acute and persistent suppression of preproenkephalin mrna expression in the striatum following...

Journal ofNrurochrmistr.v Raven Press, Ltd., New York 0 1994 International Society for Neurochemistry

Acute and Persistent Suppression of Preproenkephalin mRNA Expression in the Striatum Following Developmental

Hypoxic-Ischemic Injury

Robert E. Burke, *Steven 0. Franklin, and *Charles E. Intunisi

Department of Neurology, Columbia University, College of Physicians and Surgeons, and *Department of Pharmacology, Cornell University Medical College, New York, New York, U.S.A.

Abstract: The striatum is vulnerable to hypoxic-ischemic injury during development. In a rodent model of perinatal hypoxia-ischemia, it has been shown that striatal neurons are not uniformly vulnerable. Cholinergic neurons and NADPH-diaphorase-positive neurons are relatively spared. However, it is unknown what classes of striatal neurons are relatively sensitive. One of the major classes of striatal neurons uses enkephalin as a neurotransmitter. We have studied the effect of early hypoxic-ischemic injury on this class of neurons using a quantitative solution hybridization assay for preproenkephalin mRNA in con- junction with in situ hybridization. Hypoxia-ischemia results in an early (up to 24 h) decrease in striatal preproenkephalin mRNA, which is shown by in situ hybridization to occur mainly in the dorsal portion of the striatum. By 14 days, whole striatal preproenkephalin mRNA and total enkephalin-containing peptide levels are normal. However, at 14 days, in situ hybridization reveals that regions of complete preproenkephalin mRNA-positive neuron loss remain in the dorsal region. Normal whole striatal levels are due to an up-regulation of preproenkephalin mRNA expression in the ventrolateral region of the injured striatum. Given the important role that the enkephalin-containing striatal efferent projection plays in regulating motor function, its relative loss may be important in the chronic disturbances of motor control observed in brain injury due to developmental hypoxic-ischemic injury. Key Words: St r i at u m- Enkephalins- Hypoxia-ischemia- Solution hybridization-In situ hybridization. J. Neurochem. 62, 1878-1 886 (1 994).

Hypoxic-ischemic (HI) injury to the developing brain is an important, identifiable cause of chronic neurologic disability. The striatum is one of the brain regions most vulnerable to this form of injury in rodent (Rice et al., 1981; Johnston, 1983) and primate models (Myers, 1977) and in human postmortem studies (Friede, 1975). Given the importance of the striatum to the regulation of motor control, its injury is likely to underlie some of the chronic disturbances of motor function that result from early HI injury to the brain. One such disturbance is dystonia, a move-

ment disorder characterized by sustained and twisting involuntary movements and postures, and a common clinical correlate of striatal pathology associated with adverse perinatal events (Carpenter, 1950).

In a rodent model of HI injury during development (Rice et al., 1981), we and others have shown that striatal neurons are not uniformly vulnerable. Both cholinergic (Johnston and Hudson, 1987; Burke and Karanas, 1990) and NADPH-diaphorase-positive neurons (which contain the peptide somatostatin) (Ferriero et al., 1988) are resistant, showing relative increases in their density in the striaturn after HI injury. These same two neuron phenotypes are resistant to excitotoxic injury mediated by the NMDA receptor in vitro (Koh and Choi, 1988), and in vivo (Beal et al., 1989), and these observations support the concept that HI injury is mediated by endogenous excitatory amino acids acting at an NMDA receptor.

There have been no data available on what striatal neuronal phenotypes are relatively sensitive to developmental HI injury. The most abundant morphologic type is the medium-sized spiny neuron, which makes up >95% of striatal neurons (Graybiel and Ragsdale, 1983). There are two major phenotypes within this group, i.e., enkephalin/GABA-containing neurons that form projections to the lateral globus pallidus (Graybiel, 1 990) and substance P/GABA- containing neurons that fom projections to the medial globus pallidus (or entopeduncular nucleus in rodent) and the substantia nigra pars reticulata (Gray- biel, 1990). Examination of Nissl-stained sections of

Received July 8, 1993; revised manuscript received September 13, 1993; accepted September 20, 1993.

Address correspondence and reprint requests to Dr. R. E. Burke at Box 67, Neurological Institute, 710 West 168th Street, New York, NY 10032, U.S.A.

Abbreviafions used: BSA, bovine serum albumin; EC, enkephalin-containing; HD, Huntington’s disease; HI, hypoxia-ischemia; NEM, N-ethylmorpholine, PPE, preproenkephalin; QA, quinolinic acid RIA, radioimmunoassay; SSC, saline-sodium citrate; TCA, trichloroacetic acid.

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STRIATAL PPE mRNA FOLLOWING HI INJURY 1879

the striatum weeks after HI injury at postnatal day 7 demonstrates loss of medium-sized neurons (Burke and Karanas, 1990). After ischemic injury in the adult rat, biochemical analysis of the striatum demonstrated a persistent depression in the activity of striatal glutamic acid decarboxylase, suggesting a loss of GABA-containing neurons (Francis and Pulsinelli, 1982). Similarly, Chesselet et al. ( 1990) demonstrated, with morphologic techniques in an adult ger- bil model of ischemia, a massive loss of striatal enkephalin- and substance P-containing neurons in the presence of relative sparing of somatostatinergic and cholinergic interneurons. However, biochemical studies of the striatum after HI injury in the immature animal have thus far not demonstrated evidence for loss of enkephalin or substance P neurons. Femero and coworkers observed no change in striatal Leu-enkephalin measured by radioimmunoassay (RIA) 7 days after injury (Femero et al., 1988). In a preliminary analysis, we observed no change in striatal enkephalin-containing (EC) peptides or substance P measured by RIA 14 days after injury (Burke et al., 1989).

The purpose of this study was to attempt to better define alterations in striatal enkephalinergic neurons after perinatal HI by measuring striatal preproenkephalin (PPE) mRNA with a quantitative in solution hybridization assay (Zhu et al., 1992, 1993) in combina- tion with the anatomic resolution provided by in situ hybridization analysis of PPE mRNA at several time points after injury. We have found that both techniques demonstrate an early depression in striatal PPE mRNA, and although total striatal PPE mRNA levels return to normal with development, there are regions in the dorsal striatum that remain devoid of PPE mRNA-positive neurons, demonstrated by in situ hybridization. Thus, there appears to be a persistent loss of the enkephalin neuronal phenotype in the striatum after early HI injury.

MATERIALS AND METHODS

Unilateral HI Female Sprague-Dawley rats were obtained 14-1 6 days

pregnant on arrival from Charles River Laboratories (Wil- mington, MA, U.S.A.). The day of delivery was defined as postnatal day 1. On day 7, the left common carotid artery was ligated permanently with 6-0 silk suture. For sham- operated controls, the artery was exposed but not ligated. After recovery from anesthesia, pups were returned to the dam to nurse for 1 h. Pups were then exposed to humidified 8% oxygen/balance nitrogen for 3.0 or 3.5 h in a plastic jar that was placed in a 37OC water bath, as described by others (Rice et al., 1981). All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals, and were approved by the Institu- tional Animal Care and Use Committee of Columbia Uni- versity, Health Sciences Division. At 14 days after injury, it was possible to classify the degree of unilateral injury ac- cording to its macroscopic appearance; i.e., brains with no apparent injury were classified as “mild” injury, those with

visible hemispheric shrinkage were classified as “moderate.” A few brains showed extensive infarction with cystic cavity formation (“severe”); these were not used for further analysis.

Solution hybridization assay for PPE mRNA Rats were killed by decapitation, and the brains were re-

moved rapidly and placed in ice-cold saline. Striata from rats killed at early time points after injury (up to 24 h) were dissected by hand with fine forceps and pooled for analysis. Striata from rats killed at 3-14 days after injury were dissected by micropunch and assayed individually. Each brain was placed in a Plexiglas rat brain matrix, and a 2-mm-thick coronal section of the central portion of the striatum was taken; the section was placed anterior surface up on a chilled glass plate, and the striatum dissected on either side with a 3.0-mm (internal diameter) tissue punch. Tissues obtained from rats up to 7 days after injury were frozen at -70°C until analysis; tissues at 14 days were immediately homogenized in RNA extraction buffer. Total RNA was isolated using a guanidine HCI-phenol extraction/ethanol precipitation procedure described previously (Intumsi et al., 1988). To promote efficient recovery of RNA from tissue samples, 50 pg of E. coli tRNA was added before homog- enization. The 32P-labeled riboprobes for the assay were transcribed in vitro from plasmids containing cDNAs for rat PPE mRNA (the pYSEAl template, a gift from Dr. S. Sobol) or human 18s rRNA (the plasmid pS/E, a gift from Drs. T. Nilsen and P. Maroney) (Intumsi et al., 1988; Zhu et al., 1992, 1993). The specific activity for PPE averaged 6.6 X lo8 dpm/pg and that for the 18s riboprobe was 1 X lo7 dpm/pg. The solution hybridization assays were based on ribonuclease protection of the labeled riboprobes that were hybridized to complementary RNAs. Riboprobes protected from nuclease activity were precipitated with trichloroacetic acid (TCA) and analyzed by scintillation counting. Comparison was made with standard calibration curves to quantify mRNA or total cellular RNA levels. The standard for PPE mRNA was the sense transcript from plasmid pYSEC 1. Values from the sense calibration standards were multiplied by 1.58 to correct for the difference in length between the sense transcript (950 bases) and the 1,500 base-long rat PPE mRNA. Rat liver RNA was the standard used to quantify total cellular RNA in each tissue extract. Duplicate aliquots of each sample were assayed for PPE mRNA and total cellular RNA.

For each assay, aliquots of total RNA extracts were dried in 1.5-ml Eppendorf microcentrifuge tubes and resuspended in 30 pl of hybridization buffer [ 10 mM EDTA, 0.3 M NaCI, and 0.5% sodium dodecyl sulfate, 10 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, pH 7.41 containing 32P-labeled riboprobe, 150,000 dpm for the PPE riboprobe, and 80,000 dpm for the 18s riboprobe. This solution was covered with two drops of mineral oil and incubated for 4 h at 75°C. After hybridization, 300 pl of 0.3 M NaCI, 5 mM EDTA, 40 pg/ml RNase A, and 2 pg/ml RNase T1 in 10 mM Tris-HC1, pH 7.4, was added to the sample. Samples were vortexed for 5 s and the unprotected, single strands of RNA were digested at 30°C for 1 h. The ribonuclease reaction was terminated with 1 ml of 5% TCA and 0.75% sodium pyrophosphate. One drop of 0.5% bovine serum albumin (BSA) was added to aid precipitation. This solution was mixed and the TCA-precipitable dpms were collected onto glass-microfiber filter paper (Reeves Angel 934AH, Brandel, Gaithersburg, MD, U.S.A.) using a

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24-place-cell harvester (Brandel). The filter was washed three times with 5% TCA, dried under an infrared light, and counted by liquid scintillation in 5 ml of hydrofluor scintillation solution (National Diagnostics, Manville, NJ, U.S.A.).

Assay of EC peptides For analysis of EC peptides, each striatum dissected by

the micropunch procedure was homogenized by use of a Sorvall homogenizer at 4°C in 1 .O ml of extraction medium consisting of 1 M acetic acid/O. 1 % 2-mercaptoethanol containing pepstatin, leupeptin, and phenylmethylsulfonyl fluo- ride, each at a concentration of 1 pg/ml. The homogenate was centrifuged at 40,000 g for 30 min. The supernatant containing the EC peptides was removed and aliquots dried by lyophilization in a Savant Speed Vac Concentrator. To- tal EC peptides were determined by RIA after treatment with trypsin and carboxypeptidase B. The enzymatic diges- tion was used to release [Metlenkephalin from precursor peptides and allowed measurement of total [Met]- enkephalin content of the striatum. Dried aliquots of the striatal extract were dissolved in 200 pl of 0.2 M, pH 8.0, N-ethylmorpholine acetate buffer (NEM buffer) containing 20 pg of trypsin. After incubation for 16 h in a heating block at 37"C, the samples were centrifuged for 3 min at 1,500 g and 0.1 pg of carboxypeptidase B was added in 20 pl of NEM buffer. The samples were incubated for 2 h at 37"C, then heated at 100°C for 0.5 h to terminate enzymatic activity. The samples were dried by lyophilization as above and resuspended in RIA buffer before analysis.

The RIA for [Metlenkephalin used an antiserum purchased from Immunonuclear Corp. (Stillwater, MN, U.S.A.). Thisantiserum ( 18R2) recognizes [Met]-0-enkephalin in a 1:l ratio with [Metlenkephalin (cross-reactivity = 100%) so that any [Met]-0-enkephalin formed during the workup procedure will not distort the final estimate of [Metlenkephalin equivalents contained in the extract. The cross-reactivity is 2% for [Leulenkephalin, 3% for [Metlenkephalin-Arg6, 4% for [Metlenkephalin-Arg6-Phe7, 8% for [Metlenkephalin-Lys6, and <O. 1% for [Met]- enkephalin-Arg6-Gly7-Leus, [Leulenkephalin-Lys6, and peptide E. The antiserum was used at a final dilution of 15,250 and the 12'I-[Met]enkephalin trace (-9,OOO dpm/ tube) purchased from New England Nuclear Corp. (Boston, MA, U.S.A.). The lower limit of sensitivity for this RIA is 5.6 pg and the interassay coefficient variation for 12 consec- utive assays averaged 6.6%.

In situ hybridization For in situ hybridization studies, rats were killed at either

24 h or 14 days after HI injury. They were perfused intracar- dially with chilled saline for 5 min, and the brains were then rapidly removed and frozen immediately by immersion in isopentane on dry ice. The brains were then serially sec- tioned on a cryostat through the striatum at 12 pm. Sections were thaw-mounted onto subbed slides and stored at -80°C until use within 1-2 months. The probe for the hybridization was a 48-mer oligonucleotide complimentary to bases 388-435 ofthe rat PPE mRNA (Howells et al., 1984) either obtained from Du Pont (NEP-202) or synthesized on a Mil- liGen/Biosearch Cyclone DNA Synthesizer. The oligonucleotide was a 3'-end labeled with "S-dATP (Du Pont, NEG- 034H) in the presence of terminal transferase (Boehringer Mannheim) to a specific activity ranging from 1,000 to 4,000 Ci/mmol probe. Labeled oligonucleotide was sepa- rated from unincorporated nucleotide using a Du Pont

Nensorb cartridge. For hybridization, sections were warmed to room temperature and fixed by immersion in 4% paraformaldehyde 0.1 M phosphate buffer (pH 7.1) for 5 min. Sections were rinsed twice in phosphate-buffered saline and delipidated by successive immersion in higher con- centrations of ethanol and then chloroform. Sections were then rehydrated and immersed in 2X SSC (saline-sodium citrate). Sections were then prehybridized at 40°C for 2 h with 1: 1 formamide/prehybridization mix. The latter con- sisted of 12 ml of 5 MNaCl, 1 .O ml of Tris at pH 7.5,330 pl of 6% Ficoll, 330 p1 of 6% polyvinylpyrrolidone, 1.67 ml of 6% BSA, 400 pl of 250 mMEDTA, 5.0 ml of salmon sperm DNA at 10 mg/ml, 2.5 ml of total yeast RNA at 20 mg/ml, and 100 pl of yeast tRNA, brought to a final volume of 50.0 ml and used in aliquots. Sections were then hybridized with 1:l formamide/hybridization mix at 40°C ( 10°C below the calculated T, for the probe) overnight. Hybridization mix was similar to prehybridization mix but contained dextran 10 g/50 ml and only 1 .O ml of salmon sperm DNA and 250 p1 of total yeast RNA. For hybridization, labeled probe was added to a final activity of -3,000 cpm/pl of the 1: 1 form- amidelhybridization solution. After hybridizations, sections were washed by immersion in 4 L of 2X SSC for 60 min at 40"C, followed by immersion in 4 L of 0.1 X SSC/ 0.05% sodium pyrophosphate/ 14 mM mercaptoethanol at 40°C for 3 h with gentle stimng. Sections remained in this wash at room temperature overnight. They were then dehy- drated in ethanol, vacuum dried, and apposed in a cassette to Amersham Hyperfilm &max at -80°C for 3-4 weeks. 14C standards embedded in plastic (Amersham) were en- closed in each cassette. After exposure to the film, selected sections were dipped in Kodak NTB2 photographic emulsion and exposed for 4-6 weeks at 4°C. These autoradiograms were developed, and the sections were counterstained for Nissl substance.

The specificity of the hybridization was assessed in the following three ways: (1) sections were hybridized with a 48-mer sense probe, 3'-end labeled as described for the antisense, (2) sections were pretreated with RNase, and (3) labeled antisense probe was diluted with a 1,000-fold excess of unlabeled probe. All of these procedures resulted in only background labeling over the striatum, supporting the specificity of labeling observed with the antisense probe.

Quantitative morphologic analysis Autoradiograms of sections from rats killed 14 days after

injury were analyzed quantitatively. The film autoradiograms were analyzed on an Amersham RAS-3000 system with Loats Associates software, using a method similar to that described by Miller et al. (1989). I4C standards were included in each cassette for the following two reasons: (1) to ensure that the optical densities recorded on the film from the sections were within the linear response range, and (2) to enable us to express optical densities on each film as 14C dpm equivalents. It must be understood, however, that no attempt was made to derive an absolute measure of the amount of hybridized mRNA on the section; the I4C dpm equivalents were used simply as a convenient expression for relative comparison of optical densities on films. On each cassette, serial sections from an animal with moderate injury were exposed alongside sections from either an animal with mild injury or a sham control, to permit comparison of optical densities between injured animals and these controls on the same film. For each animal, two sections representing each of four Paxinos-Watson planes (10.2,9.7,9.2,8.7)

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II 3Days 7Days

h e p HI

FIG. 1. The effect of HI injury on PPE mRNA expression in the striatum. On postnatal day 7, rat pups were subjected to a unilateral (left) carotid ligation and subsequent exposure to 8% oxygen, as described in Materials and Methods. At the times indicated, pups were killed, their striata removed and processed for in-solution hybridization assay of PPE mRNA. expressed as picograms per microgram of total cellular RNA. At 1 and 24 h, assay was performed on pooled striata from n = 4 pups. At 3 and 7 days, assay was performed on individual striata from n = 5 pups. Data are shown for the ligated side (HI) and the contralateral control striatum. In other experiments, the contralateral control did not significantly differ from sham-operated controls. On the contralateral control side, there is a progressive increase in expression of PPE mRNA with development. After HI, expression of PPE is reduced approximately fivefold at 1 h, and fourfold at 24 h. By 3 and 7 days, although there is a trend for diminished PPE expression on the HI side, the differences are no longer significant. A similar relative effect of HI was observed in a second experiment on n = 8 pups at 1 h, n = 8 at 2 h, and n = 6 at 24 h (data not shown).

(Paxinos and Watson, 1982) were analyzed. The specific I4C dpm equivalents (total minus background over the corpus callosum) over the entire striaturn were measured on each side of the same section. These values were averaged for the two sections representing each plane, and the value on the experimental (left) side was then expressed as a percentage of the control (or right) side. This percentage for each of the four planes was averaged to give a final expression for the experimental side as a percentage of the control side for each brain. A similar procedure was followed to examine effects within striatal quadrants in plane 9.2 for each animal. The striaturn on each side was divided into four quadrants along the greatest dorsal-ventral and medial-lateral axes. Specific I4C equivalents within each cursor-defined quadrant on the experimental side were measured and expressed as a percentage of the corresponding quadrant of the control side. This was done for two sections, and the results averaged.

RESULTS

Early effects of HI Solution hybridization assay demonstrated that by

1 h after HI injury to the immature brain, striatal levels of PPE mRNA were diminished (Fig. 1). This effect persisted at 24 h after injury, but by 3 days, although there was a persistent trend for diminished

levels on the injured side, the difference was not significant.

In situ hybridization analysis of n = 5 rats at 24 h after HI injury revealed diminished expression of striatal PPE consistent with the quantitative observations made by the hybridization assay. However, the anatomical analysis revealed a pronounced variability of this effect in this model of HI. Two animals showed no alteration in striatal PPE expression on the injured side, showing a hybridization pattern similar to that observed on the contralateral uninjured side, and bi- laterally in n = 3 sham-operated controls. The three other animals showed defects in striatal PPE expression ranging from modest (Fig. 2A) to profound (Fig. 2B). In both of these examples, PPE expression was diminished on the injured side in the dorsal region of the striatum, whereas expression was relatively pre- served in the ventral regions.

Late effects of HI Consistent with the apparent normalization of

whole striatal PPE levels at 3 and 7 days after HI injury, levels at 14 days (3 weeks postnatal) were also unchanged (Table 1). Levels were found to be unchanged even when it became possible, at 14 days, to classify brains subjected to HI as showing no hemispheric shrinkage (mild HI) or visible shrinkage (moderate HI) (Table 1). Levels of striatal EC peptides were also normal at 14 days after injury (Table 2).

In spite of the apparent normality of striatal PPE mRNA and peptide levels at 14 days after injury, in situ hybridization studies showed clear abnormalities in the anatomical distribution of PPE mRNA within the striatum. Four of six rats with hemispheric shrinkage showed regions devoid of PPE mRNA hybridization in the dorsal portion of the striatum. A typical example is shown in Fig. 3. The apparent inconsis- tency between the results obtained by quantitative solution hybridization and the in situ analysis appears to be due to an altered distribution of PPE mRNA expression after HI injury. Like the solution hybridization assay, performed on whole striatal homogenates, the relative optical density of the in situ hybridization autoradiograms on the injured side did not change relative to controls when the whole striatum was examined as the region of interest. For the n = 6 rats with moderate injury, the optical density on the injured side was 102 k 1.4% that of the contralateral control, similar to the relative density on the experimental (left side) in animals with mild injury (100 k 4.3%, n = 4) and shams (102 +. 2.2’74 n = 4). How- ever, when the optical densities were examined within striatal quadrants, it was apparent that, whereas reductions in optical densities had occurred in the dorsomedial, dorsolateral, and ventromedial quadrants, a significant increase had occurred in the ventrolateral quadrant (Fig. 4). It thus appeared that, although visual reductions in PPE expression were present predominantly in the dorsal striatum, these were offset

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FIG. 2. Color-coded autoradiograms of in situ hybridization for PPE mRNA in coronal sections containing the striatum from two rat pups at 24 h after unilateral HI injury. A. This animal shows a modest degree of reduction of hybridization signal in two small regions (white arrows) in the dorsal striatum on the experimental side (EXP). B A second animal shows a profound degree of reduction of hybridization signal in the dorsal striatum. No such reductions in signal were observed on the contralateral control (CON) side in the n = 5 animals subjected to ligation/ hypoxia or on either side in n = 3 sham-operated controls.

FIG. 3. Color-coded autoradiogram of in situ hybridization for PPE mRNA in a coronal section containing the striatum in a rat 14 days after HI injury. There is a loss of hybridization signal in the middorsal region of the striatum on the experimental (EXP) side. No reductions like this were observed on the contralateral control (CON) side in n = 6 animals with moderate injury or on either side in n = 4 animals with mild injury or n = 4 sham-operated controls.

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TABLE 1. Striatal levels of PPE mRNA at 14 days ajer HI on postnatal day 7

Sham-operated Mild HI Moderate HI (n = 10) (n = 12) (n = I I )

L R UE) R(C) L(E) R(C)

PPE mRNA (pglpg RNA) 46.0 f 3.1 43.7 & 3.9 34.0 k 4.0 36.2 k 4.3 38.9 k 6.2 38.0 f 2.8

At 14 days after HI, there was no effect on whole striatal levels of PPE mRNA on the experimental (E) side ofanimals with moderate injury in comparison with the contralateral control (C) side, or other controls. ANOVA indicated that no two groups were significantly different at the 0.05 level. Pilot data in two preliminary experiments comparing a total of 19 normal rats with 16 sham-operated controls revealed no difference in striatal levels of PPE mRNA at 2 1 days of age (data not shown). L, left, R, right.

by increases in the ventrolateral quadrant, such that whole striatal measures of PPE mRNA were unchanged.

At a cellular level, dorsal regions of the injured striatum with diminished PPE hybridization showed an absence of neurons expressing PPE mRNA, rather than a diminished level of expression per neuron (Fig. 5) . Although it remains possible that neurons of the enkephalinergic phenotype are still present in regions devoid of hybridization, and express mRNA at an un- detectable level, it appears more likely, in view of the robust expression in adjacent regions, that enkephalinergic neurons have been eliminated from these regions after HI.

DISCUSSION Within the first 24 h of HI injury, striatal levels of

PPE mRNA, measured both by quantitative solution hybridization assay and by in situ hybridization, were diminished. The pronounced variability in the extent of injury induced by this model, noted by many ob- servers (Ikonomidou et al., 1989; Towfighi et al., 1991), was made especially apparent by the in situ hybridization studies in which no effect was observed in some animals, and a pronounced decrease in mRNA expression was obsemed in others. It is unlikely that the decrease in levels of PPE mRNA de- tected by the solution hybridization assay at 1 and 24 h, but not at 3 or 7 days, was in some way due to the differences in handling the tissue between these two pairs of time points (striata from early time points were dissected by hand and pooled, whereas striata

from latter time points were punched and processed individually). The decreases at the early time points were demonstrated in relation to the contralateral control sides, which were processed in parallel with the experimental sides. In addition, the decrease at 24 h was confirmed by the in situ hybridization.

There are two possible interpretations of the observed decrease in PPE expression. One is that there has been a functional decrease in expression of the enkephalin phenotype in neurons that ultimately will survive. Alternatively, the decrease could be due to death of EC neurons. Although the lasting deficits in PPE expression lead us to conclude that some enkephalin neuron loss has taken place, as discussed below, several considerations lead us to conclude that some of the decrease in expression at these early time points is functional. First, whole striatal PPE mRNA levels have returned to normal by 3 days after injury. Sec- ond, no animal examined by in situ hybridization at 14 days after injury showed an absence of striatal expression as widespread as that illustrated at 24 h in Fig. 2B. If the diminished expression at 24 h is entirely due to enkephalin neuron loss, then we must envision some mechanism whereby the striatum can become extensively repopulated with enkephalin neurons in a short period of time. Although the formation of new enkephalin neurons or the alteration of existing neurons to the enkephalin phenotype are theoretical possi- bilities, they seem unlikely within the short time required. It therefore seems more likely that some of the decrease in expression is due to a functional change in otherwise viable enkephalin neurons, that is reversed by 3 days after injury.

TABLE 2. Striatal levels of ECpeptides at 14 days after HI on postnatal day 7

Normal Sham-operated HI (n = 5 ) (n = 5) (n = 5)

L R L(E) R(C) L(E) R(C)

22.0 f 3. I EC peptides (pmol/mg) 23.6 k 2.0 23.2 k 4.5 33.0 k 2.6 25.5 & 2.9 27.0 k 2.3

At 14 days after HI performed on postnatal day 7, there was no apparent effect on striatal levels ofEC peptides on the experimental (E) side in comparison with the contralateral control (C) side, or other controls. Within the HI group, two animals showed moderate injury; their values, at 18.6 and 28.8, were 105 and 12 1%, respectively, oftheir contralateral control sides, and not outside the range observed in controls. L, left; R, right.

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FIG. 4. The effect of HI injury on the relative density of PPE in situ hybridization within striatal quadrants, determined at 14 days after injury. For six animals with moderate injury, the specific optical density in a quadrant is expressed as a percentage of the density on the contralateral, uninjured side in Paxinos-Watson plane 9.2. Densities were determined on two autoradiographs and the mean determined. Although whole striatal levels of PPE were unchanged at this time point on the injured side, both by solution hybridization assay (Table 1) and densitometry of in situ hybridization autoradiograms. there has been a significant alteration in the distribution of hybridization signal within the striatum. Relative decreases in the dorsomedial (DM), dorsolateral (DL), and ventromedial (VM) quadrants have been offset by a relative increase in the ventrolateral (VL) quadrant.

With subsequent development, whole striatal levels of PPE mRNA, measured by solution hybridization, returned to normal. At 14 days after injury, striatal levels of EC peptides were normal as well. However, the following two persistent alterations in the striatal expression of PPE were noted by in situ hybridization: (1) absence of expression in dorsal regions and (2) augmented expression in the ventrolateral region. Analysis of the dorsal regions at the cellular level indicated that these regions lacked labeled cells. We again must consider whether there has been a functional decrease in expression or a loss of enkephalin cells.

Several considerations lead us to conclude that in the chronic setting, the loss of cellular expression of PPE mRNA is probably due to the loss of these neurons. First, the location and configuration of these dorsal regions devoid of PPE mRNA hybridization signal corresponded precisely to regions of focal neuron loss identified by Nissl counterstaining of hybridized sections. Second, the emulsion autoradiograms failed to identify neurons expressing low levels of PPE mRNA in these dorsal regions. Hybridization signal was either robust, as it was in adjacent, intact striatum, or completely absent, as in the regions of signal void. This all-or-none pattern suggests to us that there has been neuron loss. Third, it is not clear what explanation would exist for a functional suppression of PPE mRNA for 14 days after the injury. We must consider whether the cortical injury that occurs in this model (Rice et al., 198 1) is relevant to the changes observed. Uhl and coworkers have shown that decortication results in a persistent decrease in striatal PPE mRNA expression (Uhl et al., 1988). However, in a model more closely related to the paradigm studied here, Sa- lin and Chesselet ( 1992) demonstrated that cortical infarction led to an increase in striatal PPE mRNA. This study suggests that alterations in cortex are unlikely to explain the decreases in expression we have observed in the dorsolateral region, but they may be related to increases in the ventrolateral area. Finally, the regions devoid of PPE mRNA are similar in their location and appearance to regions that remain devoid of dopamine D, receptor binding for up to l l weeks after injury (Kostic et al., 1991). This persistent loss of D, receptor binding is likely to be due to the loss of intrinsic neurons that synthesize these receptors. Although the cellular localization of striatal D, receptors in relation to neuron phenotype is contro- versial (Surmeier et al., 1993), there is some evidence

FIG. 5. Dark-field photomicrograph of an emulsion autoradiogram of the same section shown in Fig. 3, showing hybridization at the cellular level in the dorsal portion of the striaturn. The region shown is ventral to the ex- ternal capsule (ec) and it contains both injured and intact striatum. On the right side of the photograph (medial) are numerous PPE mRNA-positive cells, indicated by clusters of deposited silver grains; on the left (lateral) there is a complete absence of positive cells. Within the injured region devoid of PPE-positive neurons, other neurons were visible on Nissl stain. Thus, the region of depressed signal in the dorsal striatum, observed at a regional level in Fig. 3, is characterized by a loss of positive cells rather than diminished expression among a normal number of cells. Bar = 50 Wm.

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STRIATAL PPE mRNA FOLLOWING HI INJURY I885

that striatal neurons that express the dopamine D, receptor are predominantly EC striatal neurons (Ger- fen et al., 1990). Thus, a parsimonious explanation for our prior demonstration that regions of the dorsal striatum show a persistent absence of D, expression, and our current finding that similar regions show a persistent absence of enkephalin mRNA expression, is that the neurons that express both of these markers have been lost as a result of the HI injury.

If we accept the interpretation that persistent losses in D, receptor and PPE mRNA expression are likely to be due to losses of enkephalin neurons, then this is the first phenotypically defined class of striatal neurons for which selective losses have been demonstrated in this model. As previously discussed, the density of cholinergic neurons, expressed as a number per unit of striatal area, was found by Johnston and Hudson (1987) to increase in this model. We found that, although HI striatal injury resulted in a decrease in the absolute number of cholinergic neurons in the striatum, this decrease was not as profound as the loss of striatal volume. Consequently, the density of striatal cholinergic neurons (number per unit of striatal volume) increased. This increase was readily observ- able in two-dimensional tissue sections as a visible clustering of these neurons in the injured striatum (Burke and Karanas, 1990). A similar increase in the density of NADPH-diaphorase in two-dimensional tissue sections (Femero et al., 1988) has been reported, although absolute quantitation of number per unit of striatal volume has not yet been performed.

We recently completed an analysis of striatal matrix neurons, defined by immunostaining of the cal- cium-binding protein calbindin-D28K in this model (Burke and Baimbridge, 1993). HI striatal injury resulted in no change in the density of calbindin-positive neurons (number/unit of area). The dorsolateral portion of the striatum, which ordinarily contains very few, faintly staining calbindin neurons, became enriched in these neurons after HI injury, suggesting that in that specific region, they may be relatively spared. In any case, there was no region of relative loss of calbindin neurons, as we have now demonstrated for enkephalin neurons. There is, as yet, no data precisely defining the relationship between enkephalin phenotype and the calbindin phenotype among striatal medium-sized neurons. However, given that the large majority of striatal matrix neurons are calbindin positive, it appears likely that there is a substantial overlap in these two phenotypes. We must ask, then, why our previous study of calbindin staining failed to reveal the loss of any enkephalin neurons. There are at least two possible explanations. It is possible that other striatal neurons that also express calbindin are relatively spared, resulting in no net change in calbindin-positive neuron density. It is also possible that, after injury, the surviving neurons of any phenotype either up-regulate or newly express calbindin, resulting in no net change in their number. In either case,

the lack of change in the density of calbindin-positive neurons is not incompatible with our conclusion that HI injury has resulted in a relative loss of enkephalinergic neurons.

We pointed out earlier that the relative sparing of cholinergic and NADPH-diaphorase (somatostatin)- positive neurons in this model of developmental HI injury resembles the sparing of these classes in models of excitotoxic injury induced by NMDA agonists (Beal et al., 1989). Our current finding, indicating a loss of enkephalin neurons, has also been reported in excitotoxic injury (Beal et al., 1989; Ferrante et al., 1993) induced by quinolinate (QA), supporting the similarity between these two forms of injury. Ferrante and coworkers reported an acute loss of calbindin-positive neurons in primates after QA lesion (Ferrante et al., 1993), but this result is not necessarily incompatible with our observations in the chronic setting in this model, where we do not see such loss, because their observations on calbindin were restricted to the acute period. There are also many parallels between the neuropathologic alterations in this HI injury model and those in Huntington’s disease (HD), including relative sparing of dopaminergic afferents (Burke et al., 199 l), sparing of cholinergic and NADPH-diaphorase neurons, and loss of EC neurons. These parallels provide support for the concept that interference with oxidative metabolism may underlie the neuropathologic alterations in HD, albeit in a chronic setting (Al- bin and Greenamyre, 1992; Beal, 1992).

The loss of EC neurons demonstrated in this model may be relevant to the functional disturbances of motor control, such as dystonia, observed after HI injury to the human brain. Recently, a model of functional disturbances of the basal ganglia has been proposed in which decreases in activity of the EC striatal efferent system relative to the activity of the substance P-containing efferent system have been postulated to underlie some forms of dyskinesia, particularly chorea (Albin et al., 1989). In that context, it will be important to know what changes take place in the substance P efferent pathway in this model. Although we have found whole striatal substance P levels to be normal (Burke et al., 1989), the results of the current study clearly show that in the developing brain, both time course and anatomical studies using either in situ hybridization for mRNA or immunohistochemistry for the peptide are required for a definitive analysis. It is important to note that, in addition to a persistent loss of PPE mRNA-positive neurons after this injury, there also appears to be an increased level of expression in the relatively spared ventrolateral segment of the striatum. Thus, disturbances of motor control after HI injury may result not only form the loss of striatal PPE neurons, but also from an imbalance between this population of neurons in one striatal region in comparison with another.

Acknowledgment: We are grateful to Andrew Karanas, Nick Kenyon, Shu-Ming Zhu, and Eileen Janec for superb

J . Netrrochem., Vol. 62. No. 5 , 1994


technical assistance. Dr. Kirk Wilhelmsen performed the oligonucleotide synthesis. We are also grateful to Ms. Pat White for her diligent preparation of the manuscript. This work was supported by NS26836, the Parkinson’s Disease Foundation, and the United Cerebral Palsy Research and Educational Foundation, Inc. (R.E.B.), and in part by DA01457 (C.E.I. and S.Q.F.).

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acute and persistent suppression of preproenkephalin mrna expression in the striatum following...

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