and PGI 2 and 6- keto-pgf1a levels in the once group while TXB 2

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1 JOURNAL OF LOW FREQUENCY NOISE, VIBRATION AND ACTIVE CONTROL Pages The Changes and Significance of TXA 2 and Metabolism After Rat Infrasonic Brain Damage Zhou Fei Ph.D 1, Xiang Zhang Ph.D 1, Zhi-Gang Li 1, En-Yu Liu 1, Hong-Min Bai 2, Jing-Zao Chen Ph.D 2 and Ke-Yong Jia 2 1 Department of neurosurgery, Xijing Hospital, Fourth Military Medical University 2 Department of Physiotherapy, Xijing Hospital, Fourth Military Medical University, Xi an , P. R. China feizhou@fmmu.edu.cn Received 4th April 2001 ABSTRACT In this paper we study the changes and significance of the TXA 2 metabolism after infrasonic damage to rats so as to probe the mechanism of infrasonic biological effects. Some 40 SD rats were randomized into control, infrasonic damage once, 7 times, 14 times, and treatment five groups. A stimulus of 8Hz 120dB infrasound was applied to the infrasonic damage groups for 2 hours once according to the planned times. Radioimmunoassay was used for the examination of brain TXB 2 keto-pgf1a (metabolites of TSA 2 and ) contents. The results showed that there were no changes in TXB 2 keto-pgf1a levels in the once group while TXB 2 level elevated significantly keto-pgf1a level decreased remarkably in the 7 times and the 4 times groups with an augmentation of TXA 2 value. In the treatment group both TXB 2 levels nearly reached normal. It is concluded that the augmentation of the TXA 2 value in the 7 times and 14 times groups suggests that an imbalance in TXA 2 production contributes to the traumatic secondary processes, which include ischemia and edema. Also, the extent of TXA 2 metabolism disturbance is closely related to the times of infrasonic injury. It is hypothesized that mglur antagonist may exert its protective properties through increasing production in the injured brain by improving the blood supply to injured blood vessels. Key words: Infrasonic brain injury TXA 2 1. INTRODUCTION Infrasound is a sound wave in the low frequency sound range below 20Hz. Recently trends have shown an increase in its role as an unfavourable factor in industrial, transport, and residential environments (Refs.1). Hence, research on the mechanism of its biological effects makes good sense. It was shown that the brain is the primary target of infrasonic damage. In our previous study it was shown that infrasound can directly damage the function and structure of cerebral ultrastructure and BBB, and that the extent of brain damage and positive HSP70 neuronal expression is closely related to the duration of infrasonic injury (Refs.2). For some time the involvement of arachidonate metabolism in the complex pathophysiology of brain injury, ischemia and vasospasm had been suggested. Secondary brain damage caused by head injury or ischemia may be aggravated by prostaglandins such as thromboxane A 2 (TXA 2 ), a labile, potent vasoconstrictor and a proaggregatory compound by leading to a focal increase in micro-circulatory resistance (Refs. 3-4), or may be Vol. 20 No

2 TXA 2 Metabolism After Rat Infrasonic Brain Damage ameliorated by prostycyclin ( ), a potent vasodilator and antiaggregator compound, working as part of a hyperaemic response mechanism providing increased levels of energy and precursors to injured tissue undergoing repair (Refs. 5). We wondered whether the infrasonic brain damage is closely related to the changes of the TXA 2 metabolism, which is central to this study. 2. METHOD 1. Animal grouping and modelling: Forty healthy male SD rats, weighing 200 ± 15 g were used. The rats were kept in a dark, warm and quiet facility with free access to food and water for one week before the experiment, and then were randomized into control, infrasonic damage once, 7 times, 14 times and MCPG treatment five groups. Each group includes 8 rats. The rats in the infrasonic damage groups and in the MCPG treatment group were exposed to infrasound of 8Hz at 120dB for 2 hours once according to the planned schedule in the infrasound pressure chamber (Ref. 6). We chose 8Hz infrasound in this study because rates are very sensitive to this frequency and the dangerous range of infrasound is db (Ref. 7). 2. Prostaglandin Measurement: At a planned time after the above-mentioned procedures the rats were decapitated and the brains rapidly frozen in liquid nitrogen. 100 mg of right frontal cortex was removed and weighed by a digital balance (Denver Instrument Company) and then homogenized, extracted and preserved for assay. The prostanoid levels, TXB 2 (the stable metabolite of TXA 2 ) keto-pgf1a (the stable metabolite of ) were measured via radioimmunoassay (RIA). This assay was carried out using commercially available RIA kits (Dupont, Wilmington, U.S.A.). The percentage crossreactivity of these assays was <0.01 for TXB 2 and 0.4 for 6-keto-PGF1a. The absolute sensitivity of the assays was 0.5 pg /0.1 ml for both TXB 2 keto-pgf1a. In the treatment group mglur1 and mglur 2 antagonist MCPG (0.8mg/kg, Ref.8) was injected via a tail vein on the first time on the 12th day of infrasonic damage and 6 and 24 hours intraperitoneanly afterwards. 3. Statistical analysis: Data are presented as means ± standard deviation (SD). A one-way analysis of variance (ANOVA) with Duncan s multiple comparison tests was used to compare significant differences in TXB 2 keto-pgf1a levels among the groups. Levels of TXB2, 6-keto-PGF1 a after infrasonic brain damage ( X +Sx, pmol/mg.pro#) Group No. TXB 2 6-keto-PGF 1 a TXA 2 Control ± ± Infrasonic damage once ± ± Infrasonic damage 7 times ± 5.22* ± 1.66* 2.12 Infrasonic damage 14 times ± 5.05* ± 1.65* 2.96 Treatment group ± 3.73 * ± 2.89 * 1.91 * p<0.01 as compared with the control values p<0.01 as compared with 14 time group #picogram per milligram protein in supernatant of 100 milligram brain tissue homogenate 3. RESULTS 1. Control: The TXB 2 and the 6-keto-PGF1a levels are shown in table I. 2. Infrasonic damage once group: There are no changes in TXB 2 keto- PGF1a levels compared with that of the control group (P>0.05, Table I). 3. Infrasonic damage 7 times and 14 times group: TXB 2 level increased sharply but 6-keto-PFG1a decreased significantly compared with that of the control group (P<0.01). The TXA 2 value in the 7 times group is 2.12, and in the 14 times group 2.96 (Table I). 34 JOURNAL OF LOW FREQUENCY NOISE, VIBRATION AND ACTIVE CONTROL

3 Zhou Fei Ph.D, Xiang Zhang Ph.D, Zhi-Gang Li, En-Yu Liu, Hong-Min Bai, Jing-Zao Chen Ph.D and Ke-Yong Jia 4. Treatment group: Both TXB 2 keto-pgf1a levels and TXA 2 value nearly reach the control levels compared with those of the infrasonic damage 14 times group (Table I). 4. DISCUSSION Research has shown that the effects of infrasound result in functional and structural disorders of the central nervous system with symptoms such as headache, dizziness and vomiting. In some severe cases, every day life and work can be affected (Ref. 9). Furthermore, infrasound can damage the cortex, limbicorticular complex, hypothalamus, and other subcortical structures, leading to infrasonic diencephalic hypothalamic syndrome. The cortex, hippocamp and hypothalamus belong to the most sensitive tissues to infrasound (Ref. 10). That is why we chose the cortex from our sampling tissues in our study. Polyunsaturated fatty acid is rich in brain tissue where active metabolism of arachidonic acid (AA) takes place. TXA 2 are biologically short-lived products of AA, and TXA 2 stimulates platelet aggregation and vasoconstriction whereas antagonizes TXA 2 activity. These eiconsanoids play biologically opposite roles in the regulation of vascular homeostasis. These two compounds are very labile and degraded into the stable metabolites TXB 2 keto-pgf1a, which can be measured in biological materials. Vasoactive arachidonic acid metabolites are postulated to play roles in the pathogenesis of head injury and ischemic brain conditions (Ref. 11). In our previous study, in a new rodent model of impact acceleration, diffuse brain injury alone and with hypotension and hyperthermia in combination, we found that the production of these vasoactive arachidonic products, especially TXA 2, is closely connected to the severity of the brain damage (Ref. 12). To evaluate the extent of infrasound as a traumatic factor, the study on the changes and significance of the TXA 2 metabolism is of great importance. We studied the changes of the TXA 2 metabolism after different periods of infrasonic damage. In our study there are no changes in TXB 2 keto-pgf1a levels compared with that of the control group in the once group (P>0.05). However, in the 7 and 14 times groups TXB 2 level increased sharply but 6-keto- PGF1a decreased significantly compared with that of the control group (P<0.01). There is a trend towards an increased TXA 2 ratio between the 7 and 14 times groups. The reason for this may be that is produced dominantly by vascular endothelial cells (Ref. 13), thus in the case of infrasonic damage, the integrity of endothelial cells is essential for synthesis and for the expression of its protective effect. It is suggested that an imbalance in cortical TXA 2 production contributes to the traumatic secondary processes, which include ischemia and edema, and BBB breakdown. The facts that the metabotropic glutamate receptor antagonist (a- methyl 4 carboxyphenlycine) protects hipocampal CA 1 neurons of the rat from in vitro hypoxia/hypoglycemia created new hopes for brain protection (Refs. 14). However its protective mechanism remained unclear. In our present study both TXB 2 keto-pgf1a levels and TXA 2 values almost reached control levels compared with those of the infrasonic damage 14 times group. Interestingly, we also found that level increased significantly for the expression of its protective effect compared with that of the 7 and 14 times groups. In our previous study the pivotal role of infrasound in brain damage had been identified by pathological studies. For example, in the control group, the cellular membrane of neurons and the ultrastructure remained intact. In the infrasonic damage once group, the neuronal structure shows slight changes in which obscure neuronal membrane and vacuolation of neurons were found. In addition, a decrease in the number of mitochondria, mitochondrial swelling, and disappearance of mitochondrial crista could be seen. In the infrasonic damage 7 times group, some vacuolation of neurons and deformation of nuclei were found. Mitochondrial swelling, disappearance of mitochondrial crista and enlargement of Gorgi s body could also be seen. And in Vol. 20 No

4 TXA 2 Metabolism After Rat Infrasonic Brain Damage the infrasonic damage 14 times group: rupture of cellular membranes, lysis of nuclei and vacuolation of mitochondrial and Gorgi s body could be seen clearly under the microscope (Refs. 2). It is shown in our recent study (unpublished) that after the administration of MCPG, the injured neurons by infrasonic damage in the hippocamp CA 1 area decreased greatly. Therefore it is hypothesized that MCPG may exert its protective properties through increasing production in the injured brain by improving the blood supply to injured blood vessels. In short, the augmentation of TXA 2 values in the 7 and 14 times groups suggests that an imbalance in TXA 2 production contributes to the infrasonic brain damage processes, which include ischemia and edema. Similarly, the extent of TXA 2 metabolism disturbance is closely related to the duration of infrasonic injury. It is hypothesized that the mglur antagonist MCPG may exert is protective properties through increasing production in the injured brain by improving the blood supply to injured blood vessels. ACKNOWLEDGEMENT This work is supported both by National Natural Science Foundation of China ( ) and Foundation for University Key Teacher by the Ministry of Education. REFERENCES 1. Kuralesin, N.A., (1997) Hygienic and biomedical aspects of the effects of infrasound. Med-Tr-Prom-Ekol, No. 5, Fei Zhou, Zhang Xiang Lu Peiling, et al., The changes and significance of cerebral ultrastructure and the expression of heat shock protein 70 in cortex after infrasonic damage. J Low Freq Noise Vib 2000; Vol. 19(2): Nishisho, T., Tonai, T., Tamura, Y., et al., (1996) Experimental and clinical studies of eicosanoids in cerebrospinal fluid after spinal cord injury. Neurosurg, Vol.39, No.5, Hamberg, M., Samuelsson, B., (1974) Prostaglandin endoperoxides- novel transformation of arachidonic acid in human platelets. Pro Natl Acad Sci (USA), Vol. 71, No. 9, Shohami, E., Shapira, Y., Cotev, S., (1988) Experimental closed head injury in rats: Prostaglandins production in a non-injured zone. Neurosurg, Vol. 22, No. 5, Xing, X.H., Wang, L., Rao, Z.R. et al., (1998) Infrasonic stimulation induced Fos expression in medullary catecholaminergic neurons in rats, J Low Freq Noise Vib., Vol. 17, No. 3, Johnson D.L., (1979). Effects of infrasound on respiration. Presented at the Aerospace Medicine Association Meeting 1979; 5:37 8. Gong-QZ., Delahunty-TM; Hamm-R.J. et al., Metabotropic glutamate antagonist, MCPG, treatment of traumatic brain injury in rats. Brain-Res Nov 27: 700(1-2): Ulf Landstrom., Laboratory and field studies on infrasound and its effects on humans. (1987) J Low Frequency Noise and Vibration, Vol. 6, No. 6, Izmerov, N.F., Suvorov, G.A., Kuralesin, N.A. et al., (1997) Infrasound: body s effects and hygienic regulation. Vestn-Ross-Akad-Med-Nauk, No. 7, JOURNAL OF LOW FREQUENCY NOISE, VIBRATION AND ACTIVE CONTROL

5 Zhou Fei Ph.D, Xiang Zhang Ph.D, Zhi-Gang Li, En-Yu Liu, Hong-Min Bai, Jing-Zao Chen Ph.D and Ke-Yong Jia 11. Katayama, Y., Shimizu, J., Suzuki, S., et al., (1990) Role of arachidonic acid metabolism on ischemic brain edema and metabolism. Advances in Neurology Vol. 52, Fei, Z., Zhang, X., Yi, S.Y., et al., (1999) Brain TXA 2 levels related to diffuse brain injury with secondary insults. J Clinical Neurosciences, Vol. 6, No. 4, Weksler, B.B., Marcus, A.J., Jaffe, E.A., (1977) Synthesis of prostaglandin (prostacyclin) by cultured human and bovine endothelial cells. Proc Natl Acad Sci USA, Vol. 74, No.9, Nicoletti, F., Bruno, V., Copani, A., et al., (1996) Metabotropic glutamate receptors: a new target for the therapy of neurodegenerative disorders. TINS, Vol. 19, No. 7, Vol. 20 No