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From the *Department of Anesthesiology and Pain Medicine, ||Department of Pediatrics, Asan Medical Center, College of Medicine, Ulsan University; NMR Laboratory, Asan Institute for Life Sciences; Department of Anesthesiology and Pain Medicine, College of Medicine, Chung-Ang University; and Department of Anatomy, College of Veterinary Medicine, Konkuk University, Seoul, South Korea.
Address correspondence and reprint requests to Pyonghwan Park, MD, PhD, Department of Anesthesiology and Pain Medicine, Asan Medical Center, College of Medicine, Ulsan University, 388-1 Pungnap-2dong, Songpa-gu, Seoul 138-736, South Korea. Address e-mail to phpark{at}amc.seoul.kr.
Abstract
BACKGROUND: The pathophysiology of brain damage from hypoxia or ischemia has been ascribed to various mechanisms and cascades. Intracellular calcium overload and a calcium excitotoxic cascade have been implicated. It has been suggested that disturbances of endoplasmic reticulum calcium homeostasis are involved in the induction of neuronal cell injury. Two types of intracellular Ca2+-release channels, involving the ryanodyne receptor and the inositol (1,4,5)-triphosphate receptor, are essential for Ca2+ signaling in cells. Dantrolene, which is used for the treatment of malignant hyperthermia syndrome, has been reported to inhibit Ca2+ release through ryanodyne receptors from the endoplasmic reticulum into the cytosol. We designed this study to investigate the neuroprotective effects of dantrolene on hypoxic-ischemic brain damage in the neonatal rat brain.
METHODS: Seven-day-old Sprague-Dawley rats were assigned into two groups; control group (n = 69) and dantrolene group (n = 60). Dimethyl sulfoxide was administered intracerebroventricularly in the control group, and dantrolene in dimethyl sulfoxide was similarly administered to the dantrolene group, before hypoxic-ischemic brain injury (HII). HII was induced by the ligation of the common carotid artery under isoflurane anesthesia, followed by exposure to about 2.5 h of hypoxia (oxygen concentration was maintained at 7%-8%). 1H magnetic resonance spectroscopy was performed 1 day after HII. This noninvasive method evaluated apoptotic processes in the brain after HII. Morphologic score analyses and the calculated percentage of infarct areas after 2,3,5-triphenyltetrazolium chloride staining 14 days after HII were also used to evaluate the effects of dantrolene on HII. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining was performed 1 day after HII using 24 more rats.
RESULTS: The lipid/creatine ratios in the right hemispheres in the dantrolene group 1 day after HII were significantly lower than those of the control group (P < 0.05). There was no significant difference between the two groups in the N-acetylaspartate/creatine ratios. The gross morphologic scores were lower in the dantrolene group than in the control group (P < 0.05), and infarct area (%) after 2,3,5-triphenyltetrazolium chloride staining was less in the dantrolene group than in the control group (P < 0.05) 14 days after HII. Further work with 24 rats showed no significant difference, however, in the number of TUNEL positive cells on the two groups.
CONCLUSIONS: Our results show that dantrolene, administered intracerebroventricularly before HII, had a neuroprotective effect in HII model of the neonatal rat brain.
Perinatal hypoxic-ischemic brain injury (HII) has a high mortality rate and is a significant cause of neurodevelopmental impairment and disability. The pathophysiology of brain damage from hypoxia or ischemia has been ascribed to various mechanisms and cascades, but has yet to be clearly elucidated. Intracellular calcium overload and a calcium excitotoxic cascade have been suggested as causes of neuronal damage. Hypoxia-ischemia triggers over-stimulation of N-methyl-d-aspartate type glutamate receptors, calcium entry into cells, activation of calcium-sensitive enzymes, such as nitric oxide synthase, production of oxygen radicals, and injury to mitochondria. All of these events lead to necrosis or apoptosis.1 Much research has been devoted to blocking or preventing the excitotoxic cascades, in efforts to salvage brain tissue.
It has been suggested that disturbances in endoplasmic reticulum (ER) calcium homeostasis play a role in the induction of neuronal cell injury.2,3 Two types of intracellular Ca2+-release channels, involving the ryanodyne receptor (RyR) and the inositol (1,4,5)-triphosphate (IP3) receptor, are essential for Ca2+ signaling in cells.2,4 Studies have indicated that IP3 may modulate ryanodyne-sensitive channel activity in some species.5,6
Dantrolene, a hydantoin derivative that acts primarily in the sarcoplasmic reticulum of peripheral skeletal muscle,7 has been used clinically to treat malignant hyperthermia syndrome and neuroleptic malignant syndrome, heat stroke, and muscle spasticity.8 Dantrolene inhibits Ca2+ release through the RyR from the ER into the cytosol9 and prevents glutamate cytotoxicity and Ca2+ release from intracellular stores in cultured cerebral cortical neurons and cerebellar granular cell cultures.10,11 In addition, dantrolene has been reported to have beneficial effects on delayed cell death and concomitant DNA fragmentation in rat hippocampal CA1 neurons subjected to mild ischemia.12 Dantrolene also afforded neuroprotection against ischemic damage after transient middle cerebral artery occlusion in adult rats by reducing ER stress-mediated apoptotic signal pathway activation.13 It has been reported that dantrolene augmented bioenergetic recovery and ameliorated poly(ADP-ribose) polymerase-related bioenergetic failure in neonatal brain tissue, but only when administered within the initial recovery phase after ischemic insult, and it has also been suggested that early mobilization of intracellular Ca2+ is important in the infant brain.14 The possible effects of dantrolene on in vivo neonatal HII, in which apoptosis is the main pathophysiologic mechanism,15–19 have not been studied, even though such work would be of value.
This study was designed to investigate the neuroprotective effect of dantrolene on HII in the neonatal rat brain using the Rice-Vannucci model.20 We analyzed the effect of dantrolene on brain tissue using histologic, morphologic investigations, and 1H magnetic resonance spectroscopy (1H MRS) as a noninvasive tool.
METHODS
Animal Model and Drug Administration
Unsexed 7-day-old Sprague-Dawley rats, weighing 12-18 g, were randomly assigned into two groups: the control group (CG: n = 69) and dantrolene group (DG: n = 60). This animal study was approved by the Animal Care and Use Committee of Ulsan University.
Rats were anesthetized with 5 vol% isoflurane and anesthesia was maintained with 1-2 vol% isoflurane. After induction of anesthesia, a 30-gauge needle on a Hamilton syringe (Hamilton, USA) was used to approach the left lateral ventricle. The puncture point of the needle was 1-2 mm lateral to bregma and 0.5-2 mm posterior to bregma along with sagittal suture, and the needle tip was penetrated 3-4 mm under the skin. Sterile dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA) or 1 mM dantrolene (Sigma-Aldrich, USA) dissolved in DMSO, in 5 µL quantities in the Hamilton syringe, was slowly injected over at least 5 min into the left lateral ventricle of rats in the CG and the DG, respectively. After injection, a cervical midline incision was made, and the right common carotid artery of each rat pup was ligated with 5-0 surgical silk. A heat lamp was used during the procedure to maintain body temperature at 37°C. The incision was then sutured, and each pup was allowed to recover in the presence of his or her mother for 1-2 h. The rats were subjected to 2.5 h of hypoxia in the humidified nitrogen-oxygen mixture within an airtight acrylic box. The oxygen concentration was maintained at 7%-8% and was monitored with an OX-90 oxygen monitor (Atom, Japan). The temperature in the acrylic box was maintained at 37°C using a heat lamp. Exhaled carbon dioxide was eliminated using a carbon dioxide absorbent placed within the breathing circuit. Rats surviving hypoxia were returned to their mothers. We estimated the survival rate of each group just after HII. 1H MRS was performed 1 day after HII on all rats. The rats were decapitated and their brains were removed for morphologic analysis, and 2,3,5-triphenyltetrazolium chloride (TTC) staining was performed 14 days after HII. An additional 24 rats were used for histologic analysis using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining. The 24 rats were randomly assigned into two groups: CG (n = 12) and DG (n = 12). The experimental HII protocol was the same as described earlier. TUNEL staining was performed just after 1H MRS.
1H MRS
1H MRS scans were obtained using a Bruker Biospec 4.7T MRI/MRS system (Bruker, Fallanden, Switzerland) and a 0.7-cm diameter circular surface coil fabricated in-house. Rats were anesthetized with 1-2 vol% isoflurane. The spectra from 1H MRS were acquired through a signal voxel (3 x 3 x 3 mm3) in the right parietotemporal region of the brain using a spin acquisition mode sequence for 128 acquisitions with a repetition time of 3000 ms and an echo time of 30 ms. The water signal was suppressed by a chemical shift selective sequence. Analysis of 1H spectra was performed using XWIN-NMR hardware and software. The relative sizes of the metabolite peaks were compared semiquantitatively. The volumes of N-acetyl aspartate (NAA) and creatine (Cr) were measured at 2.02 and 3.03 ppm, respectively and lipid (Lip) was measured at 0.9 and 1.3 ppm (Fig. 1). To distinguish the peaks of Lip and lactate, which appear together at 1.3 ppm, 1H spectra were obtained with an echo time of 135 ms. The Lip peak does not invert with this echo time.
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TUNEL Staining
Additional work was performed for the TUNEL staining procedure.21 The same procedure as described in Animal Model and Drug Administration was applied to 24 pups. Twelve pups were treated with intracerebroventricular DMSO and another 12 with dantrolene in DMSO. After HII, the pups were anesthetized with isoflurane and perfused with 2.5 mL/g of normal saline containing 2 U/mL heparin (Hanlim, Korea) at 4°C, followed by the same volume of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. After perfusion, the brains were removed and postfixed overnight in the same paraformaldehyde-phosphate buffer. TUNEL staining on paraffin-embedded sections was done to identify DNA fragments from cells dying of apoptosis. To detect DNA breaks, the TUNEL assay was conducted with a TUNEL reaction mixture kit (Roche, Germany). Briefly, sections were dewaxed, rehydrated, and washed in phosphate-buffered saline (PBS). The sections were subjected to proteolytic pretreatment with 1.0 M citrate buffer (pH 6.0) in a microwave for 5 min to increase labeled nucleotide binding and washed in PBS buffer for 15 min at room temperature. Terminal deoxynucleotidyl transferase (TdT) and fluorescein incorporated in TdT buffer were added in a volume sufficient to completely cover the sections, which were then incubated in a humid atmosphere at 37°C. The reaction was terminated after 1 h by transfer of the slides to PBS for 5 min. TUNEL-positive cells were detected under a fluorescent microscope (Olympus, BX-51, Japan), with excitation at 450 nm.
Morphologic Score
Fourteen days after HII, individual brains were scored to a scale from 0 to 4 based on the gross morphologic appearance of the right cerebral hemisphere.22 A rating of 0 indicated no size difference between two hemispheres. A rating of 1 was given when the right side was smaller than the left side, but when no visible infarct was noted. A rating of two indicated a difference in hemisphere size and a cystic change due to a slight infarct. A rating of 3 was given when there was a marked discrepancy in size, and a cystic atrophy due to a large infarct, with some preservation of the right hemisphere. Finally, a rating of 4 indicated an extensive infarct with almost total destruction of the right hemisphere. The brains were scored by a researcher who was blinded to the treatment groups.
Measurement of Infarct Area After TTC Staining
The brain was cut into 2-mm-thick coronal blocks after gross morphologic scoring. The brain slices were immersed in a 2% (w/v) solution of TTC in normal saline, for 30 min and then fixed in 10% (v/v) phosphate-buffered formalin at 4°C (Fig. 3). TTC-stained brain slices were scanned using a Hewlett-Packard Scanjet (Model 4100C) and measured as pixel areas after manual delineation of bright red images with a computer mouse. This was performed by a researcher blind to the treatment groups. Infarct areas (IAs) were obtained by subtraction of stained areas of right hemispheres from stained areas of left hemispheres.23 The percentage of IA (%) in each group was calculated as follows.
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Statistics
All data are expressed as the means ± sd values. The survival rates were compared using 
2 tests. Unpaired Student’s t-test was performed to assess statistical differences in the Lip/Cr ratios, NAA/Cr ratios, and TUNEL positive cells. Morphologic changes and percentages of infarct areas after TTC staining were compared using the Mann-Whitney U-test. A probability (P) value of P < 0.05 was regarded as statistically significant.
RESULTS
1H MRS
The Lip/Cr ratios in the right hemisphere 1 day after HII were 5.5 ± 1.8 and 4.4 ± 2.1, in the CG and in the DG, respectively. The Lip/Cr ratio in the DG was thus significantly less than in the CG (P < 0.05). There was no significant difference between the NAA/Cr ratios of the two groups; however, these ratios were 1.2 ± 0.3 and 1.1 ± 0.3 in the CG and the DG, respectively (Fig. 1).
TUNEL Staining
The number of TUNEL-positive cells in the DG 1 day after HII were 132.8 ± 29.9 and 198.5 ± 45.5 in the right hippocampal and the right cortical areas, respectively. The numbers of TUNEL-positive cells in the CG 1 day after HII were 126.5 ± 36.2 and 189.8 ± 49.2 in the right hippocampal and the right cortical areas, respectively. The number of TUNEL-positive cells in the DG 1 day after HII were 12.8 ± 3.5 and 20.7 ± 10.1 in the left hippocampal and the left cortical areas, respectively. The number of TUNEL-positive cells in the CG 1 day after HII were 11.5 ± 3.3 and 18.8 ± 9.2 in the left hippocampal and the left cortical areas, respectively (Fig. 2). There were no significant differences between the two groups in any of these values.
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Morphologic Scores
The mean morphologic score of the DG was significantly lower than that of the CG (P < 0.05). The morphologic scores were 3.5 ± 0.9 and 2.6 ± 1.3 in the CG and the DG, respectively.
IAs After TTC Staining
The IA in the DG was significantly less than that in the CG (P < 0.05). The area in the DG was 45.0% ± 21.9% and that in the CG was 60.2% ± 18.0% (Fig. 3).
Correlations
The Lip/Cr ratio correlated with the morphologic score (r = 0.55; P = 0.00), and the morphologic score correlated with the IA after TTC staining (r = 0.73; P = 0.00). In addition, the IA after TTC staining correlated with the Lip/Cr ratio (r = 0.45; P = 0.00).
Survival Rate
Thirty-six of 69 pups in the CG survived, and 40 of 60 pups in the DG survived at least until the end of HI. The survival rates were 52.0% ± 16.2% and 67.0% ± 11.5%, in the CG and the DG, respectively. There was no significant difference in survival rate between groups.
DISCUSSION
We have shown here that dantrolene administered into the intracerebral ventricles before HII had a neuroprotective effect in the rat neonatal HII model, in which the main pathophysiologic mechanism is apoptosis.15–19
Both apoptosis and necrosis play a significant role in neuronal death after HII. The balance between these modes of death may be influenced by the severity of the insult, the cell phenotype and location, and the maturational stage. Apoptotic morphology after HII occurs within 12 h and progresses for 2 wk in neonatal rats.16,17 These chronologic apoptotic processes offer a window for intervention before neuronal death occurs.
TUNEL staining is a useful tool for detection of DNA fragments resulting from the apoptotic process.21 This technique uses TdT to add labeled nucleotides to the 3'-OH end of DNA fragments. The technique cannot, however, be used for long-term follow-up, because the experimental animals are killed before TUNEL staining. We therefore used 1H MRS, a noninvasive analytic method, to evaluate apoptosis in this study.24,25 1H MRS gives specific chemical information on numerous intracellular metabolites such as NAA, Cr, choline-containing compounds, myoinositol, glutamate, and lactate. NAA is second in concentration only to glutamate in the human central nervous system, and has been proposed to be an ideal indicator of intact central nervous tissue.26 Reduced NAA levels in visible infarcts were related to infarct extents and clinical outcomes.27 Cr levels do not change during ischemia and remain stable with brain maturation. The NAA/Cr ratio is thus useful in assessment of cellular metabolic integrity in neonatal brain injury.26,28 Previous studies have shown that 1H MRS detects polyunsaturated fatty acids (0.9 and 1.3 ppm lipid peaks) and the technique is useful in both in vivo and in vitro detection of apoptosis during tumor therapy.29,30 It has been suggested that phospholipase A2 is an essential element in tumor necrosis factor-mediated apoptosis, and that this might be related to the liberation of polyunsaturated fatty acids from membrane phosphatidylcholines.30–33 Earlier, we showed high Lip signal peaks in MRS spectra 1-14 days after HII and decreases in NAA levels 1 wk after HII in newborn rat brains.22,23 We therefore used the NAA/Cr ratio and the Lip/Cr ratio, both obtained from 1H MRS to estimate the integrity of cell metabolism and the relative extent of apoptosis, respectively.
Animals in the DG showed lower Lip/Cr ratios than did those in the CG, indicating that dantrolene may have protected against apoptosis after HII. Dantrolene did not, however, affect NAA/Cr ratios 1 day after HII. This means that the neuronal cells ultimately destined to die were still alive at this time, if the main pathophysiologic process was apoptosis rather than necrosis. Because NAA levels in apoptotic cells decrease only 7 days after HII,25 there was no significant difference between the two groups in TUNEL staining. Although this may mean that dantrolene does not have any beneficial effect on apoptosis, we consider it more likely to be a false negative finding arising from the small sizes (3-5 µm) of brain slices used for TUNEL staining and/or the small sample size (12 animals in each group). At 14 days after HII, the DG had lower gross morphologic scores and lower relative IAs, compared with the CG. These results indicate that dantrolene had a neuroprotective effect, even though beneficial effects on apoptosis were not clearly shown in our model. Additional work is required to resolve this issue.
It is believed, however, that an excessive increase in cytoplasmic or mitochondrial calcium activity is toxic, and that depletion of ER calcium is lethal to neuronal cells.2,3,34 Prolonged depletion of the ER calcium pool was a critical component of neuronal damage in some studies2 and there is some evidence of apoptotic crosstalk between the ER and mitochondria.35 Our finding that the intraventricular dantrolene had neuroprotective effects may be explained by the mechanism of action of dantrolene, which involves inhibition of calcium release from the ER. Thus, the action of dantrolene on the RyR of the ER may play a crucial role in explaining our data.
Several studies have found that dantrolene has beneficial effects on neuronal ischemia. Intracerebroventricular dantrolene had a neuroprotective effect against ischemic, delayed neuronal death after transient bilateral occlusion of the common carotid artery in the gerbil brain.36 Intracerebroventricular dantrolene also afforded neuroprotection against ischemic damage after transient middle cerebral artery occlusion in adult rats.13 In addition, dantrolene augmented bioenergetic recovery and ameliorated poly(ADP-ribose) polymerase-related bioenergetic failure only when administered within the initial recovery phase after ischemic insult in neonatal brain tissue. The early mobilization of intracellular Ca2+ is important in normal infant brain development.14 Our study is significant because we show the neuroprotective effect of dantrolene in an in vivo HII model using neonatal brains.
IV dantrolene showed no protective effect, however, in a complete cerebral ischemia model in dogs.37 This may have been related to dantrolene’s poor permeability of the blood-brain barrier. We administered dantrolene intracerebroventricularly in the present study to avoid this problem.
The survival rate just after HII did not differ between groups. Any such difference would have been related to the action of dantrolene on cardiac tissue, not on brain tissue. Thus, 1 mM of intracerebroventricular dantrolene did not show a cardioprotective effect. By contrast, another group found that dantrolene had a protective effect on ischemic rat heart.38 We consider that the dose of 1 mM of intracerebroventricular dantrolene used in the present study was too small to have systemic consequences or to produce a cardioprotective effect.
We administered 0.2 mM dantrolene intracerebroventricularly during a pilot study and found that this dose did not show any beneficial effect in our model. Intracerebroventricular dantrolene at 1 mM was valuable, as we have described earlier, but we could not prove that dantrolene specifically inhibited apoptosis. Further study, with higher dantrolene doses, is therefore required.
In conclusion, dantrolene was a useful neuroprotectant in our neonatal rat HII model. This finding suggests that dantrolene may be of value as a preventive or therapeutic drug in brain damage arising from neonatal HII.
Footnotes
Accepted for publication August 23, 2007.
Supported by Grant from the Asan Institute for Life Sciences.
There was no financial relationships between any of the authors and commercial organizations with a vested interest in the outcome of the study.
REFERENCES
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