Patent Application: US-33431302-A

Abstract:
the present invention provides the visualization of the fiber architecture in the nervous tissue without staining . the major principle of the method is to make the neural tissue transparent in normal light , and to utilize the ability of neuronal fibers to deflect and deviate light directed from the side to render them visible . the method involves the preparation of thick sections of the nervous tissue , their fixation in paraformaldehyde and dehydration in ethanol . oil of wintergreen is utilized to make the tissue transparent under normal light . dark - field illumination is used to create illuminating rays of light arriving at an angle exceeding the collecting angle of the objective lens , thus causing only the axonal pathways to be visible as a bright silver silhouette against a dark background .

Description:
the method was originally developed using fixed brains samples of calf ( purchased in a local food store ), male adult wistar rats ( institute for experimental medicine , russia ) and postmortem human brain samples ( 1 st medical institute , st . petersburg , russia ). later its usefulness was also verified on adult female and neonatal ( 16 days ) wistar rats ( charles - river , canada ) and adult c 3 h / hej mice ( the jackson laboratory , bar harbor , me .). the experiments also employed fixed tissue pre - used in electrophysiological or morphological experiments as described elsewhere ( senatorov et al ., 1993 , 1995 ). briefly , during electrophysiological experiments , 400 or 500 μm slices were maintained for up to 8 hours at room temperature in oxygenated artificial cerebrospinal fluid , and then fixed by the immersion in 4 % paraformaldehyde in 0 . 1 m phosphate buffer , ph = 7 . 4 ( ppb ). in morphological experiments , rats or mice were over - anaethetized with sodium pentobarbital and perfused transcardially with 200 - 300 ml of ppb and then brains were postfixed by the immersion in ppb . calf brains and postmortem human brains were fixed by the immersion in 10 % formalin . following fixation , 200 - 600 μm or thicker sections were cut with a set of parallel blades . alternatively , and upon availability , a chopper or any device with a vibrating blade , such as a vibratome or vibrating blade microtome , might be used . after fixation , sections were dehydrated in the ascending series of ethanol — 70 , 95 and 100 %— 10 min each , and were left in a second change of 100 % alcohol for 1 - 2 h . finally , sections were immersed in methyl salicylate ( also known as oil of wintergreen ) for about 10 - 20 min , until they became visually transparent . for observation under a microscope , the slices were mounted on glass slides and coverslipped . fiber architecture was observed using a low - power objective , e . g . 2x , 5x or 10x with a numerical aperture lower than the numerical aperture of the dark - field condenser . transparency of the slices was achieved through clearing with methyl salicylate , which together with other organic solvents such as xylene , chloroform and dimethyl sulfoxide , is known for dissolving neuronal lipids and causing neural tissue to become more transparent ( becker et al ., 1991 ; hermes et al ., 1996 ; grace and llinás , 1985 ). however , for this method , xylene , chloroform and dimethyl sulfoxide do not work . as a result of methyl salicylate exposure , lipids and some other organic molecules are removed from the brain tissue slices , which make them transparent and practically ‘ invisible ’ under normal illumination . however , when slices are illuminated by a hollow cone of light striking from the dark - field condenser at an angle exceeding the collecting angle of the objective lens , the axonal bundles become clearly visible in the form of a bright silver - white substance against a dark background ( fig1 ). in the experiments , frontal , sagittal , and horizontal sections of the brain have been successfully used without any difference in results ( fig1 a and 1b ). in the brain regions containing large axonal bundles running within a surrounding mass of gray matter such as the forebrain , the three - dimensional course of axonal bundles can be clearly visible due to gray matter transparency ( fig1 a ). when neural slices were immersed in ethanol without prefixation in paraformaldehyde , the preparation should be viewed immediately after a very short ( few minutes ) incubation in methyl salicylate . otherwise , the portion of thinner fiber bundles quickly become darker and less visible . the brain slices prefixed with paraformaldehyde required longer methyl salicylate immersion and could be kept there for days or even weeks . the current technique is usable for verification of axonal pathways in slice preparations previously used in electrophysiological experiments , e . g . cortico - striatal and thalamic slices , and brain slices containing embryonic transplants . for example , fig1 b shows a photo of a horizontal slice through the rat thalamus . i has used this slice in an electrophysiological experiment to study the thalamo - cortical connection , and found the technique to be very successfully to choose the best angle to cut a 400 μm horizontal brain slice , which contained axons running all the way through the auditory thalamo - cortical pathway . in addition to brain tissue , spinal cord slices have also been used , and it is found that the technique works equally well ( fig1 c ). methodologically , the results obtained from either adult or very young rats are not different . the technique works equally well in the brain tissue of mice , calf , and postmortem human brain . to find the optimal conditions , brain slices of different thickness have been cut and it has been found that a thickness of 300 - 500 μm is best for the visualization of neural bundles in small animals . 200 μm is a minimal thickness for slices that could be successfully used , as axonal fiber bundles are not visible in thinner slices . on the other hand , thicker slices are more difficult to view in the small animals because of the accumulating volume of white tissue . considering that in large animal and human brains , large fiber bundles extend beyond the slice thickness of 400 - 600 μm , it should be noted that there is no immediate limitation on maximal slice thickness in technique per se . depending on the microscope specifications and anatomical organization of the observed brain region , sometimes thicker slices ( 1 mm and more ) might serve better . another peculiarity of this method is that fiber architecture can be effectively observed only under low magnification , such as with 2x , 5x or 10x objectives . choosing the proper distance between the condenser and the specimen to provide appropriate illumination is very important . while i needed some practice for setting up good dark - field illumination , obtaining results is not that difficult . the first successful experiments have been carried out in home laboratory conditions . anderson j . how to stain the nervous system , e . livingstone : edinburgh , 1929 . becker d . l , dekkers j , navarrete r , green c . r , cook j . e . enhancing the laser scanning confocal microscopic visualization of lucifer yellow filled cells in whole - mounted tissue . scanning microsc ., 1991 ; 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