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Oxygen changes in tissues of germfree and conventional mice after inoculation of bacterial endotoxin
Lüe Sciences Vol. 7, Part I, pp. 1075-1081, 1988. Printed in Great Britain .
Perga.mon Press
OXYGEN CHANGES IN TISSUES OF GERMFREE AND CONVENTIONAL MICE AFTER INOCULATION OF BACTERIAL ENDOTOXIN` Raphael Wilson and Taiju Matsuzawa~`~` Lobund Laboratory, University of Notre Dame, Notre Dame, Indiana
(Received 1 July 1968; in final form 17 July 1988) Bacterial endotoxin injected into mice prior to irradiation will give protection against wholebody exposure to ionizing radiation. The greatest degree of protection is conferred when it is administered 24 hours before irradiation (1). Smith, Alderman and Gillespie found that the LD50(30) for endotoxin treated mice was approximately 200 R higher than for untreated controls when both groups were exposed to X-radiation in the dose range of hematopoietic failure.
In contrast, when endotoxin treated and untreated mice
were exposed to X-ray doses within the range of gastrointestinal damage, the only difference noted between the two groups was an increase in survival time in the treated mice . Germfree mice withstand higher doses of X-rays than conventional mice ; the LD50(30) X-ray dose is 705 R for, germfree Swiss-Webster mice and 660 R for their conventional counterparts (2).
When germfree mice are exposed to
doses of X-radiation in the "gut death" dose range between 1, 000 and 3, 000 R, they live twice as long as com~entional control mice given the same doses (2) . Germfree mice can withstand still higher doses of X-rays when administered endotoxin before irradiation (3).
That a reduction in oxygen tension ( p02) in
Supported by U. S. Public Health Service Grant RH-00239-01, 02 . Present address : Sendai University, Sendai, Japan.
1075
1076
OXYGEN CHANGES
Vol . 7, No . 19
tissues results in a concomitant reduction of tissue damage from irradiation has been well documented (4); therefore, we undertook this study to determine if the injection of bacterial endotoxin into mice can effect an alteration of the p02 of the animals' tissues and consequently afford the animals some degree of radioprotection. Material and Methods In this study 52 germfree and 52 conventional (non-germfree) CFW mice, 11 weeks old, and of both sexes were used . The germfree mice were maintained in plastic cages within Trexler flexible film isolators (5) under the routine procedures established at the Lobund Laboratory for germfree animals. Conventional mice were housed in plastic cages, given sterilized food, Purina lab chow 5010 C, and water comparable to the germfree mice but were not maintained in isolators.
Sterilized Salmonella typhosa endotoxin (Difco) in
isotonic saline solution was administered intraperitoneally to all mice : half of the germfree and conventional mice received 10 Fig and the other half received 20 Fig of endotoxin.
Thereafter the p02 was measured with a Beck-
man physiological gas analyzer with oxygen microelectrodes inserted in the following tissues : subcutaneous tissues, gluteus muscle, liver, and the marrow cavity of the femur . The mice of each set were paired randomly and each pair was used as the source of tissues for p02 determinations at one of following times after inoculation: 0, 7, 15, and 30 minutes; 1, 2, 6, and 20 hours; and 1, 2, 3, 4, and 5 days . Results In Table 1 are the p02 values of the four tissues of germfree and conventional mice examined over a period of 5 days after endotoxin administration. The pattern is qualitatively the same for each type of tissue : (a) a drop in
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ORYGEN CHANGES
Vol. 7, No. 19
p02 immediately after endotoxin injection reaching a low in 7-15 minutes, (b) a subsequent recovery to a supranormal high value in about an hour, then (c) a gradual decline in p02 to a new low 24 hours after injection, and finally (d) a gradual return to normal during the next four days . The changes in p02 in the subcutaneous tissue of germfree and conventional mice after receiving 10 ug endotoxin are depicted in Figure 1, which
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FIG . 1 Changes in E02 in the subcutaneous tissue of germfree and comrentonal mice after receiving 30 hg Escherichia coli endotoxin intraperitoneally. The bars at each point extend 1 standard error above and below the plotted means. is representative of the data comparing the p02 changes of the various tissues of germfree and comrentonal mice after the administration of endotoxin. Generally, throughout the period studied the p02 values of germfree mouse tissues were significantly lower than those of conventional mice . The greatest
Vol. 7, No . 19
OXYGEN CAANGES
1079
differences occurred during the first period of p02 depression and during the 5 day recovery period . The p02 values of the subcutaneous, muscle, liver, and bone marrow tissues of both germfree and conventional mice as determined at daily intervals up to 5 days after inoculation with endotosi.n are represented in Figure 2 .
FIG . 2 10 ug 20 ~g Changes in p02 in subcutaneous tissue, bone marrow, liver, and muscle of germfree and conventional mice determined daily after intraperitoneal injection of 10 pg and 20 kg Escherichia coli endato~n . _____o_____
In both germfree and conventional mice 20 F+g endoto~n depressed the
P2
more than 10 pg . However, the recovery period showed the most significant differences : at the end of 5 days the values for germfree mice were still
OXYGEN CHANGES
1080
Vol . 7, No . 19
subnormal after both dose levels . In conventional mice there is complete recovery in 5 days after the administration of 10 ug endotoxin, but incomplete recovery in the same period after 20 ug endotoxin. The subcutaneous tissue and the bone marrow showed a more pronounced reduction in p02 than muscle and liver. This prolonged recovery period in germfree mice may reflect an underdeveloped detoxification system ; conventional mice that are constantly under the stress of bacterial antigens may be better equipped for detoxification than the germfree mice . Discussion The radiosensitivity of various mammalian cells imrestigated is positively correlated with the availability of dissolved molecular oxygen in the medium (6). After endotoxin administration there is a decreased blood flow m capillary beds resulting from vasoconstriction (7) . Increased arteriolar resistance would reduce capillary blood pressure .
The reduced capillary blood pressure
accounts for a reduced net loss of fluid from the blood to the tissues, thus explaining the observed hemodilution and tissue dehydration. As previously reported, the most pronounced hemodilution occurs 24 hours after endotoxin administration (8).
Diffusion of blood oxygen into tissues is consequently
limited resulting in a tissue hypoxia; the period of greatest hemodilution coincided with the period of the greatest reduction in p02 after endotoxin injection. These results corroborate those of Hornsey (9) and Storer and Hempelmann (10), who concluded that anoxia is a very important factor in increased survival of mice irradiated under hypothermic conditions .
Thus it seems probable that
protection against wholebody ionizing radiation is conferred on mice by bacterial endotoxin by a mechanism that reduces the p02 of radiosensitive tissues to a level associated with protection .
Vol . 7, No . 19
OXYGEN CHANGES
1081
References 1.
W. SMITH, I. ALDERMAN and R. GILLESPIE, Am . J . Physiol, 191, 124 (1957) .
2,
R. WILSON, Radiation Res . 20, 477 (1963) .
3.
R. WILSON and T. MATSUZAWA, Radiation Res, 19, 231 (1963),
4.
J . THOMSON, Radiation Protection in Mammals, pp . 45-50, New York (1962),
5,
P. C. TREXLER and L . I. REYNOLDS, Appl . Microbiol. 5, 406 (1957),
6.
A . WHITING, Radiatian Protection and Recovery , edited by A. HOLLAENDER, pp, 133-140, Pergamon, New York (1960),
7,
L. THOMAS, Physiol, Rev. 16, 467 (1954) .
8,
R. WILSON, G. D. LEDNEY and T . MATSUZAWA, Progress in Biochemical Pharmacology, edited by R. PAOLETTI and R. VERTUA, pp . 622-626 . Butterworths, Washingtan (1965),
9.
S. HORNSEY, Proc . Royal Soc . London 147, 547 (1957),
10,
Reinhold,
J . B. STÖRER and L. H . HEMPELMANN, Am . J. Physiol. 171, 341 (1952),
Perga.mon Press
OXYGEN CHANGES IN TISSUES OF GERMFREE AND CONVENTIONAL MICE AFTER INOCULATION OF BACTERIAL ENDOTOXIN` Raphael Wilson and Taiju Matsuzawa~`~` Lobund Laboratory, University of Notre Dame, Notre Dame, Indiana
(Received 1 July 1968; in final form 17 July 1988) Bacterial endotoxin injected into mice prior to irradiation will give protection against wholebody exposure to ionizing radiation. The greatest degree of protection is conferred when it is administered 24 hours before irradiation (1). Smith, Alderman and Gillespie found that the LD50(30) for endotoxin treated mice was approximately 200 R higher than for untreated controls when both groups were exposed to X-radiation in the dose range of hematopoietic failure.
In contrast, when endotoxin treated and untreated mice
were exposed to X-ray doses within the range of gastrointestinal damage, the only difference noted between the two groups was an increase in survival time in the treated mice . Germfree mice withstand higher doses of X-rays than conventional mice ; the LD50(30) X-ray dose is 705 R for, germfree Swiss-Webster mice and 660 R for their conventional counterparts (2).
When germfree mice are exposed to
doses of X-radiation in the "gut death" dose range between 1, 000 and 3, 000 R, they live twice as long as com~entional control mice given the same doses (2) . Germfree mice can withstand still higher doses of X-rays when administered endotoxin before irradiation (3).
That a reduction in oxygen tension ( p02) in
Supported by U. S. Public Health Service Grant RH-00239-01, 02 . Present address : Sendai University, Sendai, Japan.
1075
1076
OXYGEN CHANGES
Vol . 7, No . 19
tissues results in a concomitant reduction of tissue damage from irradiation has been well documented (4); therefore, we undertook this study to determine if the injection of bacterial endotoxin into mice can effect an alteration of the p02 of the animals' tissues and consequently afford the animals some degree of radioprotection. Material and Methods In this study 52 germfree and 52 conventional (non-germfree) CFW mice, 11 weeks old, and of both sexes were used . The germfree mice were maintained in plastic cages within Trexler flexible film isolators (5) under the routine procedures established at the Lobund Laboratory for germfree animals. Conventional mice were housed in plastic cages, given sterilized food, Purina lab chow 5010 C, and water comparable to the germfree mice but were not maintained in isolators.
Sterilized Salmonella typhosa endotoxin (Difco) in
isotonic saline solution was administered intraperitoneally to all mice : half of the germfree and conventional mice received 10 Fig and the other half received 20 Fig of endotoxin.
Thereafter the p02 was measured with a Beck-
man physiological gas analyzer with oxygen microelectrodes inserted in the following tissues : subcutaneous tissues, gluteus muscle, liver, and the marrow cavity of the femur . The mice of each set were paired randomly and each pair was used as the source of tissues for p02 determinations at one of following times after inoculation: 0, 7, 15, and 30 minutes; 1, 2, 6, and 20 hours; and 1, 2, 3, 4, and 5 days . Results In Table 1 are the p02 values of the four tissues of germfree and conventional mice examined over a period of 5 days after endotoxin administration. The pattern is qualitatively the same for each type of tissue : (a) a drop in
OXYGEN CHANGES
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107 8
ORYGEN CHANGES
Vol. 7, No. 19
p02 immediately after endotoxin injection reaching a low in 7-15 minutes, (b) a subsequent recovery to a supranormal high value in about an hour, then (c) a gradual decline in p02 to a new low 24 hours after injection, and finally (d) a gradual return to normal during the next four days . The changes in p02 in the subcutaneous tissue of germfree and conventional mice after receiving 10 ug endotoxin are depicted in Figure 1, which
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~OfT - INOCULATION ( ~leo
1
FIG . 1 Changes in E02 in the subcutaneous tissue of germfree and comrentonal mice after receiving 30 hg Escherichia coli endotoxin intraperitoneally. The bars at each point extend 1 standard error above and below the plotted means. is representative of the data comparing the p02 changes of the various tissues of germfree and comrentonal mice after the administration of endotoxin. Generally, throughout the period studied the p02 values of germfree mouse tissues were significantly lower than those of conventional mice . The greatest
Vol. 7, No . 19
OXYGEN CAANGES
1079
differences occurred during the first period of p02 depression and during the 5 day recovery period . The p02 values of the subcutaneous, muscle, liver, and bone marrow tissues of both germfree and conventional mice as determined at daily intervals up to 5 days after inoculation with endotosi.n are represented in Figure 2 .
FIG . 2 10 ug 20 ~g Changes in p02 in subcutaneous tissue, bone marrow, liver, and muscle of germfree and conventional mice determined daily after intraperitoneal injection of 10 pg and 20 kg Escherichia coli endato~n . _____o_____
In both germfree and conventional mice 20 F+g endoto~n depressed the
P2
more than 10 pg . However, the recovery period showed the most significant differences : at the end of 5 days the values for germfree mice were still
OXYGEN CHANGES
1080
Vol . 7, No . 19
subnormal after both dose levels . In conventional mice there is complete recovery in 5 days after the administration of 10 ug endotoxin, but incomplete recovery in the same period after 20 ug endotoxin. The subcutaneous tissue and the bone marrow showed a more pronounced reduction in p02 than muscle and liver. This prolonged recovery period in germfree mice may reflect an underdeveloped detoxification system ; conventional mice that are constantly under the stress of bacterial antigens may be better equipped for detoxification than the germfree mice . Discussion The radiosensitivity of various mammalian cells imrestigated is positively correlated with the availability of dissolved molecular oxygen in the medium (6). After endotoxin administration there is a decreased blood flow m capillary beds resulting from vasoconstriction (7) . Increased arteriolar resistance would reduce capillary blood pressure .
The reduced capillary blood pressure
accounts for a reduced net loss of fluid from the blood to the tissues, thus explaining the observed hemodilution and tissue dehydration. As previously reported, the most pronounced hemodilution occurs 24 hours after endotoxin administration (8).
Diffusion of blood oxygen into tissues is consequently
limited resulting in a tissue hypoxia; the period of greatest hemodilution coincided with the period of the greatest reduction in p02 after endotoxin injection. These results corroborate those of Hornsey (9) and Storer and Hempelmann (10), who concluded that anoxia is a very important factor in increased survival of mice irradiated under hypothermic conditions .
Thus it seems probable that
protection against wholebody ionizing radiation is conferred on mice by bacterial endotoxin by a mechanism that reduces the p02 of radiosensitive tissues to a level associated with protection .
Vol . 7, No . 19
OXYGEN CHANGES
1081
References 1.
W. SMITH, I. ALDERMAN and R. GILLESPIE, Am . J . Physiol, 191, 124 (1957) .
2,
R. WILSON, Radiation Res . 20, 477 (1963) .
3.
R. WILSON and T. MATSUZAWA, Radiation Res, 19, 231 (1963),
4.
J . THOMSON, Radiation Protection in Mammals, pp . 45-50, New York (1962),
5,
P. C. TREXLER and L . I. REYNOLDS, Appl . Microbiol. 5, 406 (1957),
6.
A . WHITING, Radiatian Protection and Recovery , edited by A. HOLLAENDER, pp, 133-140, Pergamon, New York (1960),
7,
L. THOMAS, Physiol, Rev. 16, 467 (1954) .
8,
R. WILSON, G. D. LEDNEY and T . MATSUZAWA, Progress in Biochemical Pharmacology, edited by R. PAOLETTI and R. VERTUA, pp . 622-626 . Butterworths, Washingtan (1965),
9.
S. HORNSEY, Proc . Royal Soc . London 147, 547 (1957),
10,
Reinhold,
J . B. STÖRER and L. H . HEMPELMANN, Am . J. Physiol. 171, 341 (1952),