Mortality Caused by Intravenous Injection of India Ink - Europe PMC

Higher Susceptibility of Mast-Cell-Deficient W/WV Mutant Mice to Brain Thromboembolism and Mortality Caused by Intravenous Injection of India Ink

Y. KITAMURA, MD, T. TAGUCHI, MD, M. YOKOYAMA, MD, M. INOUE, MD, A. YAMAIODANI, H. ASANO, BA, T. KOYAMA, MD, A. KANAMARU, MD, K. HATANAKA, MD, B. K. WERSHIL, MD, and S. J. GALLI, MD

From the Institute for Cancer Research and Department of Pharmacology, Osaka University Medical School, Kita-ku, Osaka, Japan; Shizuoka Laboratory Animal Center, Hamamatsu, Shizuoka, Japan; the Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; the Department of Etiology and Pathogenesis, National Cardiovascular Center Research Institute, Suita, Osaka, Japan; the Combined Pediatric Gastroenterology/Nutrition Program, Harvard Medical School, Boston, Massachusetts; the Department of Pathology, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts; and the Charles A. Dana Research

Institute, Boston, Massachusetts

(WB x C57BL/6)FI-W/WV mice possess a genetic defect in multipotential hematopoietic stem cells; the mice are anemic and lack mast cells. The authors injected diluted India ink intravenously into W/WV mice and congenic normal + /+ mice and searched for genetically determined differences in the development of complications of the injection. In both W/WV and + /+ mice, intravenous ink resulted in thrombocytopenia and markedly prolonged bleeding times, as well as prolonged partial thromboplastin and prothrombin times and reduced fibrinogen concentrations. These effects were similar in W/WV and +/+ mice, although the reduction in platelet counts was greater in W/WV mice. In addition, the mortality associated with ink injection was significantly higher in W/WV mice than in congenic +/+ mice. Most W/WV mice which died first exhibited paralysis, and examination under the dissection microscope revealed that ink injection resulted in significantly more cerebral thromboemboli in W/WV mice than in +/+ controls. Bone marrow transplantation from + /+ mice corrected both the mast cell deficiency and the anemia of W/WV mice and protected the W/WV recipients from the adverse consequences of ink injection. By contrast, +/+ mice rendered as anemic as W/WV mice by breeding did

exhibit increased morbidity and mortality after ink x C57BL/6)F1-Si/Sid mice, which are anemic and lack mast cells because of a genetic defect different from that of W/WV mice, also exhibited increased morbidity and mortality after intravenous ink. mixture of ink with commercial heparin prior Finally, to intravenous injection markedly reduced the incidence of cerebral thromboembolism and death in WIWV mice. Taken together, these findings suggest that the increased morbidity and mortality exhibited by W/WV and mice that received injected ink might be related toSi/Si their mast cell deficiency rather than to their anemia. But measurement of the histamine content of the blood and various tissues ofWBB6F -+/+ mice injected with ink, and examination of their tissues in 1-y sections, indicated that intravenous ink did not cause substantial mast cell degranulation. As a result, the possibility that mast cells protect + /+ mice from the adverse effects of intravenous ink by a mechanism other than degranulation and release of heparin, or that the differences in the response of WIWV or Si/Si mice and their + /+ littermates are due to defects other than their lack of mast cells, cannot be excluded. (Am J Pathol 1986, 122:469-480)

MAST CELLS are especially abundant in the vicinity of blood vessels. 1-3 Many populations of mast cells contain heparin,-112 a well-characterized source of anticoagulant activity.6-12 These facts have suggested that mast cells might help to prevent intravascular clotting.1 Perhaps the most direct way to evaluate this possibility

Supported by grants from the Ministry of Education, Science and Culture, the Ministry of Health and Welfare, the Mitsubishi Foundation, the Princess Takamatsu Cancer Research Fund, the Japanese Foundation for Multidisciplinary Treatment of Cancer, and the Takeda Science Foundation and by United States Public Health Service Grants Al 20292 and CA 28834. 469

not

injection. (WC

470

KITAMURA ET AL

would be to study intravascular coagulation in animals which differ solely in the presence or absence of mast cells. An experimental model system which approximates this ideal was reported by Kitamura et al. in 1978.13 Kitamura et al found that a double gene dose of mutant alleles at the Wlocus had a profound effect on the production of mast cells.13 The number of mast cells in the skin of adult WBB6F1-W/Wv mice is < 0.37o that in congenic WBB6F1- + /+ mice, and no mast cells whatsoever are detectable in other organs of adult W/WV mice, including stomach, mesentery, peritoneal cavity, and brain.13 In addition to lacking mast cells identifiable microscopically in tissue sections stained with toluidine blue, the tissues of W/Wv mice have low or undetectable levels of the mast-cell-associated mediators histamine and heparin. Yamatodani et al measured the concentration of histamine in various tissues of W/WV mice and reported that the value in the skin was approximately 2Vo that in congenic +/+ mice," a finding which has been confirmed by others. 15-17 Nakamura et al8 and Straus et al9 isolated glycosaminoglycan fractions from the skin of W/WV mice and congenic + /+ mice. The skin of W/WV and +/+ mice contained similar amounts of hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate; but W/WV mouse skin contained no detectable heparin. Furthermore, Straus et al'9 demonstrated that an extract of W/WV mouse skin exhibited no anticoagulant activity. In the present study, we searched for differences in the responses of W/WV and + /+ mice to intravenous injection of India ink, a maneuver known to result in thromboembolic complications in the rabbit,20 rat, and mouse.2 1We found that intravenous ink caused more cerebral thromboemboli and resulted in a higher proportion of deaths in W/WV mice than in the congenic + + littermates.

AJP * March 1986

termates (WCB6F,-Sl/Sld or - + /+ mice) were purchased from the Jackson Laboratory, Bar Harbor, Maine. Experiments with these mice were performed in Boston. The mice were 3-4 months of age at the start of the experiments.

Injections of India Ink, Endotoxin, and/or Heparin Commerical India ink for fountain pens (Pelikan AG, Hanover, Germany) was used. When carbon was precipitated by concentrated NaOH, and the proteins hydrolyzed by boiling,21 the ink contained 80 mg carbon/ml. The ink was diluted in 0.9% NaCl; 2.5, 5, 10, 20, and 50%o (vol/vol) suspensions were made; and the injection volume of the diluted ink was adjusted according to the body weight of recipients, ie, 0.04 ml/g body wt in most experiments. In one experiment, heparin sodium (Kodama Co. Ltd., Tokyo, Japan) was mixed with the diluted ink just before injection. Endotoxin (lipopolysaccharide B from Escherichia coli 055:B5, Difco Laboratories, Detroit, Mich) was suspended in 0.9%o saline. Intravenous injections were made into the lateral tail vein. Clearance of India Ink At various times after the injection of ink, 44.7 I1. of blood was obtained from the retroorbital sinus with the use of heparinized hematocrit tubes (Drummond Scientific Co., Broomall, Pa). After centrifugation, 20.6 ,ul of plasma was absorbed into a piece of filter paper. The amount of carbon in the resulting black spot was measured by a densitometer (TLC Scanner CS-900, Shimazu Inc., Kyoto, Japan).

Mice (WB x C57BL/6)F1-W/Wv, or -+/+ (WBB6F1W/WV, or - + /+) mice raised in the Shizuoka Laboratory Animal Center and sent to the Osaka University Medical School, where all the experiments using these mice were done. Mast-cell-deficient (WC x C57BL/6)F1-Sl/Sld mice, and their congenic (+ /+) lit-

Counting of Cerebral Thromboemboli WBB6F1-W/Wv or -+/+ mice were anesthetized with ether and killed by decapitation. Both frontal and both parietal bones were gently removed, and the heads were fixed in 10% formalin. Thromboemboli, which appeared black because of their content of carbon particles, were detectable both on the surface and in the deeper regions of the cerebral hemispheres. The superficial thromboemboli were more easily recognized and enumerated; we therefore counted only those thromboemboli which were identifiable on the surface of cerebral hemispheres upon examination under the dissection microscope (10x).

Accepted for publication October 11, 1985. Address reprint requests to Yukihiko Kitamura, MD, Institute for Cancer Research, Osaka University Medical School, Nakanoshima 4-3-57, Osaka, Kita-ku, 530 Japan.

Hematologic Tests of WBB6F1-W/Wv or -+/+ Mice For counting platelets, blood samples were obtained from the retroorbital sinus by using a heparinized

Materials and Methods

Vol. 122 * No. 3

hematocrit tube (Drummond). Platelets were counted by the method of Brecher and Cronkite.22 The bleeding time was measured according to the method described by Holland with a slight modification.23 The mouse was kept in a restraint cage devised by Yano and Harada24; the terminal 2-mm portion of the tail was severed with a sharp blade. The bleeding time was measured by a stopwatch to the nearest second with the use of a filter paper disk intermittently applied to the adherent drop of blood. For clotting tests, blood samples were obtained from the jugular vein with a plastic syringe. Activated partial thromboplastin time and prothrombin time were measured by standard methods with the Clotek System (Hyland Division, Travenol Laboratories, Inc., Costa Mesa, Calif). The concentration of fibrinogen was estimated by measuring the thrombin time after addition of a known amount of bovine thrombin (Becton, Dickinson & Co., Rutherford, NJ). The thrombin time was also measured with the Clotek System. Fibrinogen-fibrin degradation products (FDPs) in the sera were assayed semiquantitatively by the latex particle agglutination method (FDPL test, Teikoku Hormone, Tokyo, Japan). The values were converted to micrograms per milliliter with reference to a human fibrinogen standard. The reagents used for the clotting tests and for measurements of FDPs were not derived from mice, but the values obtained from normal control mice were similar to those of normal humans25 and rats.26 Bone Marrow Tfansplantation Bone marrow cells of WBB6F1- + /+ mice were suspended in Eagle's solution as previously described in detail27; 2 x 107 cells were injected intravenously into each WBB6F1-W/Wv mouse. The number of erythrocytes was examined 15 weeks after the injection. Only rescued animals (erythrocytes > 8.0 x 106/cu mm28) were used for further study. Rescued W/Wv and untreated W/WV mice of the same age were given injected ink 2 weeks after the last examination of the blood. The presence of mast cells in the skin and stomach of the rescued W/WV mice was confirmed histologically after the experiment. Histamine Content Samples of the skin, stomach, meninges, and blood of WBB6F1- + / + mice were removed at various times after intraveneous injection of ink solution and were kept at - 80 C. Histamine concentration was assayed by high-performance liquid chromatography coupled with fluorometry as reported by Yamatodani et al.14

BRAIN THROMBOEMBOLISM IN W/WV MICE

471

Histologic Studies In two experiments, W/Wv and +/+ mice were killed 4 hours after intravenous injection of 10%76 ink or 0.9%o NaCl. Specimens of skin and glandular stomach were removed, fixed in 2.0% paraformaldehyde, 2.5% glutaraldehyde, and 0.025% CaCl2 in 0.1 M sodium cacodylate buffer at pH 7.4, and processed for 1 pi Epon-embedded, Giemsa-stained sections as previously described.29 The slides were coded and then were examined by an observer (S.J.G.) who was not aware of which specimens were from which treatment group.

Statistical Methods Values such as platelet counts and prothrombin times were compared with the use of the Student t test. Proportions of deaths were compared by the chi-square test. When the number in a group was less than 3, Yates' correction for continuity was applied to the chi-square test.30

Results

High Susceptibility of W/Wv Mice to Morbidity and Mortality After Intraveneous Injection of Ink Different concentrations of ink (0.04 ml/g body wt) were injected into WBB6F,-W/Wv or -+/+ mice, using either the intravenous or intraperitoneal route. The proportion of deaths occurring within 24 hours of the injection was determined. The 24-hour mortality of the WIWv mice was significantly higher than that of the +/+ mice when 10%o or 20%o ink was injected intravenously (Table 1). However, no significant differences were detectable when ink solutions of either lower (ie, 2.5% and 5%76) or higher (ie, 50%) concentrations were injected intraveneously. Furthermore, no significant differences were observed when 10%o or 50%o ink was injected intraperitoneally (Table 1). When the observation period was extended until 72 hours, only a few additional W/Wv mice died after 24 hours (Table 2). In contrast to the striking difference in mortality seen in WBB6F,-W/WVor -+/+ mice injected with ink intravenously, the clearance of carbon particles from the circulation of W/WV or + /+ mice injected with 2.5%o or 10%o ink (0.04 ml/g body wt) was similar (Figure 1). Moreover, the results for the mice used in our experiment were in accord with those reported by Halpern et al.21 When W/WV mice were given intravenous injections of 10% or 20% ink, most of them exhibited paralysis within 2 hours of the injection (Figure 2A). In addition, many of the mice exhibited unilateral or bilateral

472

KITAMURA ET AL

AJP * March 1986

Table 1 -Mortality of WBB6F,-W/Wv or - + / + Mice Within 24 Hours of the Injection of Ink: Effects of Injection Route and Dose Proportion of deaths in mice of each genotype Amount of carbon Liquid injected Injection wt) body (mg/g W/WV +/+ wt) body (0.04 ml/g route 0/9 (0%) 0/10 (0%) 0 Saline Intravenous Intravenous Intravenous Intravenous Intravenous Intravenous

Intraperitoneal Intraperitoneal *

0/11 0/10 2/35 3/23 1/21 0/12 1/11

0/12 (0%) 2/10 (20%) 22/27 (81%)* 8/11 (73%)* 2/15 (13%) 0/9 (0%) 1/9 (11%)

0.08 0.16 0.32 0.64 1.6 0.32 1.6

ink ink ink ink ink 10% ink 50% ink

2.5% 5% 10% 20% 50%

(0%) (0%) (6%) (13%)

(5%) (0%) (9%)

P < 0.01, when compared with the value for the control + /+ mice.

exophthalmus (Figure 2B). Autopsies were performed as soon as possible after death. Cerebral thromboemboli were observed macroscopically (Figure 2C). Examination of the orbits revealed that exophthalmus was due to bleeding from the retroorbital sinus. Extensive intestinal bleeding was observed in most W/Wv mice that died 4-24 hours after the injection. Because of the striking appearance of the brains of WBB6F1-W/Wv mice after injection of ink, and because most WBB6F1-W/Wv mice that died as a result of ink injection first exhibited paralysis, we measured the number of cerebral thromboemboli observed after ink injection in WBB6F1-W/Wv and -+/+ mice. WBB6F1-W/Wv and - + /+ mice were given intravenous injections of 2.5%/ or 10% ink (0.04 ml/g body wt), and all the mice were killed 4 hours after the injection. The number of cerebral thromboemboli was counted under the dissection microscope at 10 x. The number of thromboemboli was significantly greater in the W/Wv mice than in the + /+ mice (Table 3). Hematologic Values in W/Wv and + /+ Mice Given India Ink Various hematologic values were determined 4 hours after intravenous injection of 10% ink (0.04 ml/g body Table 2-Duration Between Intravenous Injection of 10% Ink (0.04 mlI/g body wt; 0.32 mg carbon/g body wt) and Death in WBB6F,-W/Wv and - + + Mice Genotype +/+ W/WV No. of mice with

injections No. of mice that died at each interval after injection* 0-8 hours

8-24 hours 24-72 hours Total

41

19

16 13 2 31

2

wt) (Table 4). Ink injection resulted in a marked reduction in the platelet counts of both W/Wv and +/+ mice, and this change was significantly greater in the W/Wv mice than in the + /+ controls. Ink injection was also followed by prolongation of the bleeding time, prothrombin time, and activated thromboplastin time, and by reduction in the concentration of fibrinogen. But in these four parameters, no significant differences between W/WV and + /+ mice were detected. Although our assay did not detect FDPs in the sera of untreated W/Wv and + /+ mice, FDPs were detected in both W/WV and + /+ mice 4 h after ink injection. We also measured FDP values 24 hours after ink injection in the + /+ mice (most W/WV mice died during this period). The values exceeded 40 Hg/ml (data not shown). The hematologic values shown in Table 4 were obtained after injection of 10% ink. Because the mortality of W/Wv mice injected with 5007o ink was sig-

105-I

2-

* 10% Ink to +/+ o 10% Ink to W/WV O

I

E c

0.2-

u

L 0.10

* 2.5% Ink to +/+ o 2.5% Ink to W/Wv

nv. nr ,VD -

0.02- O 0 0 2

P < 0.01, when distributions of deaths between W/Wv and + / + mice were compared.

1

2

3

4

Time after Injection (h) Figure 1-Clearance of carbon from the plasma of WBB6F,-W/WV or mice after intravenous injection of either 10% or 2.5% ink (0.04 ml/g

-+/+

body wt). Each point represents mean of 4-6 mice.

BRAIN THROMBOEMBOLISM IN W/WV MICE

Vol. 122 * No. 3

473

Table 3-Number of Thromboemboli in Superficial Veins of Cerebrum 4 Hours After Intravenous Injection of Ink No. of thromboemboli in mice of each genoLiquid Amount of type (mean ± SE)* carbon injected W/WV (0.04 mug body wt) (mg/g body wt) +/+ 2.5% ink 0.08 21 ± 6 (13)t 5 ± 1 (19) 10% ink 0.32 54 ± 9 (20)t 14 ± 2 (22)

,71

*

Number of mice is shown in

t P




>

!

.,?@

0- sJ ,0 @ ffi;'§s d :-e''at

f fP Sl; f_ , .-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~41

_- f , 0''llaiS^7, 4 40~~~~~~~~~~~~~~~~~~~ ',I I_ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I .....

:

and~~~ D 01 B S i : S * CX -1'0 + Figure 4-Histologic findings in tissues of WBB6F,-+/+ mice 4 hours after intravenous injection of 10% ink (0.04 ml/g body wt). ASkin showing four intact mast cells (arrowheads) and carbon particles within a small blood vessel (arrow) near a hair follicle. (x 250) B and C-Higher power views of A, to illustrate mast cells and carbon-containing blood vessel. (x 630) D-Glandular stomach, showing an intact mast cell (solid arrow) within four intact mast cells (arrowheads)

~~~~~~~~~~~~~.......

the muscularis propria and two intact mast cells (solid arrowheads) in the submucosa. Carbon particles (open arrowhead) are present beneath an endothelial cell of an adjacent blood vessel. (x 630)

..

476

KITAMURA ET AL

AJP * March 1986

Table 7-Concentration of Histamine in Various Tissues of WBB6F1-+/+ Mice After Injection of Either 10% Ink or Saline (0.04 ml/g body wt) Liquid Time after Concentration of histamine (nmol/g wt, mean ± SE) Tissues injected injection 0.17 ± 0.01 (8)t Blood* Saline 4 hours 10% ink

Skin Stomach Meninges

Saline 10% ink Saline 10% ink Saline 10% ink

1 10 30 4 4 4 4 4 4 4

minute minutes minutes hours hours hours hours hours hours hours

0.15 0.17 0.12 0.16 197 288 77 80 297 308

± ± + ± + ± ± ± ± ±

0.01 (7) 0.01 (7) 0.01 (4) 0.01 (8) 23 (5) 21 (4) 6 (5) 6 (5) 43 (4) 67 (5)

One milliliter of whole blood.

t Number of mice is shown in parenthesis.

in the second and third experiments, the proportion of deaths within 24 hours was determined. The number of cerebral thromboemboli was fewer and the proportion of deaths was lower in the rescued W/Wv mice than in the nontreated W/Wv mice (Table 8). Indeed, the values for rescued W/Wv mice were statistically indistinguishable from those of untreated +/+ mice. Since bone marrow transplantation rescues not only the mast cell deficiency but also the hypoplastic anemia of WIWv mice,36 we wondered whether the beneficial effect of bone marrow transplantation observed in our experiments might be due to correction of the anemia of the WIWv recipients. We therefore rendered +/+ mice anemic by bleeding from the retroorbital sinus and then gave the anemic + /+ mice an intravenous injection of 100/0 ink (0.04 ml/g body wt). Anemic +/+ mice were no more susceptible to ink injection than were untreated control +/+ mice (Table 9).

High Susceptibility of SJ/Sid Mice to Morbidity and Mortality After Intravenous Injection of Ink WBB6F1-W/Wv mice are mast-cell-deficient because of mutations on Chromosome 5 which result in an abnormality of hematopoietic stem cells. 13,28.37 WCB6F1SJ/Sid mice are mast-cell-deficient because of mutations on Chromosome 10 which result in an abnormality of microenvironmental factors necessary for development of mast cells in the tissues.35 If intravenous ink caused adverse consequences in WBB6F1-W/Wv mice because of the mast cell deficiency of these mice, we reasoned that ink injection into WCB6F,-SJ/Sld mice should produce similar effects. But if WBB6F1WIWv mice were highly susceptible to the toxic effects of ink injection because of an effect of the Wlocus mutations other than mast cell deficiency, WCB6F1-SI/Sld mice might not exhibit adverse effects of intravenous ink. We therefore gave WCB6F1-SJ/Sld and littermate + /+ mice intravenous injections of 20/o ink (0.02 ml/g body wt, providing 0.32 mg carbon/g body wt) and determined the mortality produced within 3 days of the injection (Table 10). All 11 of the WCB6F1-SJ/Sld mice died within the first 24 hours of ink injection. Most of these mice exhibited evidence of neurologic deficit before death. By contrast, none of the congenic WCB6F1-+/+ mice died, and none of them exhibited evidence of neurologic impairment. Discussion It has been recognized for many years that the intravenous injection of India ink can result in platelet aggregation,38'39 activation of the clotting system,21 33.34 and death.20'21 33'34 These toxic effects are most readily produced by commercial preparations of ink containing shellac; injections of carbon particle suspensions lack-

Table 8-Effect of Bone Marrow Transplantation on Number of Cerebral Thromboemboli and Mortality of WBB6F,-W/Wv Mice Given 10% Ink (0.04 ml/g body wt) No. of emboli at 4 hours Proportions of deaths Experiment (mean + SE)* within 24 hours Mice no. 1 36 ± 5 (13) NEt Untreated W/lV Rescued W/WV Untreated +/+ Untreated WWV Rescued W/WV Untreated +/+ Untreated W/Wv Rescued W/Wv Untreated +/+

2

3

Number of mice is shown in parenthesis.

t NE, not examined.

t P < 0.02, when compared with the value of nontreated W/Wv mice.

15 ± 4 (18)t1 11 3 (12)1: NE NE NE NE NE NE

NE NE 21/25 (84%) 2/15 1/17 (6%)T 7/13 (54%) 2/22 (9%)t 1/11 (9%)t

(13%)t

BRAIN THROMBOEMBOLISM IN

Vol. 122 * No. 3

Table 9-Effect of Bleeding on Mortality of WBB6F1-+/+ Mice Given 10% Ink (0.04 ml/g body wt) Mean hematocrit Mice given Proportion of deaths within 24 hours ink value (%) 7/8 (88%) Untreated W/WV 40 Bled +/+ Nontreated + /+ *

40 48*

0/8 (0%)* 0/9 (0%)*

P < 0.01, when compared with the value of nontreated W/WV mice.

ing this ingredient cause little or no morbidity.33 Furthermore, different animal species vary in their susceptibility to the adverse effects of intravenous ink; mice being notably more resistant than either rats or rabbits.21'3334 In the present study, we found that the intravenous injection of 10% or 207o ink resulted in far greater mortality in mast-cell-deficient WBB6F1WI/Wv mice than in congenic controls (WBB6F,-+/+ mice). This observation raised at least two questions which required further investigation: 1) What effects of ink injection were responsible for the death of W/Wv mice? 2) Why were these effects seen more frequently in W/Wv mice than in their + /+ littermates? Necropsy revealed many more cerebral thromboemboli in W/Wv mice injected with ink than in +/+ controls (Table 3). Although the superficial thromboemboli were most readily quantitated, the lesions occurred in deeper regions of the cerebrum as well. These findings, and the frequent signs of paralysis exhibited by W/WV mice later dying of ink injection, suggest that the central nervous system lesions contributed importantly to the mortality produced by intravenous ink. Foot20 proposed a similar mechanism to account for the death of rabbits given intravenous injections of ink. Most of the mice succumbing within 24 hours of ink injection also exhibited extensive intestinal hemorrhage, which may have contributed to their death. Several experiments were performed in an attempt to understand why ink injection produced more morbidity and mortality in W/Wv than in + /+ mice. The clearance of carbon from the circulation of W/Wv and + /+ mice was similar (Figure 1), which suggests that this aspect of mononuclear phagocyte function does not significantly differ in these mice. Measurements of hematologic values after ink injection demonstrated that the bleeding time, prothrombin time (PT), and activated partial thromboplastin time (PTT) were markedly prolonged, the number of circulating platelets and the plasma concentration of fibringogen were markedly reduced, and fibrin degradation products appeared in the blood (Table 4). Although these changes occurred in both W/Wv and + /+ mice, injection of 10% ink resulted in a significantly greater reduction in the platelet count in the W/Wv mice (477 ± 18 to 85

W/WV MICE

477

Table 10-Mortality of WCB6F,_SI/S/d and -+/+ Mice Within 72 Hours After Injection of 20% Ink (0.02 ml/g body wt; ie, 0.32 mg carbon/g body wt) Genotype No. of mice given ink No. of mice that died at each interval after injection 0-8 hours 8-24 hours 24-72 hours Total

SI/SId

+I+

11

9

5 6 0 11

0 0 0 0

P < 0.01, when distributions of deaths were compared between SI/SId and + / + mice.

± 9 X 103 platelets/ml before or 4 hours after ink) than in the + /+ controls (454 ± 16 to 152 ± 14 x 103/,l). The more marked thrombocytopenia observed in W/Wv mice might reflect increased consumption of platelets in association with the thromboembolic complications of ink injection in these animals. Alternatively, it might reflect differences between W/Wv and + /+ mice in other factors affecting platelet numbers. Whatever the explanation for its occurrence, the response of platelet counts to ink injection in W/Wv or + /+ mice is of interest. The mortality seen after iv. injection of 50% ink was less in both W/WV mice (13 % mortality, Table 1) and + /+ mice (5 % mortality) than the mortality observed after 10% or 20% ink (81 or 73% in W/WV mice, 6% or 13 % in +/+ mice). When the hematologic changes associated with intravenous administration of 0%, 2.507, 100o, or 50% ink were examined 4 hours after injection (Figure 3), the lowest platelet counts were observed after 10% ink, and the platelet counts recorded in W/Wv mice given 10% ink were lower than the corresponding values in the +/+ mice. By contrast, no differences in the PT of W/Wv and +/+ mice were observed at any concentration of ink, and the greater changes in PT occurred after injection of 50%o ink. That injection of 50% ink was better tolerated by the mice than injection of 10% ink is curious, but not unprecedented. In the experiment model of DIC reported by Yoshikawa et al,40 intravenous infusion of 200 mg endotoxin/kg in the rat produced effects on the PT, the fibrinogen concentration, and the platelet count which were similar to those seen after 100 mg endotoxin/kg. But 200 mg endotoxin/kg had no significant effect on the PTT, whereas the PTT in rats given 100 mg endotoxin/kg was more than double the control value. The higher dose of endotoxin also resulted in roughly half the number of glomerular fibrin thrombi that resulted with the lower dose. The reasons for the unusual dose-response effects seen with ink (or with

478

KITAMURA ET AL

endotoxin4O) may be complex, and we did not investigate them further. We did test the effect of injecting three different doses of endotoxin into W/Wv and +/+ mice, however, and found little or no difference in the response of the mast-cell-deficient animals and the congenic controls (Table 5). W/Wv mice virtually lack mast cells and are anemic."3 Bone marrow transplantation from congenic + /+ littermates repairs both problems'3'28; so we tested the susceptibility of bone-marrow-rescued W/Wv mice to ink injection. Rescued W/Wv mice given 10% ink exhibited no more cerebral thromboemboli or deaths than did similarly treated congenic + /+ mice (Table 8). Furthermore, + /+ mice rendered as anemic as their W/Wv littermates by bleeding were no more susceptible to ink-related mortality than were control + /+ mice (Table 9). Taken together, these findings suggest that the protective effect of bone marrow rescue in W/WV mice was not due to correction of the anemia. But rescued W/Wv mice theoretically may exhibit changes in the number and/or function of other bone-marrow-derived cellular elements beside mast cells, and these elements or their products might influence the response of the rescued mice to ink. We therefore examined the effect of ink injection in WCB6F,-SJ/Sld mast-cell-deficient mice and their normal +/+ littermates.35 Although the phenotypes of SJ/Sid and W/WV mice are similar, the responsible mutations are on different chromosomes and result in distinct defects (W/Wv mice have a defect in stem cells which give rise to mast cells, whereas SI/S1d mice have a defect which prevents normal mast cell development in the tissues37). SI/ISd mice exhibited much greater mortality after injection of ink than did their + /+ littermates (Table 10). The occurrence of increased mortality after intravenous ink in two different mutants whose mast cell deficiency is due to two distinct mechanisms and the correction of this problem in W/Wv mice whose mast cell populations have been restored by bone marrow transplantation suggest that mast cells might protect the host against the adverse consequences of ink injection. On the other hand, we were not able to confirm this possibility by determining how mast cells might function to inhibit the effects of ink injection. Heparin is used in the clinical treatment of DIC,31 and heparin protected W/Wv mice from the lethal effects of ink injection (Table 6). Yet injection of ink did not result in detectable mast cell degranulation in + /+ mice: the mice did not exhibit clinical evidence of an anaphylaxislike response, examination of their tissues in 1-,I Eponembedded sections did not reveal evidence of significant mast cell degranulation, and there was little or no change in the histamine content of their tissues or blood.

AJP * March 1986

Although these findings indicate that intravenous ink did not produce marked mast cell degranulation, they do not necessarily exclude a role for mast cells in protecting +/+ mice from the effects of ink injection. First, rough calculations of the amount of heparin present in mouse mast cells suggests that a relatively small amount of mast cell activation might provide sufficient heparin to counteract the effects of ink. W/Wv mice were protected from the lethal effects of 10% ink when the ink was mixed with 0.08 IU heparin/g body wt prior to injection. Straus et al'9 found that the skin represented a major source of heparin in the mouse, and that this tissue contained approximately 20 IU of heparin/g dry wt. Because the mean skin dry weight of 5 male WBB6FI- + /+ mice was 2.68 g and the mean body weight was 36.1 g, release of 5% of the heparin of all the cutaneous mast cells would provide rv2.7 IU of heparin, or "-0.08 IU/g body wt. This level of mast cell activation might be very difficult to detect histologically or by measurement of changes in histamine levels. Furthermore, mast-cell-associated heparin is present in mouse tissues other than the skin,'8"l9 which suggests that even more modest levels of mast cell activation (less than 5% if one considers the body as a whole) might provide sufficient heparin for a protective effect. But calculations such as these may be misleading. First, the consequences of mixing commercial heparin glycosaminoglycans together with ink prior to intravenous injection may differ in many ways from the effects produced by release of endogenous heparin from mast cells. "I In vitro studies have demonstrated that mast cell heparin proteoglycan is poorly soluble under physiologic conditions of pH and ionic strength.2"-24' In addition, the commercial heparin we employed had immediate access to both the ink and the intravascular compartment, whereas mast-cell-derived heparin would not. These considerations suggest that much more mast cell activation may be required to mimic the effects of intravenous injection of commercial heparin than is indicated by the rough calculations given above. Second, recent work by Marcum and his colleagues indicates that mast cells do not represent the only tissue source of glycosaminoglycans with heparinlike anticoagulant activity.42"5 Such molecules are found in association with vascular endothelial cells derived from calf retinal microvasculature, preparations devoid of mast cells according to microscopic examination or histamine assay.42 Furthermore, vascular endothelial cells of WIWv and congenic + /+ mice express similar levels of heparin-like anticoagulant activity, whether assayed in perfused hind limb preparations or in vascular endothelial cells grown in vitro.45 In short, investigating the possible role of endogenous mast cell heparin, or heparin-like glycosaminoglycans not derived from mast cells, in protecting + / +

Vol. 122 * No. 3

BRAIN THROMBOEMBOLISM IN W/WV MICE

mice from the adverse consequences of ink injection will not be simple. And another point should be made. Our experiments clearly demonstrated that commercial heparin given intravenously protected W/Wv mice from ink-induced morbidity. But this result does not prove that endogenous heparin is responsible for the relative resistance of + /+ mice to the thromboembolic complications of ink injection. Until these issues are clarified, we cannot exclude the possibility that our experiments have revealed a mast-cell-dependent effect other than those related to heparin. An alternative possibility is that our results are due to consequences of the mutations in W/Wv and SJ/Sid mice other than mast cell deficiency. To our knowledge, however, no defect which might have such an effect has yet been identified. W/Wv mice have decreased numbers of megakaryocytes in the bone marrow,46 but the size and number of circulating platelets47 and the platelet production rate in response to experimentally induced thrombocytopenia48are equivalent to those of the congenic + / + littermates. Similarly, SJ/Sid mice have fewer bone marrow megakaryocytes than the congenic +/+ mice, but normal blood platelet number, mass, and turnover.49 Furthermore, the WIWv and congenic +/+ mice used in our experiments did not differ in baseline values for platelet count, bleeding time, fibrinogen concentration, PT, or PTT. Nevertheless, it is conceivable that these tests were not able to detect differences in platelets or clotting factors which contribute to the increased susceptibility of W/WV mice to the complications of ink injection. In conclusion, we have shown that mast-cell-deficient mice exhibit increased susceptibility to the adverse effects of intravenous ink. The explanation for this observation is not yet clear. Similar findings were obtained in W/Wv and SJ/Sid mice, which have different mutations that produce mast cell deficiency by very distinct mechanisms. This result suggests that mast cells may protect + / + mice against the adverse consequences of ink injection. If mast cells have such a role, it is unclear whether they function by releasing small amounts of heparin or other mediators, or by other mechanisms yet to be defined. Alternately, both W/Wv and SJ/Sid mice might be more susceptible to ink-induced mortality because of independent genetically determined defects other than mast cell deficiency.

4. Jorpes E, Hormgren H, Wilander 0: Ueber das Vorkommen von Heparin in Gefasswanden und in den Augen. Mikrosk Anat Forsch 1937, 42:279-301 5. Wilander 0: Studien uber Heparin. Skand Arch Physiol 1938, 81 (Suppl 15) 6. Jaques LB, Waters ET: The identity and origin of the anticoagulant of anaphylactic shock in the dog. J Physiol (Lond) 1941, 99:454-466 7. Yin ET, Wessler S, Stoll PJ: Identity of plasma-activated factor X inhibitor with antithrombin III and heparin cofactor. J Biol Chem 1971, 246:3712-3719 8. Rosenberg DR, Damus PS: The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem 1973, 248:6490-6505 9. Abildgaard U: Highly purified antithrombin III with heparin cofactor activity prepared by disc electrophoresis. Scand J Clin Lab Invest 1968, 21:89-91 10. Miller-Andersson M, Borg H, Andersson LO: Purification of antithrombin III by affinity chromatography. Thromb Res 1974, 5:439-452 11. Jaques LB, Mahadoo J, Riley JF: The mast cell/heparin paradox. Lancet 1977, 1:411-413 12. Schwartz LB, Austen KF: Structure and function of the chemical mediators of mast cells. Prog Allergy 1984, 34:271-321 13. Kitamura Y, Go S, Hatanaka K: Decrease of mast cells in W/WVmice and their increase by bone marrow transplantation. Blood 1978, 52:447-452 14. Yamatodani A, Maeyama K, Watanabe T, Wada H, Kitamura Y: Tissue distribution of histamine in a mutant mouse deficient in mast cells. Biochem Pharmacol 1982, 31:305-309 15. Grzanna R, Schultz LD: The contribution of mast cells to the histamine content of the central nervous system: A Regional analysis. Life Sci 1982, 30:1959-1964 16. Hough LB, Khandelwal JK, Goldschmidt RC, Diomande M, Glick SD: Normal levels of histamine and telemethylhistamine in mast cell-deficient mouse brain. Brain Res 1984, 292:133-138 17. Orr EL, Pace KR: The significance of mast cells as a source of histamine in the mouse brain. J Neurochem 1984 42:727-732 18. Nakamura N, Kojima J, Okamoto S, Kitamura Y: Absence of heparin in glycosaminoglycan fractions isolated from the skin of genetically mast cell-depleted W/WV mice. Biochem Int 1981, 3:449-456 19. Straus AH, Nadar HB, Dietrich CP: Absence of heparinlike compounds in mast-cell-free tissues and animals. Biochim Biophys Acta 1982, 717:478-485 20. Foot NC: Studies of endothelial reactions: VII. Changes in the distribution of colloidal carbon noted in the lungs of rabbits following splenectomy. J Exp Med 1923, 37:139-152 21. Halpern B-N, Biozzi G, Mene G, Benacerraf B: Etude quantitative de l'activite granulopexique du systeme reticulo-endothelial par l'injection intraveineuse d'encre de Chine chez les diverses especes animales: I. Methode d'etude quantitative de l'activite granulopexique du systeme reticulo-endothelial par l'injection intraveineuse de particules de carbone de dimension connues. Ann Inst Pasteur 1951, 80:582-604 22. Brecher G, Cronkite EP: Morphology and enumeration of human blood platelets. J Appl Physiol 1950,3:365-377 23. Holland JM: Serotonin deficiency and prolonged bleeding in beige mice. Proc Soc Exp Med 1976, 151:32-39 24. Yano S, Harada M: A method for the production of stress erosion in the mouse and related pharmacological studies. Jpn J Pharmacol 1973, 32:57-64 25. Bauer JD, Ackerman PG, Gelson T: Clinical Laboratory Methods. 8th edition. St. Louis, C. V. Mosby, 1974 26. Yoshikawa T, Murakami M, Furukawa Y, Kato H, Takemura S, Kondo M: Lipid peroxidation and ex-

References 1. Selye H: The Mast cells. Washington, DC, Butterworths, 1965 2. Metcalfe DD, Kaliner M, Donlon MA: The mast cell. CRC Crit Rev Immunol 1981, 3:23-74 3. Galli SJ, Dvorak AM, Dvorak HF: Basophils and mast cells: Morphologic insights into their biology, secretory patterns, and function. Prog Allergy 1984, 34:1-141

479

480

AJP * March 1986

KITAMURA ET AL

perimental disseminated intravascular coagulation in rats induced by endotoxin. Thromb Haemost 1983, 49:214-216 27. Kitamura Y, Kawata T, Suda 0, Ezumi K: Changed differentiation pattern of parental colony-forming cells in F1 hybrid mice suffering from graft-versus-host disease. Transplantation 1970, 10:455-462 28. Kitamura Y, Shimada M, Go S, Matsuda H, Hatanaka K, Seki M: Distribution of mast-cell precursors in hematopoietic and lymphopoietic tissues of mice. J Exp Med 1979, 150:482-490 29. Dvorak HF, Dvorak AM, Simpson AM, Dickerson HM, Leskowitz S, Karnovsky MJ: Cutaneous basophil hypersensitivity: II. A light and electron microscope description. J Exp Med 1970, 132:558-582 30. Fisher RA: Statistical Method for Research Workers. Edinburgh, Oliver and Boyd, 1963 31. Marder VJ: Consumptive thrombohemorrhagic disorders, Hematology. Edited by WJ Williams, E Beutler, AJ Erslev, MA Lichtman. New York, McGraw-Hill Book Co., 1983, pp 1433-1461 32. Hopps HC, Dent TE: India ink and reticuloendothelial blockade. Arch Pathol 1962, 74:285-291 33. Halpern B-N, Benacerraf B, Biozzi G: Quantitative study of the granulopectic activity of the reticulo-endothelial system: I. The effect of the ingredients present in India ink and of substances affecting blood clotting in vivo on the fate of carbon particles administered intravenously in rats, mice and rabbits. Br J. Exp Pathol 1953, 54:426-440 34. Biozzi G, Benacerraf B, Mene G, Halpern B-N: Etude quantitative de l'activit6 granulopexique du systeme reticulo-endothelial par l'injection intraveineuse d'encre de Chine chez les diverses especes animales: II. Relations entre les modifications de la coagulation du sang in vitro sous l'effet de l'injection intraveineuse de doses croissantes d'encre de Chine et sa repartition dans l'organisme. Ann Inst Pasteur 1951, 81:164-172 35. Kitamura Y, Go S: Decreased production of mast cells in SI/Sid anemic mice. Blood 1979, 53:492-497 36. Russell ES: Hereditary anemias of the mouse: A review for geneticists. Adv Genet 1979, 20:357-459 37. Kitamura Y, Sonoda T, Yokoyama M: Differentiation of tissue mast cells, Haematopoietic Stem Cells. Edited by SA Killmann, EP Cronkite, CN Muller-Berat. Copenhagen, Munksgaard, 1983, pp 350-361 38. Wright HD: Experimental pneumococcal septicimia and anti-pneumococcal immunity. J Pathol Bacteriol 1927, 30:185-252

39. Dudgeon LS, Goadby HK: The examination of the tissues and some observations on the blood platelets of rabbits at intervals of five minutes, and later, after intravenous inoculations of Staphylococcus aureus and Indian ink. J Hyg (Lond) 1931, 31:247-256 40. Yoshikawa T, Furukawa Y, Murakami M, Takemura S, Kondo M: Experimental model of disseminated intravascular coagulation induced by sustained infusion of endotoxin. Res Exp Med (Berl) 1981, 179:223-228 41. Yurt RW, Leid RW, Jr., Spragg J, Austen KF: Immunologic release of heparin from purified rat peritoneal mast cells. J Immunol 1977, 118:1201-1207 42. Marcum JA, Fritze L, Galli SJ, Karp G, Rosenberg RD: Microvascular heparin-like species with anticoagulant activity. Am J Physiol 1983, 245:H725-H733 43. Marcum JA, McKenney JB, Rosenberg RD: Acceleration of thrombin-antithrombin complex formation in rat hindquarters via heparinlike molecules bound to the endothelium. J Clin Invest 1984, 74:341-350 44. Marcum JA, Rosenberg RD: Heparinlike molecules with anticoagulant activity are synthesized by cultural endothelial cells. Bichem Biophys Res Commun 1985, 126:365-372 45. Marcum JA, McKenney JB, Rosenberg RD: Anticoagulantly active heparinlike molecules from cultured endothelial cells and microvasculature of mast cell deficient mice (Abstr). Fed Proc 1985, 44:1655 46. Chervenick PA, Boggs DR: Decreased neutrophils and megakaryocytes in anemic mice of genotype W/WV. J Cell Physiol 1969, 73:25-30 47. Ebbe S, Phalen E: Regulation of megakaryocytes in W/WV mice. J Cell Physiol 1978, 96:73-79 48. Threatte GA, Ebbe S, Phalen E: Measurement of thrombocytopoiesis in W/WV mice. Proc Soc Exp Biol Med 1981, 167:576-580 49. Ebbe S, Phalen E, D'Amore P, Howard D: Megakaryocytic responses to thrombocytopenia and thrombocytosis in S1/Sld mice. Exp Hematol 1978, 6:201-212

Acknowledgments We thank Drs. Nobuo Aoki, Kazuhiro J. Mori, Justine M. Carr, Ann M. Dvorak, Harold F. Dvorak, James A. Marcum, Jan McDonagh, Stephen Robinson, and Robert D. Rosenberg for valuable discussions and Seiko Kawawake, Gary Weitzman, and Linnea Wiberg for excellent technical assistance.