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Annals of Clinical & Laboratory Science 36:307-311 (2006)
© 2006 Association of Clinical Scientists

Effect of Sodium Hydroxybutyrate on the Expression of Hippocampal N-Methyl-D-Aspartate Receptor 2B Subunit mRNA in Neonatal Rats with Hypoxic-Ischemic Insult

Zheng-Liang Ma1, Xiao-Ping Gu1, Yin-Ming Zeng2 and Yan Zhang2
1 Department of Anesthesiology, Drum Tower Hospital, Medical Department of Nanjing University, Nanjing; and 2 Affiliated Hospital of Xuzhou Medical School, Anesthesia Institute of Jiangsu Province, People’s Republic of China

Address correspondence to Dr. Zehng Liang Ma, Department of Anesthesiology, Drum Tower Hospital, Medical Department of Nanjing University, Nanjing 210008, People’s Republic of China; tel 86 25 8330 4616; fax 86 25 8331 7016; e-mail mazhengliang1964{at}yahoo.com.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The objective of this research was to test whether sodium hydroxybutyrate (GHB-Na) protects rat neonatal brain against hypoxia-ischemia (HI). Specifically, the objective was to determine the effect of GHB-Na administration on the expression of N-methyl-D-aspartate subunit (NR2B) mRNA in the rat hippocampus. Seven-day-old Sprague-Dawley rats were subjected to ligation of the left carotid artery and were randomly assigned to 5 groups: sham operated (S), saline treated (C), and those treated with GHB-Na (G1, G2, G3), at 3 dosages (50, 100, or 200 mg/kg, ip, thrice daily). NR2B mRNA levels in the left hippocampus were assayed at 2, 6, 12, 24, 72, and 168 hr after HI (exposure to 8% O2/92% N atmosphere for 2 hr). The results suggest that HI insult increased NR2B mRNA gene expression in the left hippocampus of the neonatal rats and that GHB-Na administration partially suppressed this effect of HI insult.

Keywords: rat brain, hypoxia, ischemia, carotid ligation, hydroxybutyrate, hippocampus, N-methyl-D-aspartate receptors, mRNA


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
N-methyl-D-aspartate receptors (NMDARs) are heteromeric complex of NR1 subunits combined with NR2A-D and/or NR3A-B subunits [1]. These receptors are ligand-gated ion channels whose NR2B subunits are associated with pain perception, memory, and learning [2]. In neonatal rats, the hippocampus normally shows high expression of NR2B subunits [3]. In mice, these subunits are vital to survival; mice with targeted disruption of NR2B subunits die shortly after birth. Moreover, mice with genetic overexpression of NR2D outperform wild-type mice in forebrain learning experiments [4]. The present investigation tested the effect of sodium hydroxybutyrate (GHB-Na) administration on NR2B mRNA expression in rat hippocampus after hypoxic-ischemic (HI) insult.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Commercial sources of products were as follows: sodium hydroxybutyrate (GHB-Na), Shanghai Xuedong Haipu Pharmaceutical Co., Shanghai, P. R. China; NR2B primer, Shanghai Biological Technological Service Ltd., Shanghai, P. R. China; G3PDH primer, Hong Kong Invitrogen Life Technological Co., Hong Kong, P. R. China; total RNA isolation system and RT-PCR system, Promega Corp., USA; Gene Cycle system, Bio-RAD Co., USA; Image Master VDS system, Pharmacia Biotech, USA.

Animal preparation.  Sprague-Dawley neonatal rats (7 days old, body wt 10–14 g), obtained from the experimental animal center of Xuzhou Medical College, were divided randomly into 5 groups (36 rats/group) as follows: sham operated group (S), saline treated group (C), and groups treated with GHB-Na at ip dosages of 50, 100, or 200 mg/kg (groups G1, G2, G3, respectively). Each group was further divided into 6 subgroups (6 rats/subgroup), corresponding to time points (2, 6, 12, 24, 62, and 168 hr) after HI insult.

Induction of hypoxia-ischemia.  The induction of HI has been described in detail elsewhere [5]. Rats were anesthetized with ether and the left carotid artery was doubly ligated. After surgery, the rats were allowed to recover for 2 hr. They were then placed in an airtight plastic jar and exposed to an atmosphere of 8% oxygen and 92% nitrogen for 2 hr. The temperature in the jar was maintained at 37°C throughout the hypoxic period. The S group (sham operation) were treated in a similar fashion as the other rats, but without carotid ligation or hypoxic insult. Saline solution was injected (0.1 ml/g, ip) immediately after reoxygenation and thrice daily for a maximum of 7 days for groups S and group C. Similarly, groups G1, G2, and G3 received immediate and thrice daily injections of GHB-Na solution at ip dosages of 50, 100, or 200 mg/kg body wt, respectively.

Tissue collection.  At 2, 6, 12, 24, 72, or 168 hr after HI, the rats were decapitated, the scalp peeled off the skull under sterile conditions, and the cerebra excised. The hemispheres were parted and approximately 25 mg of the left hippocampal tissue was removed and stored at –80°C.

Primer design.  The following primers for NR2B and the housekeeping gene, G3PDH, (treated as an internal standard) were designed by referring to the literature: NR2B: 5'-GAG AAG AGG ACC CTG GAT ATT C-3' and 5'-GGG AAC TAC TGA GAG ATG ATG G-3'; G3PDH: 5'-ATG GTG AAG GTC GGT GTG AAC-3' and 5'-GCT GAC AAT CTT GAG GGA GT-3'. PCR amplification from the primers for NR2B and G3PDH produced fragments of 295 and 437 bp, respectively.

Reverse transcription-polymerase chain reaction (RT-PCR).  All glass- and plastic-ware were treated with DEPC prior to use. Hippocampal tissues, which were prepared in advance, were put into 2-ml tissue grinders. Immediately after homogenization, cooled denaturant was added. Total RNA isolation was performed according the manufacturer’s protocol (Total RNA Isolation System, Promega Corp.). The isolated total RNA was dissolved in 30 µl of RNase-free H2O and prepared for RT-PCR. Residual RNA was kept at –80°C. The A260/A280 ratios of the isolated RNA samples were between 1.7 and 1.9. PCR amplification was performed with the Access RT-PCR System (Promega Corp.). The RT-PCR reaction system comprised: RNase-free H2O, 24 µl; AMV/ Tfl 5x cushion liquid, 10 µl; dNTPS, 1 µl; 25 pmol of forward and reverse primers for NR2B or G3PDH, 2 µl (12.5 µmol/ L); 25 mM MgSO4, 2 µl; AMV RTase, 1 µl; Tfl DNA polymerase, 1 µl; RNA sample, 3 µl; the final volume was 50 µl. The positive control used the control primers and positive control RNA in the Access RT-PCR System. RNase-free H2O replaced the RNA sample in the negative control. After mixing, 40 µl of oil was dropped onto the surface of the reaction mixture. The reaction tube was placed in the Gene Cycle system (Bio-RAD Corp.) for RT-PCR, comprising 48°C for 45 min for RT; 94°C for 2 min to denature; followed by 35 cycles that included 94°C for 30 sec to denature, 55°C for 1 min to resume, and 68°C for 1 min to extend; and lastly 68°C for 7 min to extend.

Gel electrophoresis and quantitation.  The polymerase chain reaction products were loaded onto agarose gels (1%) containing ethidium bromide. Electrophoresis was performed at 80 mA for 45 min. Images of the gels were placed in the Image Master VDS system (Pharmacia Biotech) and analyzed by Image Master 1D software (version 2.0). PCR products were assayed semi-quantitatively based on the relative intensity of each band. The relative quantity of NR2B mRNA was expressed as the ratio of intensity of the band of NR1 cDNA (295 bp) to that of G3PDH cDNA (437 bp).

Statistics.  Data were expressed as mean ± SD. Statistical analysis was performed by ANOVA with SPSS software (version 11.0 for Windows), followed by S-N-K multiple comparisons. A p value <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of post-natal age on NR2B mRNA expression in the hippocampus.  In sham-treated rats (group S), NR2B mRNA expression in the hippocampus tissue of 7-day-old rats was relatively high and gradually decreased thereafter. The NR2B mRNA levels in 7-day-old rats (at 2, 6, or 12 hr after sham treatment) and in 8-day-old rats (at 24 hr after sham treatment) were not significantly different (p >0.05). In 10-day-old rats (at 72 hr after sham treatment) and 17-day-old rats (at 168 hr after sham treatment), significant decreases of NR2B mRNA expression were noted (p <0.05) (Fig. 1Go, Table 1Go).


Figure 1
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  		Fig. 1. The expression of hippocampal NR2B mRNA in the sham-treated S group at serial time intervals after sham hypoxia-ischemia. The 295 bp band corresponds to NR2B; the 437 bp band corresponds to the internal standard (G3PDH). The left hand pattern is the bp calibration ladder.

 

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  		Table 1. Effects of sodium hydroxybutyrate on NR2B mRNA expression in neonatal rat hippocampus after hypoxia-ischemia.
 
Effect of HI on NR2B mRNA expression in the hippocampus.  Compared to the sham-operated S group, the expression of NR2B mRNA in the left hippocampus of rats in group C (saline treated) was greatly reduced (about 20%, p <0.05) after 2 hr of recovery, but did not change significantly after 6, 12, or 24 hr of recovery (p >0.05). Compared to the S group, the NR2B mRNA expression in the left hippocampus of the C group appeared to be slightly increased after 72 hr of recovery, but the difference was statistically insignificant (p >0.05); after 168 hr of recovery, the expression was greatly increased (about 94%)(p <0.05) (Fig. 2Go, Table 1Go).


Figure 2
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  		Fig. 2. The expression of hippocampal NR2B mRNA in the saline-treated C group at serial times after hypoxia-ischemia. The 295 bp band corresponds to NR2B; the 437 bp band corresponds to the internal standard (G3PDH).

 
Effect of GHB-Na on NR2B mRNA expression in the hippocampus after HI.  Compared to the saline-treated C group, NR2B mRNA expression in the left hippocampus of rats in groups G1, G2, and G3 did not change after 2 hr of recovery, but decreased substantially in groups G2 and G3 after 12 hr of recovery (p <0.05). In group G2, which received 100 mg/kg dosages of GNHB-Na, NR2B mRNA expression in the left hippocampus was significantly less than in the C group throughout the subsequent period of study (after 24, 72, and 168 hr of recovery) (p <0.05). In contrast, in groups G1 and G3, which received the 50 or 200 mg/kg dosages of GNHB-Na, NR2B mRNA expression in the left hippocampus was not significantly different from the corresponding values in the C group after 24, 72, or 168 hr of recovery (Figs. 3Go, 4Go, and 5Go, Table 1Go).


Figure 3
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  		Fig. 3. The time-course of expression of hippocampal NR2B mRNA in the GHB-Na-treated G1 group (injections of 50 mg/ kg GHB-Na) after hypoxia-ischemia. The 295 bp band corresponds to NR2B; the 437 bp band corresponds to the internal standard (G3PDH).

 

Figure 4
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  		Fig. 4. The time-course of expression of hippocampal NR2B mRNA in the GHB-Na-treated G2 group (injections of 100 mg/ kg GHB-Na) after hypoxia-ischemia. The 295 bp band corresponds to NR2B; the 437 bp band corresponds to the internal standard (G3PDH).

 

Figure 5
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  		Fig. 5. The time-course of expression of hippocampal NR2B mRNA in the GHB-Na-treated G3 group (injections of 200 mg/ kg GHB-Na) after hypoxia-ischemia. The 295 bp band corresponds to NR2B; the 437 bp band corresponds to the internal standard (G3PDH).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We observed that expression of NR2B mRNA in the left hippocampus decreased gradually in sham-treated rats (group S) during the period from postnatal days 7 to 17 days. This finding is similar to a previous report that the NR2B subunit was almost ubiquitous in rat brain at birth and became confined to forebrain structures during the first 3 postnatal weeks [6]. In our study, the expression of NR2B mRNA was significantly reduced in saline-treated rats (group C) at 2 hr after HI, compared to group S, but was unaffected at 24 hr, consistent with previous observations of HIBD in neonatal rats [7]. We found that expression of NR2B mRNA in group C was significantly greater than in group S at 168 hr, which was widely divergent from the effect of hypoxic brain damage of adult rat [8]. This discrepancy may reflect differences in developmental period and experimental methods.

We speculate that the concentrations of excitatory amino in brain increased during the late recovery period after HI, increasing the expression of NR2B mRNA, augmenting NR2B protein expression, stimulating extrasynaptic NMDARs, and triggering apoptosis. During the period from 6 to 24 hr after HI, no significant changes of hippocampal NR2B mRNA expression were seen in group C, which suggests that HI did not affect the number of NR2B subunits, but might have changed tyrosine phosphorylation of NR2B, similar to changes observed in an adult rat model of hypoxic brain damage [9].

In hypoxic-ischemic brain damage (HIBD), the cytotoxicity of excitatory amino acids (EAA) has always been emphasized. The immature brain is very sensitive to the cytotoxicity of EAA, but the curative effect of EAA receptor antagonist is not as anticipated. Recently, scholars have proposed the theory of "excite-suppress," which holds that important factors in hypoxic brain damage are gamma-aminobutyric acid (GABA) dependent progression, reduced inherent oppression, increased excitotoxicity, and disturbed balance of "excite-suppress" [10]. It has been reported that GABA may protect against neuronal damage caused by EAA that is released after ischemia [11]. Gamma-hydroxybutyric acid (GHBA) is a metabolic product of GABA that can inhibit transmission in the nerve center and cross the blood-brain barrier. GHB-Na, the sodium salt of gamma-hydroxybutyric acid (GHBA), has been used in anesthesia since 1861. Recently, investigators have found that GHB-Na can reduce the consumption of energy in cerebral and peripheral tissues, protecting them from hypoxic impairment [12].

The differences in short-term vs long-term effects of GHB-Na on the hippocampal expression of NR2B mRNA may reflect the interactions of neonatal brain growth, hypoxia-ischemia, and GHB-Na levels. These 3 factors affect jointly the expression of NR2B mRNA. In our experiment, systemic administration of GHB-Na after hypoxia-ischemia appeared to change the expression of NR2B mRNA in neonatal rat hippocampus, which may potentially offer a new avenue for clinical treatment of hypoxic-ischemic brain damage. Because of the limited data that have been obtained to date, we cannot yet evaluate their significance. Since we measured the hippocampal expression of NR2B mRNA, but not the NR2B protein levels, we do not know if the alterations of mRNA expression resulted in corresponding alterations of NR2B protein levels. Expression of NR2B may be structurally and functionally asymmetric in rat brain, but the asymmetries between the left and right hippocampus have not yet been investigated.

In our study, we observed that GHB-Na could counteract the changes of NR2B mRNA expression after HI. After 2 hr of recovery from HI, the hippocampal NR2B mRNA levels in groups G1, G2, and G3 appeared to increase slightly compared to the saline-treated C group, although the changes were not statistically significant. At the remaining 5 time points, the hippocampal NR2B mRNA expression was decreased significantly in group G2 (100 mg/kg GHB-Na) compared to group C; the expression was also decreased (but not statistically significant) in group G1 (50 mg/kg GHB-Na) except at 6 hr after HI. In group G3 (200 mg/kg GHB-Na) the expression was significantly less than in group C at 12 hr, but did not differ significantly at 24, 72, or 168 hr of recovery after HI.

It seems possible that the low dose of GHB-Na influenced the GABA receptor in group G1, and reduced the release of dopamine [13]. A previous study showed that injury caused by dopamine after brain ischemia may stimulate NMDAR and that low dose GHB-Na inhibits the stimulation of NMDAR, inhibiting the expression of the predominant subunit of NMDAR by reducing dopamine release, and to some degree inhibiting apoptosis [14]. In further studies, we plan to include another dosage level of GHB-Na (150 mg/kg), which may enable us to delineate better the dose-response and time-response relationships.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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