Volume:6 Number:1 JANUARY 1995TABLE OF CONTENTS
SUMMARY : This study is carried out to investigate the effect of electric field applied in different directions on collagen synthesis by determining the quantitative level of hydroxyproline in lung tissue. Ten guinea pigs were exposed to vertical electric field, while another 10 being exposed to horizontal eletric field with the intensity of 1.9 kV/m. Twenty guinea pigs were used as control, keeping in the same conditions without being exposed to any electric field. Exposure period was 9 hours / day for 3 days.A statistically significant increase was found in the lung tissue hydroxyproline level of vertical electric field applied group (p<0.001) and horizontal field applied group (p<0.05) compared to non-exposed controls. It was also observed that the increase in vertical electric field applied group was more than the horizontal field applied ones suggesting that the vertical electric field might be more effective than the horizontal.Key Words : Electric Field, Lung Tissue, Tissue Hydroxyproline. INTRODUCTION A very active period was between 1925 and the Second World War when a number of biophysicists applied potential theory originally developed for dielectrics to biological material (blood, tissue, nerve cells). About 1935, investigations began of the electrical properties of protein molecules by application of the concept of polar molecules originated by Debye (41). Of more fundemantal importance are studies of the electrical properties of tissue and cell suspensions related to : a. Structural analysis of cellular organism (properties of cell membranes and cytoplasm).b. Analysis of characteristics of protein molecules, such as dipole moment, shape, hydration. All of the apparatus and devices necessary in modern life such as power lines and electrical machinery are the sources of electric (E) fields which cause important effects on living organisms. Behavioral, cellular and pyhsiological functions of animals can be affected by e.m. stimuli (1, 12, 26). In recent years a body of data on the interactions of exogenous and endogenous e.m. fields with biological system has accumulated. As a result of these findings our understanding of biological functions has started to change.The electric field of the Earth consists of a static component, which is dominant, and a time varying component, which is smaller than the static component by several orders of magnitude in the 50-60 Hz frequency range (38). The natural electric field near the Earth's surface is a static field of about 130 V/m (24). Daily changes in the natural electric field are attributed to factors, such as thunderstorms, which have magnitudes of 3-20 kV/m (20).Man-made sources of electric fields are high voltage transmission lines and all devices containing current - carrying wire, including equipment and appliances in the home and in industry. Measurements of electric fields in a home ranges from 2 V/m to about 50 V/m at 30 cm from the appliances and can vary to 250 V/m near some appliances (36, 47). Living organisms interact with this environment of electric and magnetic fields. The question is did they adopt to this environment of electric and magnetic fields. I think, they did not yet, at least they didn't accomplish the process of adoptation. The connective tissue protein, collagen, is the most abundant protein in higher animals. Collagen also provides the framework for parenchymal organs such as the liver, kidney, and spleen, either in its fibrous form or organized in basement membranes. Some tissues contain much more collagen than others : the collagen content of the skin accounts for about 70 % of the dry weight of skin whereas the collagen content of abdominal viscera is much lower and, in the case of the liver, amounts to about 4 % of the dry weight (32). Hydroxyproline is found almost exclusively in collagen, constituting about 14 % of the dry weight of the protein, and the amounts of this amino acid are relatively constant in collagens from various tissues in humans. The rate of hydroxyproline formation is therefore considered to be a good indication of the rate of collagen biosynthesis (22). The collagen content of a tissue is determined by measuring the content of protein bound hydroxyproline, an amino acid that is a marker of collagens (45). The aim of this study is to examine protein synthesis under the effect of exogenous electric fields in different directions. Electric field was chosen with the magnitude in the order of electric fields of man made apparatus. For this aim 1.9 kV/m electric field in two different directions was applied to 20 guinea pigs and the effect of electric field was observed by determining the change in hydroxyproline level of lung tissue. MATERIALS AND METHODS Guinea pigs (10-12 weeks old) were exposed continuously to uniform electric fields of 1.9 kV/m generated between the parallel plates of a capacitor. The animals were separated into three groups. In Group I, 10 guinea pigs were exposed to vertical electric field while another 10 in Group II were exposed to horizontal electric field. Twenty guinea pigs were also kept in the cages at the same laboratory conditions and studied as the Control Group without any vertical and horizontal electrical field application. For 3 days the experimental animals were housed in wooden cages of 50 cm x 50 cm x 14 cm dimensions.The simplified equation for the electric field between two parallel plates, the potantial difference per distance, is widely used in biological experiments. This is, however, only true when the plate spacing is small enough in comparison with the dimensions of the plates. For that reason the plate spacing was 14 cm whereas the dimension of the plates was 50 cm x 50 cm x 0.1 cm. For 10-12 weeks old guinea pigs, this much spacing between the capacitor plates was the minimum possible distance with respect to dimensions of the animals.Vertica l Field Exposure Circuit : Copper plates 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 Circuit : Copper plates were mounted to the left and right faces of the cages. Positive probe of the power supply was always connected to the left plate and negative probe to the right plate for all of the cages.Vertical and horizontal electric fields with the magnitude 1.9 kV/m were obtained from power supplies with 300 V DC potential and applied to the plates. Exposure period was 9 hours/day for 3 days throughout the study. Duration of electric field exposure was from 800 am to 500 pm, 9 hours/day. During the study, all guinea pigs were housed in a room having a temperature of 23ûC, and a light-dark cycle of 12 : 12. Determination of Tissue Hydroxyproline : Lung tissue hydroxyproline contents of animals were determined with Stegemann-Stalder's Method (43). Among the methods for the determination of hydroxyproline the oxidation of this amino acid to a compound reacting with p-dimethylaminobenzaldehyde to form a chromophore is regarded as a relatively specific and sensitive assay. In the oxidation phase of hydroxypoline only Stegemann-Stalder and Woessner used chloramine-T, while others chosed hydrogen peroxide. It was shown that by using chloramine-T the same sample's hydroxyproline amount gave more sensitive result (23). At the end of exposure period the lung was rapidly removed from guinea pigs after decapitation, and cut to small slices. The sample is placed in a pyrex tube; then heated so as to maintain a temperature of at least 70ûC for at least 30 minutes. Four grams of the sample was taken into the hydrolysis flask. Hydrolysis was carried out in 6 N HCl at 107ûC for 16 hours. Hot hydrolysate was filtered through filter paper into a 200 ml one-mark volumetric flask. Flask and filter paper were washed three times with 10 ml portions of hot hydrochloric acid solution and the washings were added to the hydrolysate.Water was added and mixed, and 5 ml of the hydrolysate was diluted to 250 ml, so that the hydroxyproline concentration will be within the range 0.5 to 2 µg/ml. Two milliliters of the sample was mixed in a test tube with 1 ml chloramine-T solution both having a temperature of about 20ûC.After 20 min, 1 ml of the perchloric acid solution is added and the mixture was shaked thoroughly. The tubes are immersed into a water bath at 60ûC. After 15 min, the rack is cooled under tap water and within 45 min the color is read in Milton Roy UV-3000 Spectrophotometer at l =560 nm. Hydroxyproline contents of the tissue samples were determined by using the standard curves which were plotted daily before each spectrophotometric reading by using the sample containing 0.5-2 µg/ml hydroxyproline (SIGMA H-1637). RESULTS Lung tissue hydroxyproline content of vertical and horizontal electric field applied groups were found from daily plotted standard curves. For each hydroxyproline determination two samples of the tissue taken from the homogenization step were studied seperately and the avarage of their spectrophotometric data was taken as the absorbance value of each sample. The avarage hydroxyproline concentration value was taken into consideration at the calculation step (Table Statistical comparisons were made in two groups : In the first group, hydroxyproline contents of lung tissues of electric field applied groups were compared with their controls with DUNCAN Test and the results were found as follows : There is statistically significant difference between hydroxyproline contents of lung tissues of vertical electric field applied and control groups (p<0.001).Vertical electric field causes an increase in lung tissue hydroxyproline level : Vertical = 2.306± 0.814Control = 1.374 ±0.539 There is statistically significant difference between hydroxyproline contents of lung tissues of horizontal electric field applied and control groups ( p < 0.05). Electric field also causes an increase in lung tissue hydroxyproline level :Horizontal =1.906± 0.722 Control = 1.374 ± 0.539 It was observed that the vertical electric field increased hydroxyproline content of lung tissue more than the horizontal electric field. In the second group, hydroxyproline contents of lung tissues of vertical electric field applied groups were compared with horizontal electric field applied group. Statistically no significant difference is observed between hydroxyproline contents of lung tissues of vertical electric field group and horizontal electric field group ( p>0.05). DISCUSSION The interaction of externally applied electric fields with living organisms has become an important subject for some decades. The fields can act only if physically present in or on tissue, and knowledge of their magnitude and distribution must be correlated with observed physiological responses before a deeper understanding of the interaction process itself can emerge. The structural complexity of even the simplest living systems, however, has prevented direct measurement of field distributions in tissue (33). Instead an approach to field distribution in tissue has been tried with modelling studies (11). The high resistivity of a cell membrane and the high conductivity of its fluid interior serve to concentrate the externally applied electric field across the portion of the membrane at right angles to the field. Some membranes carry structural surface charge which arises from the presence of ions on lipids, lipopolysaccharides, or proteins. Experimental results support the view that some properties of the bilayer membanes are influenced by electrostatic potential at the membrane. Such is the case with regard to membrane excitability, permeability, adsorption, adhesion and even membrane structure. The understanding and the theoretical description of these membrane properties, however, all depend on detailed knowledge of the electrostatic potential and of the electric field at and near the membrane surface (44).Cellular organization and function can be described in terms of structural and regulatory interactions. On a molecular level interactions between proteins, lipids, carbonhydrates and other compounds can be described in terms of electromagnetic fields effects between participating molecules. Although the characteristics of the associated electromagnetic fields may be complex (particularly for large molecules, such as proteins), biological reactions are essentially electromagnetic. From this perspective, it is reasonable to consider that biological reactions may be influenced by electromagnetic fields (25). In vitro studies have shown that exposure to electric fields can increase cell proliferation and the results suggest that electric field may be transduced at the level of the plasma membrane (25). Electromechanical force generated in the cell membrane by rapidly increasing electroforces can lead to temporary reversible electronic collapse of the membrane (40). DC fields of 103 V/m have been shown to move protein molecules along the surface of the membrane and through gap junctions (21, 39).The localized electric field in saline-field scale models of human and animals are measured by Kaune and Forsythe. The vertical currents dominate and are fairly uniform across cross sections of the body. However there are small horizontal current components and in the axillae region the horizontal currents dominate. Measurements in saline-filled pig and rat models indicated increased horizontal current components arising from the horizontal orientation of the animal bodies (11).In experimental studies on vertical and horizontal electric field applications on animals, since induced currents are not possible to measure ; mortality percentage, the changes in body weight and blood protein fractions, lipid peroxide levels, and protein synthesis are investigated (9, 10, 28, 29, 30, 31, 34, 35). In vivo studies on man, animal and bacteria with DC and AC applications also exist (2, 3, 4, 5, 6, 7, 8, 13, 14, 15, 16, 17, 18, 19).The electrical properties of dried collagen and bone have been studied and both were shown to be piezoelectric (27). Nessler reported an increase in proline and hydroxyproline levels in rabbit flexor tendon by applying 7 µA DC along 7,14,21 and 41 days (37). DNA synthesis in epiphyseal cartilage cells increased in an electric field of 166 V/cm (40). Fitzsimmons observed the changes in hydroxyproline level in embryonic chick tibia with, 10-5 V/m the effect of electric field (25).Marino found alterations in body weight, mortality, and blood protein fractions of mice under the effect of vertical and horizontal directions of exogenous electric field (34, 35). He found vertical E field application is more effective to blood protein fractions than the horizontal one although the difference was not found significant.In this study vertical and horizontal application of E field with the intensity of 1.9 kV/m had been found to increase hydroxyproline content of lung tissue. The increase in hydroxyproline content of vertical E field application was more than the horizontal field although it was statistically insignificant, suggesting that the vertical E field might be more effective than the horizontal application.Since the tissues like lung, kidney and liver contain 20 % - 25 % protein and 70 % - 80 % water of their weights, they exhibit much more lower resistance and higher dielectric constant with respect to fatty tissues. Furthermore, increasing water percentage, increases conductivity which is a result of polar structure of water. This structure of lung might also been effective on the influence of E field on lung tissue
SUMMARY : The external development of rat embryos was investigated with the help of a stereomicroscope. Gestation days between 11 and 20 were described and photographed in the study, since the most prominent changes occur in this period. Moreover, fetal skeleton was demonstrated by alizarin red staining.Key Words : Alizarin Red, Rat Embryo, Stereomicroscope INTRODUCTION Detailed information of the external development of the embryos of laboratory animals has become a necessity during recent years because of the increased interest in the effects of drugs on the developing embryo (3, 4, 8, 11). Teratological studies are now an important part of the toxicity testing of drugs, and it is impossible to assess the effects of drugs on the embryo without having a knowledge of normal development (6, 7, 10). By comparison with normally developing embryos it is often possible to discover the origin of an abnormality and the developmental stage reached by resorbing fetuses before death occured : a drug might retard the development of the embryo, and the extent to which retardation occured could be assessed by comparison with normal embryos of known age (2, 9, 12).A method of designating embryos to defined stages based on external features, without using chronological age or somite number, is therefore clearly needed. Since few authors have described the external development of the laboratory rat, we planned this study in a way which would be of practical use in teratological work. MATERIALS AND METHODS Previously unmated female albino rats were placed in cages with males and watched for 12 hours. Those observed in copulation were isolated and regarded as zero days pregnant. The pregnant rats were sacrificed at intervals from 11 to 20 days post coitus and the uteri removed. All stages were large enough to be easily dissected from the uterus immediately. Early stages were examined as fresh embryos, but larger fetuses were fixed in formalin to reveal surface detail. The fetuses were examined grossly, detailed descriptions of each developmental stage were made, and photographs were taken under an Olympus stereomicroscope.Skeletal Examination : After the removal of the skin, body fat, viscera and brain; the 20 days old fetuses were fixed in FAA (formalin, 1; acetic acid, 1; 70 % alcohol, 8) and cleared in 1 % aqueous solution of KOH and then stained with alizarin red (2-3 drops of 0.1 % alizarin red per 100 ml of distilled water) so that skeleton could be examined macroscopically in the whole animal (1). RESULTS Detailed features of each stage are summarized with corresponding photographs :11th day (Fig 1) : This stage begins with the closure of the anterior neuropore, followed by closure of the posterior neuropore and the otic vesicles. The optic vesicles also oppear towards the end of the stage. Ventro-caudal to these vesicles, the mandibular arches are quite prominent. The heart tube is visible through the thin-walled pericardium. The process of "rolling over" of the embryo progresses; by the end of the stage the dorsal surface of the embryo is convex.12th day (Fig 2) : The mandibular arches which are growing towards the ventral mid-line have not yet fused. Two other pairs of branchial arches and clefts are now clearly differentiated. The anterior limb buds appear as swellings on either side of the trunk. Finally the liver anlage becomes visible just above the level of the anterior limb buds.13th day (Fig 3) : In this stage, the maxillary process has grown round underneath the eye to meet the lateral nasal process. Fusion of the mandibular arches to form the lower jaw commences and is almost completed during this stage. Fusion begins at the caudal margins of the arches and extends cranially. Later during this stage the ventral ends of the hyoid arches extend ventrally and almost meet in the mid line. At the end of this stage, the otic vesicles disappear from view owing to the thickening of the overlying tissues and the lens vesicle closes. Fusion of the maxillary process with the lateral nasal process is complete, and there is a slight elongation in the nasal region giving rise to the earliest appearance of a snout. The posterior limb buds appear as swellings on either side of the caudal end of the embryo. Further differentiation of the anterior limb buds takes place; they differentiate into upper and lower regions. The heart is still clearly visible through the pericardial wall. 14th day (Fig 4) : The constant feature marking the beginning of this stage is the appearance of the first vibrissary papilla on the maxillary region. This papilla is sometimes difficult to ee in an unstained specimen. The number of papillae increases during the stage. Further development of the ear occurs. The mandibular and hyoid arches are no longer distinguishable and they have fused beneath the auditory cleft. It is during this stage that the first sign of digital condensation appears in the fore - limbs and towards the end of the stage webbing between the rays is distinguishable.Further development of the posterior limb leads to the differentiationof the paw, and towards the end of the stage digital condensation begins. Throughout development the progress of the hind-limbs lags slightly behind that of the fore-limbs.15th day (Fig 5) : Two gut loops have appeared externally at the beginning of the stage. Four distinct rows of papillae appeared on the snout region. As development proceeds, the number of vibrissary papillae increases and more of them invaginate. The external ear-flap increases in size and begins to cover the auditory meatus. The shape of the eye appears to change due to the gradual growth of the eyelids. The development of the limbs continues. The degree of webbing in the fore-paws is now considerable, giving the paw a rather angular shape, and the same process begins in the hind-limbs.16th day (Fig 6) : This stage does not show many advances over the previous one. At the beginning of the stage there are six rows of vibrissary papillae. The digits of the fore-paws are just beginning to separate, while on the hind-limbs, webbing is completed during this stage. There is an increase in the number of gut loops visible externally.17th day (Fig 7) : The beginning of this stage is marked by the first appearance of papillae on the trunk region. These are not easy to see at first since they are somewhat flattened and merely more dense than the surrounding tissue. At the beginning of the stage there are seven rows of vibrissary papillae, and the number of papillae increases throughout the stage. The ear-flap continues to grow and the eyelids become rather more prominent.The digits of the fore-paws continue to separate and this process is quite well advanced towards the end of the stage; late in the stage digital separation in the hind limb commences. By the end of the stage, nine rows of maxillary papillae are present and the trunk is extensively covered with papillae. The number of gut loops visible externally still continues to increase.18th day (Fig 8) : This stage begins when the digits on the fore-paws are fully separated; they are partially separated on the hind-paws.The external auditory meatus is almost covered by the ear pinna, and the palpebral fissure narrows during this stage concomitant with the growth of the eyelids. Limb development shows some further progress. The digits of the fore-paws are completely subdivided and the terminal phalanges which are the future claw-bearing areas are demarcated. The complete subdivision of the hind-paw digits is effected by the end of the stage and the formation of the terminal phalanges of hind-paw digits begins. By the end of the stage,reduction of the umbilical hernia has begun.19th day (Fig 9) :This stage begins when the umbilical hernia is just totally reduced and ends when the palpebral fissure and the auditory meatus are completely closed. The development of the limbs is completed so that by the end of the stage both fore-and hind-limbs bear claws. Between the end of this stage and parturition, there is further growth but no further externally visible developmental progress occurs.20th day (Fig 10) : Since ossification begins on the sixteenth day, and there is no further externally visible developmental progress after the 19th day; we used this stage to show the skeleton by staining the embryos with alizarin red. DISCUSSION The rat from the time of fertilization, has a gestation period of about 22 days. Implantation begins about the seventh day. The primitive streak appears at 8.5 to 9 days, the heart begins to beat about the ninth day, the tailbud appears at 11.5 days, and the "end of metamorphosis" appears at 16 to 17 days (16). Ossification begins on the sixteenth day (17 Our results with the external features of the developing rat embryo and the skeletal formation are in good agreement with these data.In the previous studies of rat development, gestation age or somite number have often been the crieria on which classification of development depends (15, 16). Gestation age is not a valid criterion since there is often considerab
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