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* From the Departments of Anesthesiology and
Pathology, Dokkyo University School of Medicine, Mibu, Tochigi, Japan.
Address correspondence to: Dr. Shinsuke Hamaguchi, Department of Anesthesiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan. Phone: 81-28-286-1111; Fax: 81-28-286-0478; E-mail: s-hama{at}dokkyomed.ac.jp
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Abstract |
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Methods: Thirty gerbils were randomly assigned to five groups: physiological saline solution (PSS) group, ischemia/reperfusion treated with PSS 2 mL•kg-1; 5% HSS group, treated with 5% HSS 2 mL•kg-1; 7.5% HSS group, treated with 7.5% HSS 2 mL•kg-1; 10% HSS group, treated with 10% HSS 2 mL•kg-1; 20% HSS group, treated with 20% HSS 2 mL•kg-1. Transient forebrain ischemia was induced by occluding the bilateral common carotid arteries for four minutes. Five days later, histopathological changes in the hippocampal area were examined, and the degenerative ratio of the pyramidal cells were measured according to the following formula: (number of degenerative pyramidal cells/total number of pyramidal cells per 1 mm of hippocampal CA1 subfield) x 100.
Results: In PSS and 20% groups, neuronal cell damage was observed five days after ischemia. In the other three groups, these changes were not observed. The degenerative ratios of pyramidal cells were as follows; PSS group: 91.6 ± 5.6%, 5% HSS group: 7.2 ± 1.6%, 7.5% group: 8.3 ± 1.4%, 10% HSS group: 6.2 ± 1.1%, 20% HSS group: 85.8 ± 8.7% (P < 0.05; PSS and 20% HSS vs three other groups).
Conclusion: This study demonstrates that 5, 7.5 or 10% HSS 2 mL•kg-1 may prevent delayed neuronal death in the hippocampal CA1 subfield after cerebral ischemia/reperfusion in gerbils.
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Introduction |
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The purpose of this study was to clarify the appropriate concentration and dose of HSS to protect against delayed neuronal death after a short period of ischemia/reperfusion in gerbils.
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Methods |
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Thirty Mongolian gerbils weighing between 65–90 g were anesthetized with 50% nitrous oxide in oxygen with halothane (end-tidal concentration: 1.0–1.5%) through a rubber mask. The animals were placed in the supine position and the common carotid arteries were exposed bilaterally. The arteries were occluded with miniature aneurysmal clips for four minutes. During this period we verified the absence of blood supply from the vertebral arteries with a microscope. Four minutes after occluding both carotid arteries, the clips were released and we visually confirmed spontaneous circulatory reperfusion with the microscope. The temperature of the tympanic membrane was monitored (Mon-a-therm Model 6510; Mallinckrodt, CA, USA) and maintained with a heating blanket at 37 ± 0.2°C during the experiment. Then, 2 mL•kg-1 of physiological saline solution (PSS; 154 mEq•L-1), 5% HSS (855 mEq•L-1), 7.5% HSS (1283 mEq•L-1), 10% HSS (1711 mEq•L-1) or 20% HSS (3422 mEq•L-1) were injected via the tail vein. Six gerbils were used in each of the five groups. The animals were kept and fed in a cage at a room temperature of 26°C.
Five days later, laparotomy and thoracotomy were performed on all gerbils under halothane anesthesia. Fixation was achieved by irrigation with a fixative solution (1:2:7 ratio of 4% formaldehyde: phosphate buffer solution: distilled water) via the heart. The bodies were stored in a refrigerator at 4°C for two hours. A craniotomy was performed and the cerebrum was extracted and embedded in paraffin which was sliced into 5 µm sections in the coronal direction and stained with hematoxylin and eosin. Histopathological changes in the hippocampal CA1 subfield were examined using a light microscope (Olympus BH-2, Olympus, Tokyo, Japan). Morphological changes were detected in the pyramidal cells and structural changes in the cell layers of the CA1 subfield of the hippocampus. In order to compare the ratios of degenerative pyramidal cells between the five groups, the total number and the number of degenerative pyramidal cells were counted over a uniform 1-mm length of the central area of the hippocampal CA1 subfield. The degenerative ratios were determined according to the following formula: Degenerative ratio (%) = (number of degenerative pyramidal cells/total number of pyramidal cells per 1-mm of hippocampal CA1 subfield) x 100.
Signs of degenerative processes such as atrophy of the pyramidal cells, deep staining of their cell bodies, vacuolation, and disappearance of the radial striated zone were taken to indicate cell degeneration, and cells in which these signs were absent were regarded as normal neurons.1 Evaluation of the cell damage was performed by a single pathologist blinded to the experimental conditions.
Data are presented as mean ± SD. Kruskall-Wallis one-way analysis of variance was used for the statistical comparisons between each group. Statistical significance was considered to be P < 0.05.
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Results |
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), almost all the pyramidal cells were atrophic, and they exhibited nuclear pycnosis, deep staining of the cell cytoplasm, vacuolation, and structural disruption of the radial striated zone due to degenerative necrosis. In the 5, 7.5 and 10% groups, the normal hippocampal pyramidal cells were well maintained, and no structural disruption of the radial striated zone was detected (Figure b–d). In the 20% HSS group, however, the pyramidal cells of hippocampal CA1 subfield showed degenerative cell damage and structural disruption of the radial striated zone similar to the PSS group (Figure e).
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Discussion |
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Following transient ischemia of the brain, extracellular sodium ions enter the neuronal cells and accumulate there, as depletion of intracellular adenosine triphosphate (ATP) inactivates Na-K ATPase and increases the pH-regulatory Na-H exchange. This intracellular accumulation of sodium ions leads to the extracellular increase of glutamate as an excitatory neurotransmitter and accumulation of intracellular calcium ions, which are associated with delayed neuronal death. Intravenous injection of HSS may compensate for decreased extracellular sodium ions after ischemia/reperfusion and correct the imbalance between extracellular and intracellular concentrations of sodium ions caused by hypoxia. Ho et al.15 reported that hypertonic perfusion minimized intracellular sodium uptake and reduced accumulation of intracellular calcium in the hypoxic rabbit myocardium. Similarly, we speculate that less than 20% HSS 2 mL•kg-1 may minimize sodium uptake in the neuronal cells, prevent extracellular increase of glutamate and reduce the accumulation of intracellular calcium in the hypoxic hippocampus.
Excessive administration of sodium ions causes acute swelling of neuronal cells and neuronal death.16 Following 90 min of hypernatremia, the sodium, potassium, and chloride ions enter the neuronal net tissues, especially the dendrite and soma. This alteration of electrolytes and osmotic pressure produces swelling and necrosis of neuronal cells. Lien et al.17 reported that severe chronic hypernatremia (serum sodium 180 mEq•L-1, seven days) induced an intracellular increase of idiogenic osmoles, which included myoinocitol, betaine, taurine, glutamine and glutamate, to reduce the transmembrane osmolarity gradient in the brain. However, the overload of sodium ions due to acute hypernatremia may not induce an intracellular increase of idiogenic osmoles. In the present study, we could not find histopathological differences between PSS and 20% HSS 2 mL•kg-1 using a light microscope although transient ischemia and excessive administration of HSS might increase intracellular sodium ions, followed by neuronal damage. Therefore, excessive elevation of extracellular sodium after iv injection of higher doses of HSS may induce neuronal damage. Additional studies are needed to examine the differences between PSS and 20% HSS 2 mL•kg-1 using an electron microscope.
In addition to the prevention of neuronal damage after a short period of cerebral ischemia/reperfusion, small doses of HSS are effective for cardiopulmonary resuscitation from hemorrhagic shock in animals.18–20 Mean arterial pressure, cardiac output, mean pulmonary arterial pressure, central venous pressure and heart rate are increased from minutes to hours after iv injection of HSS, and total peripheral vascular resistance and pulmonary vascular resistance decrease because of improvement of microcirculation.21
Petito et al.13 reported a clinical case of hippocampal delayed neuronal death after cardiopulmonary arrest. The iv administration of an appropriate dose of HSS may not only improve hemodynamic changes but, possibly, also prevent neuronal damage such as postresuscitation encephalopathy in shock patients.
In conclusion, we have shown that administration of 2 mL•kg-1 of 5%, 7.5% and 10% HSS prevents delayed neuronal death in the hippocampal CA1 subfield after a short period of cerebral ischemia/reperfusion in gerbils.
Revision received May 27, 2002. Accepted for publication March 11, 2002.
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