hippocampal neurons and of a persistent disappearance of immunolabeling in highly vulnerable CA1 cells are in accord with these previous data. Both diminution of cytoplasmic NSE visualized by immunohistochemistry and elevation of NSE serum levels measured by EIA were demonstrable before necrosis became evident in either the cerebral cortex or the hippocampus, respectively.

Review of the literature revealed several experimental studies on variations of NSE levels in the CSF after global ischemia and in stroke or trauma models.^{25 37 38} Moreover, CSF NSE has also been assayed in humans suffering from stroke, transient ischemic attacks, intracerebral hematoma, subarachnoid hemorrhage, head injury, or after cardiac arrest and in those in coma due to pediatric encephalitis, acute encephalopathy, or Reye's syndrome.^{19 38 39 40 41 42} The essential findings of both experimental and clinical studies are that CSF NSE is a sensitive and reliable indicator of even minor brain injuries⁴³ and shows good correlation with the severity of the resulting lesion, eg, in the relation between stroke and infarct size.³⁷

However, since usually initial CSF samples are available only in a small number of patients, the more pressing demand in the clinical field is to obtain sufficient information by basic and, if necessary, serial serum samples. Accordingly, there are few clinical reports on elevated NSE serum levels in stroke^{19 20 22} and in patients resuscitated from cardiac arrest.^{21 23} In addition, NSE has been reported to increase in the cerebral and systemic circulation after either transient or permanent occlusion of the middle cerebral artery in the rat.²³ Most similar to results from CSF NSE studies, serum NSE is described as a potent indicator of cerebral ischemia with special reference to early prognostic assessment of cardiac arrest victims.

In conclusion, since early indicators of cerebral ischemia are urgently needed to initiate therapeutic intervention without delay, serological detection of NSE may provide a valuable diagnostic and prognostic tool in clinical management of the early stages of cerebral ischemia and during the postischemic course. Recently reported promising clinical findings concerning the relevance of elevated serum NSE titers in various metabolic and inflammatory disorders of the brain could be corroborated by the present experimental data.

	Acknowledgments

 
The authors gratefully acknowledge Dr Kerstin Bode-Greuel (Tropon-Werke, Köln, FRG) and Dr Christiane Kiefert (Max Planck Institute for Brain Research, Frankfurt/Main, FRG) for helpful suggestions concerning management of experimental gerbils, Ursel Rech for technical assistance with histological preparation, and Dr Gerhard Oremek (Department of Clinical Chemistry, University of Frankfurt/Main, FRG) for NSE detection in serum samples. We also wish to express our appreciation to Dr Brooks Ferebee (Institute for Applied Mathematics, University of Frankfurt/Main) for performing statistical analysis and for reading the manuscript. The antibody 18E5 was a generous grant by Dr Andreas Maurer (Hoffmann–La Roche Ltd, Switzerland).

Received April 11, 1994; revision received October 4, 1994; accepted October 25, 1994.

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