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Incorporation of thymidine by injured lens epithelium
Solutions of colchicinr give ~narrosco~~icall~ a faint tlccl)-t’c(ltlisll Iluor~~sccnc~. Unfortunately, its intensit>- \vithin the muscle cells is loo weak (0 IJCtlc(cctctl I)> the microscope.
REFERENCES
1. 2. 3. 4. 5.
6.
DE BRUYS, P. I’. H. and SMIIX, N. Ii. ~.c~)//. (Xl k’ese(ir(.h 17, 182 (1M~l). KLINGENBEIIC;, H. G., %. Bid. 108, 312 (1956). __ ibid. 108, 330 (1956). ibid. 109, 161 (1957). K~LBEL, H., Z. Satnr/orsch. 3h, 442 (194X). ZEIFEK, I<., HAKDERS, H. und MLILLE~, \V.. I’rotoplctsmu 40, 56 (1951).
INCORPORATION
OF THYMIDINE
BY INJURED
LENS
EPITHELIUMl C. V. HARDING, Departments
A. DONN and B. DOBLI
SRINIVASAN
of Ophthalmology and PhysioEogy, College of Physicians Columbia University, New York, N.Y., U.S.A.
und Surgeons,
Received September 29, 1959
A
SINGLE layer of epithelial cells covers the anterior surface of the crystalline lens. Mitotic figures appear in this layer of cells but they occur almost exclusively in the germinative zone at the far periphery; they are rarely seen in the central region [4]. When, in the course of another study, the central lens epithelium was accidentally injured, large numbers of mitotic figures were discovered in the epithelium adjacent to the wound. Thymidine has been shown to be incorporated into the nuclei of cells in proliferating tissues [a], and it is generally assumed that this is an indication of DNA synthesis by the cell in preparation for division. Thymidine incorporation, as visualized by means of autoradiography, has been used as a means of studying cellular proliferation in various tissues 131. In this respect it becomes of interest to study the reaction of tissues to injury; and, in the present paper, the incorporation of thymidine into the cells of injured lens epithelium is reported. Materials and i\/lethods.-Healthy rabbits of 8-10 weeks of age (approximately 1 kg) were studied. After the rabbits were anesthetized, 0.3 cc of Heparin (1000 units/cc) was injected intravenously to prevent fibrin formation in the anterior chamber. The lens was injured by inserting a 27 gauge needle through the cornea near the limbus into the anterior chamber. When the point of the needle was over the central 1 This investigation was supported in part by the Knapp acknowledge the help and advice of Dr. Virginia Weimar. Experimenfai
Cell Research 18
Memorial
Fund. We should like to
Thymidine
uptake in lens epithelium
Fig. l.-Autoradiogram of 24 hour wound. Thymidine was injected at 22 hours after injury, and the eye was fixed at 24 hours. Harris hematoxylin. Approximately 32 x magnification. Note the large numbers of radioactive nuclei adjacent to the wound. Fig. 2.-Autoradiogram of small field adjacent to the 24 hour wound shown in Fig. 1. Harris hcmatoxylin. Approximately 300 x magnification. Note the high per cent of radioactive nuclei.
of the lens, a single puncture was made through the lens capsule, and the needle was immediately withdrawn. At different periods of time after injury the aqueous humor was removed and approximately 4 yc of tritium-labelled thymidine was injected into the anterior chamber. The tritium-labelled thymidine had a specific activity of 0.36 c/mM and it was made up in Eagle’s basal medium with a final concentration of 20 &ml. Two hours after injection the eyes were enucleated and fixed in acetic acid : alcohol (1: 3). Whole-mount preparations of lens epithelium were prepared for autoradiography by a method which has recently been described [l]. The autoradiograms were developed at the end of seven days exposure. Results and Discussion.-The results demonstrate that many epithelial cells in the vicinity of the wound incorporate thymidine following injury. Fig. I shows the large numbers of radioactive cells surrounding a 24-hour wound. In some restricted areas as many as 80 per cent of the nuclei are radioactive, Fig. 2. The per cent of cells showing incorporation, however, is time-dependent, with maximum values at one to two days following injury. None of the epithelial cells in the vicinity of the wound incorporate thymidine by 4.5 hours after injury. A maximum per cent was portion
Experimental
Cell Research
18
obtained at 21 J~ours, and a high per ccnl was I’ountl at 48 hours. \.cr>. I’(*\Y NCI’~ found at 72 hours. The distribution of radioactive nuclei also changed with tilnc. At 24 hours, as is seen in Fig. 1, thymidinc uptake was confined to cells in the immediate vicinity of the wound, whtreas at 3X hours the radioactive nuclei wcrc distributed in two separate bands about the wound. Pig. 3 shows a preparation 4X hours after injury. There is an outer zone of radioactive nuclei (C) in addition Lo the band (II) immediately adjacent to the wound. The distribution is, however, \-ariable. At 48 and 72 hours after injury, cells appeared within the wound, and many of these cells incorporated thymidine. Some preparations at 24 hours also showed cells, but there were no cells evident within the wounded area at 4.5 hours. It is possible that some cells are lost from the wounded arca during preparation of lhe wholcmount. Control experiments without thymidine injection did not show silver grains concentrated over cell nuclei in any of the autoradiograms. The results demonstrate that injury stimulates many epithelial cells adjacent to the wound to take up thymidine. The possibility that this distribution corresponds to the diffusion of an injury substance(s) remains to be explored. The very high per cent of cells incorporating thymidine raises the question of the relationship betwern thyExperimental
Cell flesurch
18
Thymidine
585
uptake in lens epithelium
midine uptake and cell division. If all the radioactive cells in the 24 and 48 hour preparations would have ultimately divided, it would appear that injury stimulates a very high rate of cellular proliferation in the surrounding epithelium. Experiments are in progress to determine the distribution of mitotic figures in lens epithelium treated with colchicine at different times after injury. REFERENCES 1. HARDING, C. V., HUGWZ, W. L., BOND, V. P. and SCH~RK, P., Arch. OphthuZ. (in press) 1959. 2. HUGHES, W. L., BOND, V. P., J~RECIIER, Cr., CRONKITE, E. P., PAINTER, R. B., QUASTLER, H. and SHERMAN, F. G., Proc. Natl. Acud. Sci. 44, 476 (1958). 3. STOHLMAN, I:., JR., The Kinetics of Cellular Proliferation. Prune and Stratton, N.Y., 1959. 4. VON SALLMANN, L., Arch. Ophthd. 47, 305 (1952).
AUTORADIOGRAPHIC
STUDIES
W-LABELED ALGAL PROTEIN EARLY SEA URCHIN
ON INCORPORATION HYDROLYSATE DEVELOPMENT1
OF
IN THE
J. IMMERS Wenner-Gras
Institute
for Experimental
Biology,
University
of Stockholm,
Sweden
Received October 1, 1959
N..a.mo and
Monroy (71 have worked out an efficient method of incorporating labeled amino acids into the ovarial eggs of sea urchins. The labeled compounds are injected into the body cavity of females which thereafter are kept for four hours in the moist chamber. Subsequently the animals are opened and after several washings in sea water an egg suspension is prepared. It seemed of interest to use the procedure of Nakano and Monroy for incorporation of labeled amino acids in connection with ensuing autoradiographic examination of unfertilized and of fertilized eggs fixed in different stages of development. Mafcrial and melhods.-Paraeenfrotus lividus has been used as material for these experiments which were carried out at the “Stazione Zoologica” of Naples. Algal protein hydrolysate IX, containing 14 different amino acids was injected into the body cavity of ripe females. Each animal received 88 PC in 0.5 ml sea water per animal which thereafter were kept in a moist chamber for four hours. Afterwards the eggs were prepared, fertilized and the larvae reared, fixed, embedded and sectioned in customary way [2, 41. Stripping-film autoradiographs were prepared of sections which after dcparaffination in xylol had been brought to water through graded alcohols. ’ Supported by grants from the Swedish Natural Anti-Cancer Society. 37 - 693709
Sciences Research
Council
Experimental
and the Swedish Cell Reseurch 18
REFERENCES
1. 2. 3. 4. 5.
6.
DE BRUYS, P. I’. H. and SMIIX, N. Ii. ~.c~)//. (Xl k’ese(ir(.h 17, 182 (1M~l). KLINGENBEIIC;, H. G., %. Bid. 108, 312 (1956). __ ibid. 108, 330 (1956). ibid. 109, 161 (1957). K~LBEL, H., Z. Satnr/orsch. 3h, 442 (194X). ZEIFEK, I<., HAKDERS, H. und MLILLE~, \V.. I’rotoplctsmu 40, 56 (1951).
INCORPORATION
OF THYMIDINE
BY INJURED
LENS
EPITHELIUMl C. V. HARDING, Departments
A. DONN and B. DOBLI
SRINIVASAN
of Ophthalmology and PhysioEogy, College of Physicians Columbia University, New York, N.Y., U.S.A.
und Surgeons,
Received September 29, 1959
A
SINGLE layer of epithelial cells covers the anterior surface of the crystalline lens. Mitotic figures appear in this layer of cells but they occur almost exclusively in the germinative zone at the far periphery; they are rarely seen in the central region [4]. When, in the course of another study, the central lens epithelium was accidentally injured, large numbers of mitotic figures were discovered in the epithelium adjacent to the wound. Thymidine has been shown to be incorporated into the nuclei of cells in proliferating tissues [a], and it is generally assumed that this is an indication of DNA synthesis by the cell in preparation for division. Thymidine incorporation, as visualized by means of autoradiography, has been used as a means of studying cellular proliferation in various tissues 131. In this respect it becomes of interest to study the reaction of tissues to injury; and, in the present paper, the incorporation of thymidine into the cells of injured lens epithelium is reported. Materials and i\/lethods.-Healthy rabbits of 8-10 weeks of age (approximately 1 kg) were studied. After the rabbits were anesthetized, 0.3 cc of Heparin (1000 units/cc) was injected intravenously to prevent fibrin formation in the anterior chamber. The lens was injured by inserting a 27 gauge needle through the cornea near the limbus into the anterior chamber. When the point of the needle was over the central 1 This investigation was supported in part by the Knapp acknowledge the help and advice of Dr. Virginia Weimar. Experimenfai
Cell Research 18
Memorial
Fund. We should like to
Thymidine
uptake in lens epithelium
Fig. l.-Autoradiogram of 24 hour wound. Thymidine was injected at 22 hours after injury, and the eye was fixed at 24 hours. Harris hematoxylin. Approximately 32 x magnification. Note the large numbers of radioactive nuclei adjacent to the wound. Fig. 2.-Autoradiogram of small field adjacent to the 24 hour wound shown in Fig. 1. Harris hcmatoxylin. Approximately 300 x magnification. Note the high per cent of radioactive nuclei.
of the lens, a single puncture was made through the lens capsule, and the needle was immediately withdrawn. At different periods of time after injury the aqueous humor was removed and approximately 4 yc of tritium-labelled thymidine was injected into the anterior chamber. The tritium-labelled thymidine had a specific activity of 0.36 c/mM and it was made up in Eagle’s basal medium with a final concentration of 20 &ml. Two hours after injection the eyes were enucleated and fixed in acetic acid : alcohol (1: 3). Whole-mount preparations of lens epithelium were prepared for autoradiography by a method which has recently been described [l]. The autoradiograms were developed at the end of seven days exposure. Results and Discussion.-The results demonstrate that many epithelial cells in the vicinity of the wound incorporate thymidine following injury. Fig. I shows the large numbers of radioactive cells surrounding a 24-hour wound. In some restricted areas as many as 80 per cent of the nuclei are radioactive, Fig. 2. The per cent of cells showing incorporation, however, is time-dependent, with maximum values at one to two days following injury. None of the epithelial cells in the vicinity of the wound incorporate thymidine by 4.5 hours after injury. A maximum per cent was portion
Experimental
Cell Research
18
obtained at 21 J~ours, and a high per ccnl was I’ountl at 48 hours. \.cr>. I’(*\Y NCI’~ found at 72 hours. The distribution of radioactive nuclei also changed with tilnc. At 24 hours, as is seen in Fig. 1, thymidinc uptake was confined to cells in the immediate vicinity of the wound, whtreas at 3X hours the radioactive nuclei wcrc distributed in two separate bands about the wound. Pig. 3 shows a preparation 4X hours after injury. There is an outer zone of radioactive nuclei (C) in addition Lo the band (II) immediately adjacent to the wound. The distribution is, however, \-ariable. At 48 and 72 hours after injury, cells appeared within the wound, and many of these cells incorporated thymidine. Some preparations at 24 hours also showed cells, but there were no cells evident within the wounded area at 4.5 hours. It is possible that some cells are lost from the wounded arca during preparation of lhe wholcmount. Control experiments without thymidine injection did not show silver grains concentrated over cell nuclei in any of the autoradiograms. The results demonstrate that injury stimulates many epithelial cells adjacent to the wound to take up thymidine. The possibility that this distribution corresponds to the diffusion of an injury substance(s) remains to be explored. The very high per cent of cells incorporating thymidine raises the question of the relationship betwern thyExperimental
Cell flesurch
18
Thymidine
585
uptake in lens epithelium
midine uptake and cell division. If all the radioactive cells in the 24 and 48 hour preparations would have ultimately divided, it would appear that injury stimulates a very high rate of cellular proliferation in the surrounding epithelium. Experiments are in progress to determine the distribution of mitotic figures in lens epithelium treated with colchicine at different times after injury. REFERENCES 1. HARDING, C. V., HUGWZ, W. L., BOND, V. P. and SCH~RK, P., Arch. OphthuZ. (in press) 1959. 2. HUGHES, W. L., BOND, V. P., J~RECIIER, Cr., CRONKITE, E. P., PAINTER, R. B., QUASTLER, H. and SHERMAN, F. G., Proc. Natl. Acud. Sci. 44, 476 (1958). 3. STOHLMAN, I:., JR., The Kinetics of Cellular Proliferation. Prune and Stratton, N.Y., 1959. 4. VON SALLMANN, L., Arch. Ophthd. 47, 305 (1952).
AUTORADIOGRAPHIC
STUDIES
W-LABELED ALGAL PROTEIN EARLY SEA URCHIN
ON INCORPORATION HYDROLYSATE DEVELOPMENT1
OF
IN THE
J. IMMERS Wenner-Gras
Institute
for Experimental
Biology,
University
of Stockholm,
Sweden
Received October 1, 1959
N..a.mo and
Monroy (71 have worked out an efficient method of incorporating labeled amino acids into the ovarial eggs of sea urchins. The labeled compounds are injected into the body cavity of females which thereafter are kept for four hours in the moist chamber. Subsequently the animals are opened and after several washings in sea water an egg suspension is prepared. It seemed of interest to use the procedure of Nakano and Monroy for incorporation of labeled amino acids in connection with ensuing autoradiographic examination of unfertilized and of fertilized eggs fixed in different stages of development. Mafcrial and melhods.-Paraeenfrotus lividus has been used as material for these experiments which were carried out at the “Stazione Zoologica” of Naples. Algal protein hydrolysate IX, containing 14 different amino acids was injected into the body cavity of ripe females. Each animal received 88 PC in 0.5 ml sea water per animal which thereafter were kept in a moist chamber for four hours. Afterwards the eggs were prepared, fertilized and the larvae reared, fixed, embedded and sectioned in customary way [2, 41. Stripping-film autoradiographs were prepared of sections which after dcparaffination in xylol had been brought to water through graded alcohols. ’ Supported by grants from the Swedish Natural Anti-Cancer Society. 37 - 693709
Sciences Research
Council
Experimental
and the Swedish Cell Reseurch 18