To reveal the relationship between the morphologic structure and the functioning of cells it is of great importance to know the molecular arrangement in cytoplasm. For this purpose the observation by polarization
microscope is one of the most exellent methods. There are several excellent works on nerve fibre done using polarized lightl,2,3,4 but there is no report concerning the nerve cell itself. It is the purpose of this paper to
show our data obtained on ganglion cells and nerve fibres with the deduced speculation on the structural arrangement of lipoprotein in the protoplasm of nerve cells and fibres.</p

Kanda, Saburo

Maki, Teruo

Seno, Satimaru

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Observations on Nerve Cells and Fibres by Polarization Microscope

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 11, Issue 1 1957 Article 1APRIL 1957Observations on Nerve Cells and Fibres byPolarization MicroscopeSatimaru Seno∗ Saburo Kanda†Teruo Maki‡∗Okayama University,†Okayama University,‡Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Observations on Nerve Cells and Fibres byPolarization Microscope∗Satimaru Seno, Saburo Kanda, and Teruo MakiAbstractTo reveal the relationship between the morphologic structure and the functioning of cells itis of great importance to know the molecular arrangement in cytoplasm. For this purpose theobservation by polarization microscope is one of the most exellent methods. There are severalexcellent works on nerve fibre done using polarized lightl,2,3,4 but there is no report concerningthe nerve cell itself. It is the purpose of this paper to show our data obtained on ganglion cellsand nerve fibres with the deduced speculation on the structural arrangement of lipoprotein in theprotoplasm of nerve cells and fibres.∗Copyright c©OKAYAMA UNIVERSITY MEDICAL SCHOOLActa Med. Okayama 11, 1, 1-9 (1957)OBSERVATIONS ON NERVE CELLS AND FIBRESBY POLARIZATION MICROSCOPESatimaru SENO, Saburo KANDA and Teruo MAKIDepartment of Pathology, Okayama University Medical SchoolOkayama, JAPAN. (Director: Pro/. S. Seno)Received for Publication Feb. 20, 1957To reveal the relationship between the morphologic structure and thefunctioning of cells it is of great importance to know the molecular arrange-ment in cytoplasm. For this purpose the observation by polarizationmicroscope is one of the most exellent methods. There are several excel-lent works on nerve fibre done using polarized lightl • 2. 3.4 but there is noreport concerning the nerve cell itself. It is the purpose of this paper toshow our data obtained on ganglion cells and nerve fibres with the de-duced speculation on the structural arrangement of lipoprotein in theprotoplasm of nerve cells and fibres.Materials and MethodsTissues: Motor nerve cells from the pons and the sciatic nerve fibresof rabbits, guinea pigs and rats served as materials. The nerve tissueswere observed in a frozen section without fixing or fixing with 5 %formalin solution in 50 (;0 ethanol and the nerve fibres were observed aftermaceration without fixing or fixing with 10 (;0 formalin.Microscope: A polarization microscope has been constructed in ourinstitute using a general optic microscope and two nicols so as to besuitable for the detection of the weak birefringence given by biologicalmaterials. The diagram of this microscope has been reported elswhere5. 6. 7.As is demonstrated in Fig. 1 the light source is a tungsten lamp of 700 Wor a high pressure mercury lamp. As the condenser a general optic micro-scope was used upside down. Two nicols were set between the objectiveand the ocular, one as the polarizer in the microscope used as condenserand the other as the analyzer in the upper microscope respectively. Forthe purpose of removing the aberrant ray which might hinder the obser-vation of the slight birefringence given by cells, eleven iris diaphragmshave been set inserting into the pathway of light as demonstrated in thediagram (Fig. 1), d 4 being of objective (x 100). Furthermore each lensof microscope has been removed of their stress carefully and coated their11Seno et al.: Observations on Nerve Cells&#12288;and Fibres by Polarization MicroscopeProduced by The Berkeley Electronic Press, 19572 S. SENO, S. KANDA and T. MAKI:- absorbing solution- polarlzer- analyzerstage - --~~-:;-FJ-;':';"test plate",d" ... _[me_~~.·Id~..L..:.~~~'/)-+--+--+-t--(~ ,11 ~~ a....-c:L.....I! ! ! !lu~en filter mirror mercurylamp lampFig. 1 Diagram of polarization microscope. dl-d9; iris diaphragmssurface to reduce the reflextion of light on the surface which might be asource of stray light. The objective and deck glasses were also removedof their stress by heating at 600 0 C in a electric furnace and cooling veryslowly. Thus removing the aberrant ray as perfectly as possible, thematrials have been observed using the polarized spot light of high illumi-nation. To decide the sign of birefringence the mica plates of )./10 to )./20have been used which were prepared by stripping the mica plate of a con-denser which is generally used for radio. These were sandwiched betweentwo objective glasses of 7 cm. in breadth using balsam and set incertinginto the holder attached to the revolver.Observations and Results1) Observation on nerve cells: -On the tissue sectioned freezing without fixation the ganglion cellsshowed no birefringence. The cells fixed with formol alcohol proved tohave the Nissle body clearly demonstrable by hematoxylin stain. Withoutstaining these cells were observed by polarized light imbibing with water.A slight birefringence appeared in cytoplasm and nucleus. This birefrin-gence became distinct when the materials were imbibed with air (Plate 1).After affecting nonpolar solvents like ether-alcohol and drying quickly, how-2Acta Medica Okayama, Vol. 11 [1957], Iss. 1, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol11/iss1/1Observations on Nerve Cells and Fibres by Polarization Microscope 3ever, a clear and distinct granular birefringence appeared on the cells(Plate 2). This birefringence appeared most distinctly when the sectionswere imbibed with air and became slight when imbibed with water andcompletely disappeared by imbibing with alcohol. Continual treatmentwith ether-alcohol (one volume to one volume) resulted in the increase ofthis granular birefringence reaching the maximum at about one hour(Plate 3). Further treatment with ether-alcohol caused the gradual de-crease of the birefringence and it became very slight after about 6 hoursbut did not disappear completely. At this stage where the granular biref-ringence was minimized there appeared a slight fibrous birefringence.Comparing these birefringent structure with the structure of basophiliaseen on the same cell stained with hematoxylin after the observation bypolarization microscope (Plate 4), it has been revealed that these birefrin-gences were of the Nissl's bodies and the chromatin nets of nucleus. Thenucleoles showed not birefringence. The affection of ribonuclease resultedin the complete loss of basophilicity of cytoplasm but gave not any effecton the birefringence.The sign of the birefringence has been decided on another cell. Ob-servation proved that the sign of the fibrous birefringence seen at firstwas psitive (Plates 5a & b) and the sign of the granular strong birefrin-gence appeared after affecting ether-alcohol is negative concerning to thelong axis of a fibrous Nissle's bodies (Plates 6a & b) and the fibrous birefrin-gence appeared after 5 to 20 hours treatment with ether-alcohol is positiveconcerning to the same axis (Plates 7a & b and Sa & b).2) ObservtLtions on nerve fibre: -The most strong birefringence has been observed on myelin sheath.This birefringence has been clearly recognized on the sciatic nerve in wetas well as fixed with formalin (Plate 10). This birefringence did notchange its intensity by changing imbibing media, air, water or ceder oil,differently from the case of ganglion cells. The sign of the birefringencewas negative concerning to the long axis of nerve fibre (Plate 11). Thetreatment with non-polar solvents resulted in the decrease of the birefrin-gene. By treating with pure alcohol for 20 hours at room temperature,this negative birefringence was reduced markedly but not completely dis-appeared. Aceton had no reducing effect on the birefringence. Ethershowed only a slight reducing effect by affecting for 36 hours. Under themicroscope the imbibition with alcohol resulted in the immediate dis-appearance of birefringence of myelin sheath. But by changing the im-bibing media to water the birefringence reappeared clearly, though3Seno et al.: Observations on Nerve Cells&#12288;and Fibres by Polarization MicroscopeProduced by The Berkeley Electronic Press, 19574 S. SENO. S. KANDA and T. MAKI:slightly reduced in its intensity.On fresh tissue the axon hardly shows the birefringence, however,the treatment with alcohol, aceton or ether gives rise to the birefringenceof granular type in water (Plate 12) and a positive homogenious intrinsicbirefringence in balsam (Plate 13).SCHWANN'S sheath has an intrinsic birefringence whose sign is alwayspositive concerning to the long axis of the fibre. This birefringence wasclearly recognized after the prolonged treatment with alcohol where thenegative birefringence of myelin sheath had been minimized.CommentOne of the important facts revealed by this experiment is that theganglion cells show a weak form birefringence in their cytoplasm andnucleus when they are fixed with formol-alcohol. Of course in these cellsthe Nissle body is formed in their cytoplasm as revealed by hematoxylinstain. The birefringence appears on the Nissl's bodies in cytoplasm and onchromatin nets in the nucleus. As the cells showed no birefringence whenthey are observed in wet without fixation, it is obvious that the molecularstructure in the cells is turned to a compact one by fixation. The effectof alcohol or alcohol-ether on the tissue section will mainly be the dehy-dration by which the micellar structure should become more compact one.The granular negative birefringence appeared after affecting ether-alcoholand rapid drying will be the result that the nonpolar tails of lipid mole-cules, which are attached to the fibrous protein with their polar groups,are arranged in a radial direction losing the imbibing media. This nega-tive birefringence is of lipid but not of ribonucleic acid, because thetreatment with ribonuclease gave not any influence on the birefringence,though it acted as to lose the basophilicity of cytoplasm perfectly and yetthis granular birefringence reduces markedly its intensity by treatingwith ether alcohol for a long period. Thus it explains the phenomenon thatthe short treatment with alcohol-ether followed by rapid drying gives riseto an intence negative birefringence. On the other hand as reportedpre-viously, the authors revealed on various cells that the basophilia likeendoplasmic reticulum agglutinate by dehydration" simple drying, ortreatment with dehydrating agent like alcohol, ethergas, hypertonicsodium azid and others, developing into a visible fibrous structure8. 9. lo.n.This is true in the case of the Nissl's bodies, too, especially in those partswhere the cytoplasm is narrowed. It is generally the case with the endo-plasmic reticulum that is arranged parallel keeping the lamellar structurenear the nucleus especially in the part having narrow cytoplasm12• Then4Acta Medica Okayama, Vol. 11 [1957], Iss. 1, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol11/iss1/1Observations on Nerve Cells and Fibres by Polarization MicroEcope 5it may be considered that the fibrous Nissl's bodies formed in such part ofcytoplasm is consisted of the compact bundle of endoplasmic reticulum orcytoplasmic fibres. Therefore, it should be said that the birefringencegiven by the fibrous Nissl's bodies gives directly the molecular structureof endoplasmic reticulum. In other words the stem molecular structure ofendoplasmic reticulum is of lipoprotein, the fibrous protein attached withlipid moleules arranged vertically to the long axis. The fact that theliving cells give no birefringence may be explained by the loose arrange-ment of each endoplasmic reticulum and the irregular arrangement ofnonpolar tails of lipid molecules.In chromatin nets of nucleus the experimental result is entirely thesame as in the case of the Nissl's bodies. Then the molecular structure ofchromonema should be also the same as with that of endoplasmic reticu-Ium. Nucleoles showed no birefringence. Then it may be said that thereis no regular molecular arrangement.Concerning the nerve fibre it can be deduced that the myelin sheathis constructed by the compact radial arrangement of lipid molecules.The structure maintaining the radial arrangement of lipid molecules willbe the lamellar protein membrane arranged as concentric cylinders asrevealed by electron microscope13• The lipid will be mainly of phospha-tides, because the most parts of lipid can be extractable with alcohol butnot with aceton. SCHMIDT1 and recently YONEZAWA3 reported that theaxon shows the positive birefringence concerning the long axis, however,we can not recognize any birefringence in wet preparates, though thegranular birefringence appeared after fixation. Then it is irrelevant tothink that there should exist the compact fibrous structure in the axon inliving tissue. The positive homogenous birefringence which appears onthe axon imbibed with balsam will not be of the axon itself but possiblybe of the axolemma. The SCHWANN'S sheath shows a distinct intrinsicpositive birefringence which is not affected by non polar solvents. Thenit must be considered that there exists a lamella of compact structure com-posed of fibrous protein molecules arranged parallel to the long axis ofthe nerve fibre.Summary1) Motor cells and sciatic nerve fibres have been observed bypolarization microscope.2) It was deduced from the findings on the fixed cells that the baso-philia, the endoplasmic reticulum, should be constructed with lipid mole-cules attached to the fibrous protein.5Seno et al.: Observations on Nerve Cells&#12288;and Fibres by Polarization MicroscopeProduced by The Berkeley Electronic Press, 19576 S. SENO, S. KANDA and T. MAKI:3) It is also deduced that the chromonema of the nucleus should beof the same structure as that of endoplasmic reticulum in the arrangementof fibrous protein and lipids. However, nucleoles has no regular arrange-ment in its molecular structure.4) In nerve fibre the myelin sheath has been proved to have a struc-ture of lipid molecules arranged radial holding the axon, mainly phospha-tides. In the axon itself the existence of a compact fibrous structure wasdenied. SCHWANN'S sheath proved to have a membrane structure com-posed of protein moleculs arranged parallel to the long axis of nerve fibre.Bibliography1 SCHMIDT, W. J.: Die Doppelbrechung von Karyoplasma, Zytoplasma and Meta.plasma. Gebriider Borntrager, Berlin, 1937. - 2 FREY·WISSLING, A.: Submicroscopicmorphology of protoplasm. Elisevier, London, 1953. - 2 ARAKI, M.: Pathomorphologicobservation on the peripheral nerve. Transact. societ. Pathol. Japon. 44, 35, 1955. --I ARAKI, M. and YONEZA W A, T.: The structure and the function of nerve fibre. Sym.posia for the societ. of cellular chem. 5, 93, 1957. - f'SENO, S.: Analysis of cytoplasmby polarization microscope. Electron microscope. 4, 22, 1955. - 6 KANDJ\, S.: Studieson the structure of the cytoplasmic basophilia. III Analysis of cytoplasm by polarizingmicroscopy. Okayama Igakkai Zassi 68, 2008, 1956. - 7 UTSUMI, K.: CytochemicalStudies on Ciliata. V. Medicine and Biology 42, 179, 1957. - 8 SENO, S., KANDA, S.and KA W AI, K.: Studies on the structure of cytoplasmd 1. Ribonucleic acid in cytoplasm.Transact. Societ. Pathol. Japon. 41, 83, 1952. - (j SENO, S. and KAZDA, S.: Studies onthe structure of cytoplasm and its relationship to RNA. Ibid. 41, 86, 1952. - JO SENO,S. KANDA, S. et al.: Maturation of reticulocytes and related phenomena V. Morpho-logical and cytochemical studies on reticulocytes. Mie Med. J. 4, Suppl. 1, 45, 1953. -II KANDA, S.: Studies on the cytoplasmic basophilia. 1. Agglutination of cytoplasmicbasophilia and its relationship to ribonucleic acid. Okayama Igakkai Zassi 68, 1989,1956. - 12 DE ROBERTIS et al. : General cytology. Saunders, London, 1955. - 13 SJOSTRAND,F.: The lamellated structure of the nerve myelin sheath. Experientia, 9, 68, 1953.6Acta Medica Okayama, Vol. 11 [1957], Iss. 1, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol11/iss1/1Observations on Nerve Cells and Fibres by Polarization Microscope 7ExpIanation of PlatesPlates I, 2 and 3. A motor nerve cell of the pons of a rabbit fixed with formolalcohol and sectioned freezing. The photos are taken by the polarization microscopeafter treating with ether-alcohol for 10 minutes, 40 minutes and 60 minutes respectively.The imbibing media are air.Plate 4. The same cell stained by hematoxylin after the observation by polariza-tion microscope. Strong birefringence appeared in the parts where the Nissle's bodiesare recognized as the strong basophilic mass and the chromatin nets in the nucleus.Nucleoles gave no birefringence.Plates 5 to 10. A motor nerve cell from the pons of guinea pig.Plate 5a. The birefringence seen in the cytoplasm. A frozen section after fixingwith 5% folumarin in 50% ethanol.Plate 5b. The birefringence disappeared after inserting mica plate vertically to thecytoplasmic membrane. But there appeared the negative birefringence of the cyto·plasmic membrane. Arrow shows the long axis of mica plate.Plate 6a. After the treatment with ether·alcohol for 3 hours. The birefringencebecame strong.Plate 6b shows that this birefringence is negative concerning to the long axis ofthe Nissle's body. Arrow shows the axis of mice plate.Plates 7a and b. After the treatment with ether. alcohol for 5 hours. The negativebirefringence decreased, and the intensity of positive birefringence increased.Plates 8a and b. After the treatment with ether.alcohol for 20 hours. The negativebirefringence is still retained but the positive birefringence increased its intensity.Plate 9. The same cell stained with hematoxylin after finishing the observationby polariza tion microscope.7Seno et al.: Observations on Nerve Cells&#12288;and Fibres by Polarization MicroscopeProduced by The Berkeley Electronic Press, 19578 S. SENO, S. KANDA and T. MAKI:t'late 1 Plate 2Plate 3 Plate 48Acta Medica Okayama, Vol. 11 [1957], Iss. 1, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol11/iss1/1Observations on Nerve Cells and Fibres by Polarization Microscope 9Plate 5 aPlate 5 bPlate 8 aPlate 6 aPlate 7 aPlate 8 bt'late 6 bPlate 7 bPlate 99Seno et al.: Observations on Nerve Cells&#12288;and Fibres by Polarization MicroscopeProduced by The Berkeley Electronic Press, 195710 S. SENO, S. KANDA and T. MAKI:Plate 10 Plate 12Plate 11 Plate 13Plate 10. A sciatic nerve fibre fixed with 10% formol. The picture seen under thecrossed nicols imbibing with 0.85% saline solution. Note the strong birefringence ofthe myelin sheath.Plate 11. The same fiber observed inserting a mica plate parallel to the long axisof nerve fibre. It shows that the sign of myelin sheath is negative concerning to thelong axis of nerve fibre. The positive intrinsic birefringence appeared on the Schwann'ssheath.Plate 12. After the treatment with 7096 alcohol for 7 days. The positive birefrin-gence of Schwann's sheath increased, and the positive granular birefringence appearedon the axon.Plate 13. Another fibre treated with pure ethanol for 20 hours, and photographedafter imbibing with balsam. The weak homogenious positive birefringence of axon(possibly be of the axolemma) and of the Schwann's sheath are recognizable,10Acta Medica Okayama, Vol. 11 [1957], Iss. 1, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol11/iss1/1

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