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Influence of temperature on wound healing in a poikilotherm
442
J. R. Reddan and H. Rothstein
bryonic chick heart inner ventricular wall is striking. The effect is stable over an extended time period. A similar conditioned medium phenomenon has been reported by Konigsberg [9] for voluntary striated muscle. In the present case, however, the source of the conditioning agents in the differentiation maintaining system is the factor or factors added to the medium by the differentiating cardiac muscle tissue fabric itself. REFERENCES 1. BURROWS, M. T., Science 2. DE HAAN, R., Carnegie 3. EAGLE, H., Science 148,
36, 90 (1912). Inst. Wash. Year 42 (1965).
Hook
63, 531
(1964).
4. ~ ibid. 130, 432 (1959). 5. GORDON, H. P., Nature 202, 1035 (1964). 6. GRAY, J. C. and POWELL, E. W., Science 116, 232 (1952). 7. HARARY, 1. and FARLEY, B., Expptl Cell Res. 29, 451 (1963). 8. JUR~ND, A., J. Embrol. Expfl Morphol. 10, 602 (1962). 9. KOXIGSBERG, I., Science 140, 1273 (1963).
INFLUENCE
OF TEMPERATURE
ON WOUND
HEALING
IN A POIKILOTHERMl J. R. REDDAN Department
of Zoology,
and H. ROTHSTEIN
University Received
of Vermont, July
Burlington,
Vt.,
U.S.A.
27, 1965
I N earlier reports we have shown that mechanical injury of the bullfrog lens epithelium stimulates the synthesis of DNA and is followed by cell division [5, 61. A similar effect of injury was reported earlier by Harding and Srinivasan in rabbit lenses [3] but the details of the responses differ [5]. Amphibians being poikilotherms, it becomes possible to use them for in uiuo experiments on the healing process as it occurs at different temperatures. The results of such experiments form the subject of this communication. Bullfrog lenses were injured with a zero (0) gauge insect needle, as described in our earlier work, and the animals were then exposed for varying periods of time to temperatures of 4, 24, or 33°C. After such exposure the eyes were enucleated, and the lenses were isolated and incubated in Eagles minimal essential medium [l] diluted with triple distilled water so as to be physiologically compatible with amphibian tissue. Tritiated thymidine was added to this medium to a final concentration of 5 PC/ml (Sp. AC. 3.0 c/mm). The incubation period was 2& hr. The lenses were fixed directly after incubation in 3:i alcoholLacetic acid and whole-mounts were then 1 This work was 2 NDEA Fellow.
Experimental
supported
Cell Research
by
40
Public
Health
Service
Grant
No.
NB-05425-01.
Temperature
and wound
Fig. Figs. l-3.-Autoradiograms 24 hr in 3H-thymidine,
of epithelial 46 hr after needle
whole injury.
healing
443
in frog lens
1. mounts from lenses which were incubated Fig. 1, 4°C; Fig. 2, 24°C; Fig. 3, 33°C.
fol
made according to the procedure of Howard [4], and prepared for autoradiography in accord with previously described methods [a]. Exposure time was two weeks. Evaluation of the autoradiograms revealed that the course of the healing process was strongly affected by variation in temperature. The point is forcibly illustrated in Table I, as well as in Figs. 1-3. Table I shows that at high temperatures (33”) TABLE
I. Effects of different
temperatures
“C
Beginning of DNA synthesis (W
4 24 33
216 44 22-32
a Experiments
at 4°C were
on wound
closure and DNA
Waning of DNA synthesis W-1
Wound closure W-1
(7 96 72 not carried
synthesis.
None’ 72-96 4s beyond
216 hr. Experimental
Cell Research
40
J. R. Reddan
and H. Rothstein
Fig.
Experimc mtal Cell Research
40
3.
Use of pronase
in tissue culfure
the onset of both DNA synthesis and wound closure are accelerated. In the 4” experiments there was no DNA synthesis near the injury until approximately 216 hr after it had been inflicted, and at that time the wound had not yet closed. That this low temperature was not suppressing DNA synthesis itself is proven by the aforementioned observation, as well as the finding that there was some random incorporation of $H-Thd throughout the course of the experiment. Figs. I, 2, and 3, which are autoradiograms of healing lenticular tissue at 48 hr, also show clearly that the rapidity of the healing process is notably affected by the high and low temperature regimens. At 4” there is no synthesis or closure; at 24” there is synthesis but no closure; and at 33” there is both closure and synthesis. The relative number of labeled cells in the 33” preparation is not as great as in the 24” specimen. The former looks, in fact, almost identical to a 24” wound at 96 hr, for at this time the number of cells in synthesis decreases and the wound begins to close. Clearly, the number of chemical reactions whose timing could have been changed by temperature fluctuations must be legion. In the cytological realm, the kinetics of proliferation appear to be most grossly affected. A cursory inspection of grain density over the nuclei in the several experiments performed is, alone, strongly suggestive of alterations in the rate of thymidine incorporation (i.e., DNA synthesis). Doubtless other phases of the cell cycle must also have been affected. Current experiments arc aimed at providing a quantitative footing for assessment of these effects. REFERENCES 1.
EAGLE,
2.
HAILING,
H., thd.
3.
HARDING,
4. 5. 6.
HOW.ZRD, ROTHSTEIN, ROTHSTEIX,
Science 122, 501 (1955). C. V., HUGHES, W. L., BOND, V. P. and Scaon~, A. B., Am. Alled. Ass. 63, 58 (1960). C. V. and SRINIVASAN, B. II., Ezpll Gel/ Kes. 25, 326 (1961). A., Stain Technol. 27, 313 (1952). H., REDDAS, J. and WEINSIEDER, A., Exptl Cell Res. 37, 440 (1965). H., WEINSIEOER, A. and BLMKLOCK, R., ibid. 35, 548 (1964).
A CAUTIONARY
NOTE
CULTURE
M. J. ASHWOOD-SMITH
and D. MI. ROBINSON
Medical Research Council Psychiatric Genetics Research Medical Research Council Radiobiological Research Received
Oph-
ON THE USE OF PRONASE
IN TISSUE J. KAHN,
Arch.
August
Unit, Maudsley Unit, Harwell,
Hospital, Rerkshire,
London, L.:.I<.
10, 1965
GWYTKIN : and Thomson [2] recommended the Streptomyces griseus protease “Pronase” as ‘The best enzyme so far discovered for general use in tissue culture’, and as a replacement for trypsin. As a result of their advocacy we acquired two amounts of pronase (Calbiochem, lot 34045 and lot 45549) but after trials in these laboratories Experimental
Cell
Research
40
J. R. Reddan and H. Rothstein
bryonic chick heart inner ventricular wall is striking. The effect is stable over an extended time period. A similar conditioned medium phenomenon has been reported by Konigsberg [9] for voluntary striated muscle. In the present case, however, the source of the conditioning agents in the differentiation maintaining system is the factor or factors added to the medium by the differentiating cardiac muscle tissue fabric itself. REFERENCES 1. BURROWS, M. T., Science 2. DE HAAN, R., Carnegie 3. EAGLE, H., Science 148,
36, 90 (1912). Inst. Wash. Year 42 (1965).
Hook
63, 531
(1964).
4. ~ ibid. 130, 432 (1959). 5. GORDON, H. P., Nature 202, 1035 (1964). 6. GRAY, J. C. and POWELL, E. W., Science 116, 232 (1952). 7. HARARY, 1. and FARLEY, B., Expptl Cell Res. 29, 451 (1963). 8. JUR~ND, A., J. Embrol. Expfl Morphol. 10, 602 (1962). 9. KOXIGSBERG, I., Science 140, 1273 (1963).
INFLUENCE
OF TEMPERATURE
ON WOUND
HEALING
IN A POIKILOTHERMl J. R. REDDAN Department
of Zoology,
and H. ROTHSTEIN
University Received
of Vermont, July
Burlington,
Vt.,
U.S.A.
27, 1965
I N earlier reports we have shown that mechanical injury of the bullfrog lens epithelium stimulates the synthesis of DNA and is followed by cell division [5, 61. A similar effect of injury was reported earlier by Harding and Srinivasan in rabbit lenses [3] but the details of the responses differ [5]. Amphibians being poikilotherms, it becomes possible to use them for in uiuo experiments on the healing process as it occurs at different temperatures. The results of such experiments form the subject of this communication. Bullfrog lenses were injured with a zero (0) gauge insect needle, as described in our earlier work, and the animals were then exposed for varying periods of time to temperatures of 4, 24, or 33°C. After such exposure the eyes were enucleated, and the lenses were isolated and incubated in Eagles minimal essential medium [l] diluted with triple distilled water so as to be physiologically compatible with amphibian tissue. Tritiated thymidine was added to this medium to a final concentration of 5 PC/ml (Sp. AC. 3.0 c/mm). The incubation period was 2& hr. The lenses were fixed directly after incubation in 3:i alcoholLacetic acid and whole-mounts were then 1 This work was 2 NDEA Fellow.
Experimental
supported
Cell Research
by
40
Public
Health
Service
Grant
No.
NB-05425-01.
Temperature
and wound
Fig. Figs. l-3.-Autoradiograms 24 hr in 3H-thymidine,
of epithelial 46 hr after needle
whole injury.
healing
443
in frog lens
1. mounts from lenses which were incubated Fig. 1, 4°C; Fig. 2, 24°C; Fig. 3, 33°C.
fol
made according to the procedure of Howard [4], and prepared for autoradiography in accord with previously described methods [a]. Exposure time was two weeks. Evaluation of the autoradiograms revealed that the course of the healing process was strongly affected by variation in temperature. The point is forcibly illustrated in Table I, as well as in Figs. 1-3. Table I shows that at high temperatures (33”) TABLE
I. Effects of different
temperatures
“C
Beginning of DNA synthesis (W
4 24 33
216 44 22-32
a Experiments
at 4°C were
on wound
closure and DNA
Waning of DNA synthesis W-1
Wound closure W-1
(7 96 72 not carried
synthesis.
None’ 72-96 4s beyond
216 hr. Experimental
Cell Research
40
J. R. Reddan
and H. Rothstein
Fig.
Experimc mtal Cell Research
40
3.
Use of pronase
in tissue culfure
the onset of both DNA synthesis and wound closure are accelerated. In the 4” experiments there was no DNA synthesis near the injury until approximately 216 hr after it had been inflicted, and at that time the wound had not yet closed. That this low temperature was not suppressing DNA synthesis itself is proven by the aforementioned observation, as well as the finding that there was some random incorporation of $H-Thd throughout the course of the experiment. Figs. I, 2, and 3, which are autoradiograms of healing lenticular tissue at 48 hr, also show clearly that the rapidity of the healing process is notably affected by the high and low temperature regimens. At 4” there is no synthesis or closure; at 24” there is synthesis but no closure; and at 33” there is both closure and synthesis. The relative number of labeled cells in the 33” preparation is not as great as in the 24” specimen. The former looks, in fact, almost identical to a 24” wound at 96 hr, for at this time the number of cells in synthesis decreases and the wound begins to close. Clearly, the number of chemical reactions whose timing could have been changed by temperature fluctuations must be legion. In the cytological realm, the kinetics of proliferation appear to be most grossly affected. A cursory inspection of grain density over the nuclei in the several experiments performed is, alone, strongly suggestive of alterations in the rate of thymidine incorporation (i.e., DNA synthesis). Doubtless other phases of the cell cycle must also have been affected. Current experiments arc aimed at providing a quantitative footing for assessment of these effects. REFERENCES 1.
EAGLE,
2.
HAILING,
H., thd.
3.
HARDING,
4. 5. 6.
HOW.ZRD, ROTHSTEIN, ROTHSTEIX,
Science 122, 501 (1955). C. V., HUGHES, W. L., BOND, V. P. and Scaon~, A. B., Am. Alled. Ass. 63, 58 (1960). C. V. and SRINIVASAN, B. II., Ezpll Gel/ Kes. 25, 326 (1961). A., Stain Technol. 27, 313 (1952). H., REDDAS, J. and WEINSIEDER, A., Exptl Cell Res. 37, 440 (1965). H., WEINSIEOER, A. and BLMKLOCK, R., ibid. 35, 548 (1964).
A CAUTIONARY
NOTE
CULTURE
M. J. ASHWOOD-SMITH
and D. MI. ROBINSON
Medical Research Council Psychiatric Genetics Research Medical Research Council Radiobiological Research Received
Oph-
ON THE USE OF PRONASE
IN TISSUE J. KAHN,
Arch.
August
Unit, Maudsley Unit, Harwell,
Hospital, Rerkshire,
London, L.:.I<.
10, 1965
GWYTKIN : and Thomson [2] recommended the Streptomyces griseus protease “Pronase” as ‘The best enzyme so far discovered for general use in tissue culture’, and as a replacement for trypsin. As a result of their advocacy we acquired two amounts of pronase (Calbiochem, lot 34045 and lot 45549) but after trials in these laboratories Experimental
Cell
Research
40