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SUMMARY :

The effect of electric fields in different directions on protein synthesis was studied on Guinea Pigs. Electric field which is obtained from 150 V potential difference applied in vertical and horizontal directions and their effects on collagen synthesis were analyzed by studying hydroxyproline level of liver tissue with Stegemann-Stalder's method.A statistically significant decrease was found in hydroxyproline levels in both of the horizontal and vertical E field applied groups. It was also noticed that the vertical E field was more effective than the horizontal.The results showed that both of the electric fields-vertical and horizontal - decreased liver tissue hydroxyproline content but the vertical one is more effective than the horizontal electric field.

Key Words : Electric Field, Hydroxyproline, Liver Tissue

In the oxidation phase of hydroxyproline only Stegemann - Stalder and Woessner used chloramin - T, while others chosed hydrogen peroxide. Dahl - Persson showed that by using chloramin - T the same sample's hydroxyproline amount gave more sensitive result (15). For this reason Stegemann and Stalder's method was used in this study.

MATERIALS AND METHODS

Experiments were performed on 40 male, Guinea Pigs with an average weight of 350-400 g. They were separated into three groups. In Group I, 10 Guinea Pigs exposed to the vertical electric field while another 10 in Group II exposed to the horizontal electric field. 20 Guinea Pigs were also kept in the cages at the same laboratory conditions and studied as Control Group without any electric field exposure. Three animals in 3 different cages were studied simultaneously each time.Vertical and horizontal electric fields with the magnitude of 580 V/m were applied to Guinea Pigs in wooden cages wih dimensions of 50 cm x 50 cm x 14 cm. Exposure period was 9 hours / day for 3 days throughout the study.Potential differences applied to the cages were 150 V and obtained from high intensity power supplies. Voltage was kept fixed and controlled continuously with a voltmeter in the exposure circuit.Vertical Field Exposure CircuitCopper plates with dimensions of 50 cm x 50 cm x 0.1 cm were mounted on the top and the bottom faces of the cages.Positive probe of the power supply was always connected to the upper plate and negative probe to the lower plate for all of the cages.Horizontal Field Exposure CircuitThe plates were mounted to the left and right faces of the cages.Positive probe of power supply was always connected to the right plate and negative probe to the left plate for all of the cages.Duration of electric field exposure was from 8ûû am to 5ûû pm, 9 hours / day. Wooden blocks (60 cm x 60 cm x 2 cm) were used under the cages for isolation.Liver tissue hydroxyproline contents of the animals of exposure groups and control group were determined with Stegemann - Stalder's Method (33). The basic principle of this method is to get the hydroxyproline of the tissue by hydrolization of the sample after homogenization and measuring the optical density of the color formed by adding p-dimethylaminobenzaldehyde, perchloric acid and propan - 2 -ol at pH=8 and at l = 560 nm. Hydroxyproline contents of the tissue samples were determined by using the standard curves which were plotted before each spectrophotometric reading by using the sample containing known concentrations of Hydroxyproline (SIGMA H-1637).

RESULTS

Two samples were studied for hydroxyproline determination after the homogenization of each liver tissue. The average hydroxyproline concentration value was taken into consideration at the calculation step in determining the hydroxyproline content of liver tissue.Statistical comparisons were made in two groups :1. For each group, hydroxyproline contents of lung tissues of the electric field applied groups and their controls were compared with DUNCAN test (Table 1) and the results were found as follows :- There is statistically significant difference between hydroxyproline contents of liver tissues of vertical electric field applied and control groups (p<0.01). Vertical electric field application led to decreased liver tissue hydroxyproline level

(Hyd.E.Vertical = 0.0779 ± 0.0228; Hyd.Control = 0.2615 ± 0.1450). - There is statistically significant difference between hydroxyproline contents of liver tissues of horizontal electric field applied and control groups (p<0.05). Horizontal electric field application resulted in significantly decreased liver tissue

hydroxyproline level (Hyd.E.Horizontal=0.1194 ± 0.032; Hyd.Control = 0.2615±0.1450). 2. A comparison was made between the vertical and horizontal field groups to specify the effective direction of the electric field application : Vertical electric field decreased hydroxyproline content of liver tissue more than the horizontal electric field although the difference was not statistically significant (p>0.05).

DISCUSSION

The effect of electric field in different directions on collagen sythesis was investigated by detecting the variations in tissue hydroxyproline content. Both the vertical and horizontal E fields created by 150 V potential difference led to decreased liver tissue hydroxyproline levels, whereas vertical E field was found to be more effective than the horizontal.

In recent years a body of data on the interactions of exogenous and endogenous electromagnetic fields (em) with biological systems has been gatherede. As a result of these findings our understanding of biological function has started to change. It would be incredible if em fields did not effect the electrochemical systems in living organisms. And since living organisms have only so recently found themselves immersed in this environment, they have not had an opportunity to adapt to it. This gives us, as biophysicists, the opportunity to use exogenous em fields to study the functioning of living systems. As in the other studies on living organisms including bacteria with em fields, the data collected is very few (2, 3, 4, 5, 6, 12, 13, 17) and are required to be In this article it has hypothesized that the exogenous electric field has effect on collagen synthesis in exposed tissues and the effect of electric field in different directions - vertical and horizontal - on collagen synthesis was investigated by detecting the changes in hydroxyroline content of liver tissues of the exposed animals. The method used for detecting hydroxyproline amount of the tissue was Stegemann - Stalder modified method (33).The first method used for hydroxyproline determination was Neuman - Logan's method (26). This method was modified by Barker et al, Martin and Axelrod, Wierbicki and Deatherage, Miyada and Tappel, Arenson and Elvehjem, Grunbaum and Glick, Antonacopoulos, Lollar, Laech Woessner, Stegemann and Stalder

There are researches that E fields are effective on tissues (6, 17, 25, 27, 28, 30, 35).Internal E fields existing in tissues and external E fields transfer energy on the tissue in two different ways (19) :

1. In the tissues under the effect of E fields, positive charges moves in the same direction with E field while the negative ones move in the opposite direction, causing the tissue polarized.

1. Dipole moment vectors of polar molecules like H2O, rotate to get the same direction with the externally applied E field. As a result an internal E field arises and its interaction with the external E field lessens the net E field in the tissue.
2. External E field applied living organisms are being examined in the senses of different E fields that arises on body surface and inside of the body, current intensity created in the body and the net electric current occuring along the whole body.
3. In the modelling studies for human and animals, it has been possible to determine the E field and electric current values arising inside and on the surface of the model by the effect of an externally applied field with the help of the developments in the biomedical electrode techniques. Using human, rat and pig models the values of surface electric fields and current densities generated with a vertical E field of 10 kV/m, and 60 Hz frequency are determined. The maximum sensitivity is localized at the head (180 kV/m) for the human, and at the upper body

for the pig (67 kV/m) and for the rat (37 kV/m). Diferent tissues have different resistances and dielectric properties, and the external E field will always be greater than the induced E field inside of the body (9, 20, 21, 22).It has been possible to direct collagen under E field. If a collagen solution soluble in acid is applied 1 µA direct current, collagen form a concave band near cathode (16, 18, 32). This finding shows that collagen acts as a cation under E field.Proteins having a net electrical charce attain a movement under the influence of electric field. Since macromolecules such as proteins have different moleculer structure and shapes, they are exerted different frictional forces in a medium. For these reasons different protein molecules have different velocities when they are inserted in the same electric field. Amino and carboxyl groups are responsible from the net charge of a protein molecule. Secondly, proteins may gain positive or negative charge or they may have no charge according to the pH value of the medium. In the latter case proteins do not have the capability of movement in a electric field. If pH of the medium is less than the isoelectric pH of protein, then it posses a net positive charge and moves in the direction of electric field, whereas it gain a negative charge and move in the opposite direction with the applied electric field in the case that the pH value of the medium is to be greater than the isoelectric pH of the protein (14). The DC electric field with the strenght of 10 kV/m has the ability to move the protein molecules along the membrane surface (8).When low intensity direct current is applied to tissues, O2 is consumed at cathode. Tissue O2 tension decreases and an increase occurs in the number of hydroxyl radicals present (7, 10, 11). Free oxygen radicals have toxic effect on proteins and DNA giving result to structural and functional defects in the cell. Amino acid compositions of proteins specify the extent of influence from free radicals. Unsaturated molecules and sulphure containing molecules are highly sensitive to free radicals. Besides the oxidation of amino acids, free radicals give rise to hydrolysation of peptide bonds, disulfide bonds, and cross bindings resulting the defects in functions of enzymes, disturbing ion seperation in and out of the cell, and causing serious injury of cell. The increase in radicals can be traced with the variation in malondialdehyde (MDA) level (37). In our another study carried out parallel to this study, it was found
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SUMMARY :
Middle latency auditory evoked potentials (MLAEPs) were studied in 30 definite multiple sclerosis (MS) patients in addition to brain-stem auditory evoked potentials (BAEPs). BAEP abnormalities were detected in 18 (60 %) patients. MLAEPs were abnormal in 22 (73 %) of them. In 15 patients BAEPs and MLAEPs were both abnormal. MLAEPs were found abnormal in 7 of the 12 patients with normal BAEPs. On the other hand in 18 patients with abnormal BAEPs only 3 had normal MLAEPS. MLAEPs abnormalities are compatible with a rostral auditory pathway involvement. Therefore they can be used in combination with BAEPs to examine the whole auditory system to improve the sensitivity.Key Words : Brain - Stem Auditory Evoked Potentials, Middle Latency Auditory Evoked Potentials, Multiple Sclerosis.

INTRODUCTION
A specific method of exploring the central auditory pathways from auditory nerve to cortex is the use of auditory evoked potentials (AEPs) recorded over the scalp after repetetive acoustic stimulation.AEPs are classified as early responses (0-10 ms), middle latency responses (10-50 ms) and late responses (50-500 ms) (7, 9).Early components depend upon the integrity of the eight nerve and the brainstem whereas the late components represent the vertex response and probably arise from cortical structures (11).Middle latency auditory evoked potentials (MLAEPs) have recieved a renewed interest in the last decade (4, 6). They are composed of several components that can be detected from 10 to 50 ms after stimulus onset. These components have been labeled by Picton et al as follows : No, Po, Na, Pa, Nb, Pb (9). Recent investigations have served to uncover the electrophysiological origins of these potentials (6).Clinical application of MLAEPs is limited because they are often interfered with myogenic potentials and the generator sites of components are still not clear (2, 6, 7, 12). No and Po potentials mostly overlap with myogenic potentials. Nb and Pb on the other hand show considerable interindividual variability. Na and Pa are the only stable components that are constantly obtained in normals between 16 and 30 ms after click stimulation (3, 12). Therefore they are taken into consideration in clinical papers.Recent studies suggest different generators for Na and Pa (3, 13). It has been proposed that Na is generated subcortically and that Pa is generated When BAEPs were compared with MLAEPs; they were both abnormal in 15 (50 %) patients. In 18 (60 %) patients with abnormal BAEPs 3 had normal MLAEPs. On the other hand, in 12 patients with normal BAEPs, MLAEPs were abnormal in 7 (58.3 %). Number of patients with normal and abnormal BAEPs and MLAEPs can be seen in Table 2.Since there was no significant difference for latency and amplitude between the right and left ears of normal individuals, the values for the two ears were pooled for control data and the mean Na and Pa latencies obtained from normal individuals were compared with the latencies obtained from the MS patients. In the patient group prolonged latencies were observed. The difference were statistically significant for both Na (p=0.001) and Pa (p=0.004) (Table 3).

MATERIALS AND METHODSMonoaural BAEPs and monoaural and binaural MLAEPs were studied in 30 definite MS patients (7 men and 23 women, ages ranging from 19 to 51, mean 33.2) who were diagnosed according to the Poser criteria (10). Both BAEPs and MLAEPs were recorded in a control group of 12 adult volunteers (10 men and 2 women, ages ranging from 18 to 65, mean 36). Stimulation and recordings were performed by a Nihon Kohden Neuropack 5200. After taking their informed consent subjects were seated comfortably in a semidarkened, quiet room with their eyes closed. Acoustic stimuli were alternating clicks with a duration of 0.1 ms. Intensity was fixed at 90 db. During monoaural stimulation -40 dB masking noise was delivered to the contralateral ear. Responses were recorded by silver-silver chloride electrodes placed on the scalp.For BAEPs ipsilateral mastoid-Cz electrodes were used for recordings. Following amplification (bandpass 100-300 Hz) 1024 individual responses were averaged with an analysis time of 10 ms. The repetetion rate was 10 Hz. I-III, III-V and I-V interpeak latencies (IPL) were taken into consideration.For MLAEPs Cz-C7 neck electrodes were used for recordings. Amplifier limits were between 5-1000 Hz 1024 individual responses were averaged with an analysis time of 50 ms. The repetition rate was 5 Hz. Na and Pa latencies and Na-Pa peak to peak amplitude were measured.
Latencies were considered abnormal when their values exceeded mean control values by more than 2 standard deviation (SD).For the MLAEPs group analysis Na and Pa latencies were taken into consideration. Statistical analysis was performed using the student's t test.

RESULTSBAEPs were abnormal in 18 (60 %) MS patients. Peak V was absent bilaterally in 3 and unilaterally in 5 patients. In 4 patients I-V IPL was prolonged bilaterally. In 4 patients unilaterally prolonged I-V IPL with contralateral absence of peak V was detected. In 2 patients I-III interpeak latency was prolonged unilaterally.Table 1 demonstrates the mean Na and Pa latencies obtained from 12 healthy volunteers by monoaural and binaural stimulation. The mean latencies of Na and Pa recorded by monoaural or binaural stimulation were not significantly different from each other. Na-Pa peak to peak amplitude was found larger after binaural stimulation. Figure 1 demonstrates MLAEPs recorded from two normal individuals. Figure 2 demonstrates MLAEPs recorded from the same individual after monoaural and binaural stimulation.MLAEPs were abnormal in 22 (73.3 %) MS patients. In Figure 3 unilateral absence of Na and Pa in an MS patient can be seen. In Figure 4 delayed Na and Pa potentials in 2 MS patients are shown. Na was prolonged unilaterally in 4 patients, bilaterally in 3 patients. It was absent unilaterally in 3 and bilaterally in 7 patients. In two patients it was prolonged on one side and absent on the other. Pa was prolonged unilaterally in 3 and bilaterally in 3 patients. It was unilaterally absent in 3, bilaterally absent in 7 patients. In 2 patients it was absent on one side and was prolonged on the other. Figure 5 and 6 demonstrates the results obtained from the patients.

DISCUSSION
MLAEP components are largely distributed over both hemispheres being maximally recorded over fronto-central scalp regions (3, 4, 9). The Na component is currently believed to originate subcortically from the midbrain, thalamus or thalamocortical radiations (3, 4, 6, 8, 9). Pa component on the other hand is thought to be mainly related to the simultaneous activation of both supratemporal auditory cortices (1, 2, 3, 4, 6, 7). A deeper midline generator system has also been suggested (5). MLAEP recording in addition to BAEPs can reflect brain activity from cochlea to temporal cortex. By this way, they can be used in the objective evaluation of various neurological disorders including MS.In our study, BAEPs were abnormal in 60 % of the patients. On the other hand MLAEP abnormalities were detected in 73.3 % of the patients. In a large series studied by Robinson and Rudge, BAEP abnormalities up to 79 % have been reported in patients with clinical evidence of brainstem involvement. In those with no clinical evidence of a brain-stem disorder the abnormality rate was 51 %. In the same study, 45 % of the definite MS patients had abnormal latenciesy of one or more MLAEP components. Similar to the BAEP studies there was a higher proportion of abnormalities in those patients with a clinically apparent lesion in the brainstem (70 %) than in those with no evidence of such involvement (11). Poser et al have reported abnormalities up to 80 % in MS patients (10). Versino et al reported BAEP abnormalities in 35 % of their MS patients where MLAEPs were abnormal in 47 % (12).In 12 of our patients with normal BAEPs 7 (58.3 %) had abnormal MLAEPs. Robinson and Rudge reported this occuring in 12 % of their cases.In conclusion, the present work demonstrates that MLAEPs are of value in detecting the occurrence of auditory impairment and can be used as a supplementary technique to BAEPs.Correspondence to : Dr.Neþe ‚ELEBÜSOY Ege Üniversitesi Týp Fakültesi Nšroloji Anabilim Dalý Bornova 35100 ÜZMÜR - TÜRKÜYE Phone : 232 - 388 09 80

REFERENCES

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3. Deiber MP, Ibanez V, Fischer C, Perrin F, Mauguiere F : Sequential mapping favours the hypothesis of distinct generators for Na and Pa middle latency auditory evoked potentials. Electroenceph Clin Neurophysiol 1988; 71 : 187-187
4. Ibanez V, Deiber MP, Fischer C : Middle latency auditory evoked potentials in cortical lesions. Arch Neurol 1989; 46 : 1325-1332.
5. Jacobson GP, Newman CW : The decomposition of the middle latency auditory evoked potential (MLAEP) Pa component into superfical and deep source contributions. Brain Topogr 1990; 2 (3) : 229-236 (Abstract)
6. Jacobson GP,Privitera M, Neils RJ, Grayson SA, Yeh HS : The effects of anterior temporal letm h deanaioy ok ti(LEP). Electroenceph. Clin Neurophysiol 1990; 75 : 230-241.
7. Kaseda Y, Tobimatsu S, Takato M, Kato M : Auditory middle latency responses in patients with nonlocalized and localized lesions of the central nervous system. J Neurol 1191; 238 : 427-432.
8. Mc Gee T, Kraus N, Comperatare C, Aical T : Subcortical and cortical components of the MLR generating system. Brain Res 1991; 544 (2) : 211-220 (Abstract).
9. PictonTW, Hillyard SA, Krausz HI, Galambos R : Human Auditory Evoked Potentials I : Evaluation of components. Electroenceph Clin Neurophysiol 1974; 36 : 179-190.
10. Poser CM, Paty DW, Scheinberg L, et al: New diagnostic criteria for multiple sclerosis : Guidelinesfor research protocols. Ann Neurol 1983; 13 : 227-231.
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12. Versino M, Bergamaschi R, Romani P, Banfi R, Callicco A, Citterio EG, Cosi V : Middle latency auditory evoked potentials improve the detection of abnormalities along auditory pathways in multiple sclerosis patients. Electroenceph Clin Neurophysiol 1992; 84 :296-299.
13. Vizioli L, Bucciero A, Quaglietta P, Mosca F : Auditory middle latency responses in patients with intracranial space occupying lesions. J Neurosurg Sci 1993; 37 (2) : 77-81 (Abstract
    

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SUMMARY :

The purpose of this study is to evaluate the effect of lidocaine, an amid group anesthetic agent, on endothelium-derived relaxing factor (EDRF).For this study, adult rabbit aorta has been used. The response of intact strips and denuded strips to acetylcholine before and after the lidocaine has been added to the media has been meausered separately.The same procedure has been carried out with aspirin, with or without the presence of procaine and prilocaine.Statistically analyzed by Student's t test, addition of lidocaine to the medium caused a significant increase in the relaxation response to acetylcholine in intact strips but not in the denuded strips.Neither procaine nor prilocaine change the relaxation induced by acetylcholine.In conclusion, long lasting analgesic effect of high dosage iv lidocaine has been attributed to the release of EDRF and analgesic effect of factor nitric oxide (NO) which is a kind of EDRF.Key Words : EDRF, Lidocaine.

INTRODUCTIONLidocaine, is one of the most widely used local anesthetic. The anesthetic role of lidocaine has been mainly limited to direct application to nervous tissue, although there have been reports of pain relief after systemic administration (5). It has been considered unlikely that analgesia after systemic administration is due to a direct effect on the excitability of peripheral nerves since it has been shown that neither afferent receptors nor peripheral nerve fibers are significantly blocked by non-toxic doses of lidocaine (9). A possible explanation for the analgesic effect of systemically administered local anesthetics is their central anticonvulsant property (1).Endothelial cells also produce a vascular smooth muscle relaxant, known as endothelium-derived relaxing factor (EDRF) (3). Although the chemical nature of EDRF is still unknown, some studies indicate that one of the EDRF might be nitric oxide (NO) (7). Recently it has been shown that NO has an analgesic effect and therefore it is one of the analgesic mediator of the organism (4).It seemed interesting to us to investigate whether lidocaine had an effect on the release of EDRF from vascular endothelium.

MATERIAL AND METHOD

Aortae were taken from adult rabbits of both sexes (1.5-2.5 kg) anesthetized with sodium pentobarbital (30 mg/kg) dissected free of fat and surrounding tissues and the strips (4 mm width and 2 cm length) were prepared. The endothelium was either left intact or removed by rubber or wooden stick. The strips were then suspended in a jacketed organ bath and isometric contractions were recorded on a GRASS POLYGRAPH (Model 7D) by force displacement transducer (FT-03) under 1.0 g initial tention. Submaximal contication of the strips (% 70 of maximum) was elicited by phenylephrine (10-7 M) and the concentration-response curve of acetylcholine were determined in both endothelium intact and denuded strips before and after adding lidocaine to the medium. Experiments were also repeated in the presence of aspirin added to the medium containing lidocaine in order to eliminate the possible interaction of cyclo-oxygenase products of arachidenic acid in vascular wall. Aortic segments were allowed to contact with lidocaine and aspirin for 15 minutes, then the tests were repeated. In another series of experiments, after control measurements on the aortic strips, procaine and prilocaine were added to the bath and the responses to acetylcholine were repeated.The bathing medium was Krebs solution at the following composition (mM) : NaCl 112; KCl 5; NaHCO3 25; NaH2PO4 1; CaCl2 2.5; MgCl2 0.5; dextrose 11.5. Krebs solution was continuously gassed with 95 % O2 and 5 % CO2 mixture and warmed at 37ûC.The results were statistically analyzed by using Student's t test and presented as mean ± SEM.

RESULTS

Acetylcholine produced a concentration-dependent relaxation in endothelium-intact rabbit aortic strips precontracted with phenylephrine.

This relaxation was completely abolished in endothelium-denuded strips.Addition of lidocaine to the incubation medium at the concentration of 10-6 M for 15 minutes caused a significant increase in the relaxation response to acetylcholine (Fig 1). Aspirin did not change these responses.In endothelium denuded strips, however, the response to acetylcholine was completely abolished and this unresponsiveness was still present in lidocaine-pretreated strips. Contractile responses to phenylephrine were slightly increased by 10 % in endothelium denuded strips.Addition of procaine and prilocaine to the medium of precontracted endothelium intact aortic strips, the relaxation induced by acetylcholine did not change.The results are summarized at Table 1.

DISCUSSION

The data presented in this study indicate that lidocaine enhanced the relaxing effect of acetylcholine in the isolated rabbit aortic strips. The potentiation by lidocaine of the relaxing effect of acetylcholine apparently originated from the vascular endothelium since acetylcholine responses completely disappeared in endothelium-denuded aortic strips. It is well known that vascular endothelial cells release the cyclooxigenase derivative of arachidonic acid, PGI2, which exerts and inhibitory influence on vascular smooth muscle (8). On the other hand, it has been shown that lidocaine causes the release of endogenous PGI2 from heart (6).Thus one can assume that the potentiation by lidocaine of the relaxing effect of acetylcholine in the aortic segment may be mediated through endogenous PGI2. But this is unlikely since the enhancement by lidocaine of the relaxing effect of acetylcholine was altered by cyclooxig
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