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Observations on the ultrastructure of experimentally altered nerve fibres, grown and injured in tissue cultures
186 OBSERVATIONS ON THE ULTRASTRUCTURE EXPERIMENTALLY ALTERED NERVE FIBRES, INJURED IN TISSUE CULTURES1
OF GROWN
AND
H. MEYER Institute
de Biofisica
da Universidade Received
do Bras& May
Rio de Janeiro,
Bras2
25, 1955
IN a previous paper the results of an electron microscopic study of nerve fibres, grown in tissue cultures, have been discussed [2]. It has been shown that the cytoplasm of these fibres has a fine longitudinal striation which can be followed up to the growing tips of the fibres. Mitochondria-like inclusions and vacuol-like holes can be found among the striations. We know that cultures of nervous tissue must be treated with special care. Slight injuries or damages during manipulation of the cultures have an immediate effect upon the aspect of the fibres [I 1. Varicosities are formed and swellings which may be reversible, but which in case of severe damage may lead to a rapid degeneration of the distal part of the fibres. Swellings or fusiform enlargements in the nerve fibres may also occur normally in the tissue cultures, and we saw that those which are thin enough for the penetration of the electron beam have the same longitudinal striation which the fibre has. There are also others which show no structure at all, and in more recent observations inclusions have been found in them in form of droplets, or mitochondria-like filaments. These formations do not always represent pathological alterations of the nerve fibres, since they are found in fibres which are in perfect condition with regard to structure and growth. For a better understanding as a whole, and to avoid future errors, we thought it interesting to injure some cultures and examine in the electron microscope the effect of the damage upon the structure of the nerve fibres, then compare the aspect of the experimentally altered fibres with that of the intact ones. Material and Technique.--In preparing the tissue cultures the samematerial and technique were used as in the earlier work for the normal fibres [2]. Spinal ganglia of 9 to 14 days old chick embryos were cultivated in a thin plasma clot on covers slips which had been coated previously with Formvar. The incubation temperature was 33-35”. When the cultures showed a good growth of fibres, a slight cut was made in r This investigation Rio de Janeiro (Dir. fellowship.
Fig. still
1. Spinal preserved,
gangl. of 14 days old chick embryo. droplets appear along the fibres
Fig. 2. Spinal gang]. membranes and fine and 3000. Experimental
has been carried out in the Instilulo de Biofisica da Universidade do Brasil, Dr. Carlos Chagas) under the tenure of a Conselho National de Pesquisas
of 14 days prolongations.
Cell Research
9
old chick embryo. In (6) terminal
7 minutes after an injury. Striation and are extruded. x 10,000. Terminal extension
extension one hour
in cytoplasm
of intact fibre after an injury.
(a) with x 1500
Ultrastructure
of experimentally
altered nerve fibres
Experimental
187
Cell 1Zt:search
9
H. Meyer the centre of the explant, seat of the nerve cells, with the point of a new cataract knife. Care was taken not to push the plasma clot too much. The effect of this injury could be observed immediately in the peripheral part of the cultures, where the nerve fibres showed the alterations which are described above. The cultures were fixed from two to 10 minutes after the injury, some after one hour, in vapour of osmic acid, with the same precautions and care which had been observed during the fixation of the normal fibres [2]. After washing in dist. water, and after removing the plasma clot the formvar film with the remaining fibres was transferred to the electron microscope grids, according to the technique of Porter, Claude and Fullam [3]. Here they were shadowed with chromium. They were examined then in the “Philips” electron microscope with 60 and 80 kV. Obseruations.-When the injury in the centre of the culture was restricted to a small area not all the nerve cells or fibres were damaged, and not all of the fibres in the periphery react. In those which are altered the aspect varies from complete destruction to more or less pronounced alterations. In all cases we see droplets which seem to be extruded from the cytoplasm, appear along the fibres (Fig. I), often at quite regular distances. They tend to flatten with time and stain with osmic acid. In some cases the fibrillar aspect of the cytoplasm becomes more pronounced by this loss of material, in others the structure is completely lost. Also in the terminal extensions of the fibres the effect is immediate. The ameboid membranes retract and enlargements appear along the thin prolongations (Fig. 2a and b). In the fusiform enlargements of the fibres which occur normally in tissue cultures the striation may be partially preserved after an injury. The enlargements may also show extrusion of droplets, or the structure may be completely lost. Results and Discussion.-The effect of damage on the tissue cultures is immediate and varies in degree on the fibres, on their terminal extensions, and on the enlargements which preexisted in the fibres. The most immediate and striking effect is the apperance and extrusion of droplets along the fibres. They are probably of lipid nature becausethey stain black with osmic acid. Judging from their rather regular distribution the droplets might be responsible for the varicosic aspect of the fibres in the light microscope immediately after an injury. The results obtained during this investigtion are only preliminary. For technical reasons we thought it necessary to examine in a rough test the alterations in the structures of the injured fibres, because similar damages are easily produced by inadequate treatment of the tissue cultures, and similar changesin the structures will also occur during the preparation of peripheral nerves or of the central nervous system for inclusion or ultrathin sectioning, since it is almost impossible to obtain such preparations without an interruption of the nerve fibres’ continuity. REFERENCES 1. LEVI G. and MEYER, H., J. Expll. Zool. 99, 141 (1945). 2. MEYER, H., Exptl. Cell Research 7, 15 (1954). 3. PORTER, K. R.&LAUDE, A., and FULLAM, E. A., J. Exptl.
Experimental
Cell Research
9
Med.
81, 233 (1945).
OF GROWN
AND
H. MEYER Institute
de Biofisica
da Universidade Received
do Bras& May
Rio de Janeiro,
Bras2
25, 1955
IN a previous paper the results of an electron microscopic study of nerve fibres, grown in tissue cultures, have been discussed [2]. It has been shown that the cytoplasm of these fibres has a fine longitudinal striation which can be followed up to the growing tips of the fibres. Mitochondria-like inclusions and vacuol-like holes can be found among the striations. We know that cultures of nervous tissue must be treated with special care. Slight injuries or damages during manipulation of the cultures have an immediate effect upon the aspect of the fibres [I 1. Varicosities are formed and swellings which may be reversible, but which in case of severe damage may lead to a rapid degeneration of the distal part of the fibres. Swellings or fusiform enlargements in the nerve fibres may also occur normally in the tissue cultures, and we saw that those which are thin enough for the penetration of the electron beam have the same longitudinal striation which the fibre has. There are also others which show no structure at all, and in more recent observations inclusions have been found in them in form of droplets, or mitochondria-like filaments. These formations do not always represent pathological alterations of the nerve fibres, since they are found in fibres which are in perfect condition with regard to structure and growth. For a better understanding as a whole, and to avoid future errors, we thought it interesting to injure some cultures and examine in the electron microscope the effect of the damage upon the structure of the nerve fibres, then compare the aspect of the experimentally altered fibres with that of the intact ones. Material and Technique.--In preparing the tissue cultures the samematerial and technique were used as in the earlier work for the normal fibres [2]. Spinal ganglia of 9 to 14 days old chick embryos were cultivated in a thin plasma clot on covers slips which had been coated previously with Formvar. The incubation temperature was 33-35”. When the cultures showed a good growth of fibres, a slight cut was made in r This investigation Rio de Janeiro (Dir. fellowship.
Fig. still
1. Spinal preserved,
gangl. of 14 days old chick embryo. droplets appear along the fibres
Fig. 2. Spinal gang]. membranes and fine and 3000. Experimental
has been carried out in the Instilulo de Biofisica da Universidade do Brasil, Dr. Carlos Chagas) under the tenure of a Conselho National de Pesquisas
of 14 days prolongations.
Cell Research
9
old chick embryo. In (6) terminal
7 minutes after an injury. Striation and are extruded. x 10,000. Terminal extension
extension one hour
in cytoplasm
of intact fibre after an injury.
(a) with x 1500
Ultrastructure
of experimentally
altered nerve fibres
Experimental
187
Cell 1Zt:search
9
H. Meyer the centre of the explant, seat of the nerve cells, with the point of a new cataract knife. Care was taken not to push the plasma clot too much. The effect of this injury could be observed immediately in the peripheral part of the cultures, where the nerve fibres showed the alterations which are described above. The cultures were fixed from two to 10 minutes after the injury, some after one hour, in vapour of osmic acid, with the same precautions and care which had been observed during the fixation of the normal fibres [2]. After washing in dist. water, and after removing the plasma clot the formvar film with the remaining fibres was transferred to the electron microscope grids, according to the technique of Porter, Claude and Fullam [3]. Here they were shadowed with chromium. They were examined then in the “Philips” electron microscope with 60 and 80 kV. Obseruations.-When the injury in the centre of the culture was restricted to a small area not all the nerve cells or fibres were damaged, and not all of the fibres in the periphery react. In those which are altered the aspect varies from complete destruction to more or less pronounced alterations. In all cases we see droplets which seem to be extruded from the cytoplasm, appear along the fibres (Fig. I), often at quite regular distances. They tend to flatten with time and stain with osmic acid. In some cases the fibrillar aspect of the cytoplasm becomes more pronounced by this loss of material, in others the structure is completely lost. Also in the terminal extensions of the fibres the effect is immediate. The ameboid membranes retract and enlargements appear along the thin prolongations (Fig. 2a and b). In the fusiform enlargements of the fibres which occur normally in tissue cultures the striation may be partially preserved after an injury. The enlargements may also show extrusion of droplets, or the structure may be completely lost. Results and Discussion.-The effect of damage on the tissue cultures is immediate and varies in degree on the fibres, on their terminal extensions, and on the enlargements which preexisted in the fibres. The most immediate and striking effect is the apperance and extrusion of droplets along the fibres. They are probably of lipid nature becausethey stain black with osmic acid. Judging from their rather regular distribution the droplets might be responsible for the varicosic aspect of the fibres in the light microscope immediately after an injury. The results obtained during this investigtion are only preliminary. For technical reasons we thought it necessary to examine in a rough test the alterations in the structures of the injured fibres, because similar damages are easily produced by inadequate treatment of the tissue cultures, and similar changesin the structures will also occur during the preparation of peripheral nerves or of the central nervous system for inclusion or ultrathin sectioning, since it is almost impossible to obtain such preparations without an interruption of the nerve fibres’ continuity. REFERENCES 1. LEVI G. and MEYER, H., J. Expll. Zool. 99, 141 (1945). 2. MEYER, H., Exptl. Cell Research 7, 15 (1954). 3. PORTER, K. R.&LAUDE, A., and FULLAM, E. A., J. Exptl.
Experimental
Cell Research
9
Med.
81, 233 (1945).