Kagoshima University Research Center for the Pacific Islands, Occasional Papers No.34, 177-183, 2001
Part 2, Section 2, Report 6. The Progress Report of the 1999 Survey of the Research Project“Social Homeostasis of Small Islands in an Island-zone”

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DYSBARIC OSTEONECROSIS (DON) IN THE FEMUR OF AN
EXPERIMENTAL DOG: PATHOPHYSIOLOGICAL ASPECTS
Motoo KITANO, Yasushi TAYA, Motohiko MOHRI, Mahito KAWASHIMAAbstract
A 32 months old male beagle dog developed a sign of limb bends in the left posterior limb aftera single 24-hour hyperbaric exposure to 3.2 atm abs. with acute decompression. It showed transient
MRI lesions in the left femur. Eight weeks after the hyperbaric exposure, histopathological examination disclosed an apparent lesion of dysbaric osteonecrosis (DON), which consists mainly of acicatrized fibrous tissue including many small necrotic foci in the bone marrow of the left femoral
lower shaft. This study supports the potential use of the dog as a model for human DON. The present
case and a review of the literature suggest that as for the pathophysiological aspect of DON, changesof the intraosseous nutrient artery and its peripheral microvasculature under stasis of bubbling bloodwith activation of both Hageman factor and blood platelets are very important.
Key words: Beagle dog, Femur, Hyperbaric exposure with acute decompression, MRI, Dysbaric
osteonecrosis (DON), Decompression sickness (DCS), Limb bends, Hemostasis, Hageman factor,Thrombosis, Plasmin, Kallikrein, Complements, Kinins, Vascular permeability, InflammatorycascadeIntroduction
Dysbaric osteonecrosis (DON), a form of aseptic bone necrosis associated with decompression sickness (DCS), is still a significant occupational hazard among divers and caisson workers
in Japan as well as in other countries. Various mechanisms, such as bubble embolism, fat embolism, endothelial damage, angiospasm, intravascular coagulation of blood, disturbance of venous
flow, increased intraosseous pressure, release of inflammatory mediators including somecytokines, etc. have been proposed regarding the pathognesis of DON.
For elucidation of the precise pathogenesis of this bone disease, experimental analysis using
some animal models is indisdpensable. Since 1990 Lanphier and Lehner have established an ex1-5
perimental model using sheep. Sheep is a particularly suitable animal to human DON model,
and we can observe apparent necrotic changes of the long bones, such as femurs and tibiae.6-8 In
1997 we found a definitive vegetation of thrombi in the branches of the intra-osseous nutrient artery of a sheep tibia which was affected by extensive necrotic changes in bone marrow tissue andcortical bone tissue.7,8 Our conclusion was that certain kinds of intra-osseous vascular diseases
are very important as a cause of DON.
In Japan, unfortunately, there are no facilities for sheep experiment. Thus, the dual purposesof this study are to clarify whether a dog can be evaluated as a possible model for human DON,
and to find evidence of mechanisms relating to the development of DON.

Material and Method
This study was based on one of the eight male beagle dogs which we have described

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Fig.1. Sign of limb bends observed in the left hind limb.

Fig.2. Abnormal findings in MRI in the lower
shaft of the left femur, 4 weeks after thehyperbaric exposure.

Fig.3. Schematic drawing of distribution of theleft femur lesions.

previously (TAYA, et al., 1997, 1998).9,10 The experimental procedure used in this study also has
9,10
been described previously. Briefly speaking, we put the dog, 32 months old, 12.2 kg bodyweight, into a hyperbaric chamber at JAMSTEC, with the atmospheric pressure raised to 3.2 atm
abs (ata) and kept the dog there for 24 hours, at the end of which the chamber was decompressed
quickly to ambient pressure. The rates of compression and decompression were approximately

Dysbaric Osteonecrosis (DON) in the Femur of an Experimental Dog: Pathophysiological Aspects

179

10m/min. At maximum pressure, the PCO2 level in the chamber air was maintained at <5 torr by
CO2 absorption (SODASORB), and air temperatures were kept at approximately 24 ℃.
The dog developed marked limb bends 20 minutes after the 24-hour hyperbaric exposure in
the left posterior limb, which persisted for two days (Fig.1).
Non-invasive diagnostic techniques using magnetic resonance imaging (MRI: Toshiba
Medical Systems Co. FREX-ART) were used to detect the early developmental stages of DON.
MRIs of long bones were taken one week before the hyperbaric exposure, and 1, 2, 4 and 8weeks after hyperbaric exposure. MRIs revealed a dyaphyseal lesion in the left femur after 2, 4and 8 weeks (Fig.2).
We euthanized the dog with an intravenous injection of pentobarbital sodium eight weeks
after the hyperbaric exposure. His femurs were cut longitudinally along the sagittal plane andfixed with 10% buffered formalin for subsequent histopathological survey.

Histopathological Analysis on the Femurs
Histopathological analysis of the left femur disclosed a characteristic lesion of bone necrosis in the shaft, while the right femur did not reveal any obvious pathological changes. The bonelesion was relatively well defined in the lower half portion of the shaft marrow, forming

Fig.4. Irregular-shaped cicatrized regions in the lower shaft of the left fermur. The cortical bone shows
irregular widening of the canal system (cutting cones).

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Fig.5. A necrotic focus with foreign body giant
cells in the cicatrized layer.

Fig.6. Descending branchs of the intra-osseous
nutrient artery without any evidence ofthrombosis.

cicatrized areas where many small foci of necrotic debris were buried (Figs.3 & 4). Some of thefoci were composed mainly of multinucleated giant histiocytic cells with a foamy cytoplasm surrounding small amounts of necrotic debris (Fig.5). Small spaces composed of fat necrosis weresurrounded by flat multinucleated giant cells. Formation of reactive new bone trabeculae was observed in the cicatrized areas.
The cortical bone near the cicatrized areas revealed features of cutting cones as a result ofactive resorption and formation of bone at the inner surfaces of the Haversian and Volksmann’s
canal systems, perhaps due to cortical bone necrosis.
The upper and lower articular cartilages and juxta-articular bone tissue were almost normal.
There were neither apparent vascular alterations nor thrombotic changes in the lumina of
the intra-osseous nutrient artery and its branches (Fig.6).

Discussion
This study supports the potential use of the dog as a model for human DON by a singlehyperbaric exposure to 3.2 atm abs which produced a persistent sign of limb bends of the left
hind limb and induced abnormal MRI changes in the left femur shaft. At the time of autopsy, we
detected an apparent change of bone marrow necrosis in the lower shaft of the left femur. We
already pointed out that the lower shaft of the femur was a predisposing site of DON in sheep
as well as humans.6,11
We have mentioned that intravascular coagulation of blood with thrombus formation is theDysbaric Osteonecrosis (DON) in the Femur of an Experimental Dog: Pathophysiological Aspects

181

most important factor for the etiology of DON and DCS-associated tissue injuries of various organs. 7,8,11-13 In the present case, therefore, we examined the intra-osseous nutrient artery and its
branches very carefully, but failed to find any thrombotic changes in the left femur.
Whether extensive necrotic changes occur even without an intra-osseous arterial thrombosis
is a very important point to discuss. During decompression nitrogen gas bubbles may create in
intra-, and extravascular spaces. The formation of extravascular gas bubbles should directly result in the destruction and alteration of cells and intercellular tissue matrices, to some extent. Fatembolism, which has been widely observed in the cadavers of DCS victims14, 15 seems caused by
fat cell disruption by direct mechanical stress of creation of nitrogen gas bubble in theircytoplasms. Fat droplets, other tissue disintegration products and extravascular gas bubbles may
enter the blood circulation system. They all may cause blood stagnation (hemostasis) with acti16
17vation of Hageman factor and aggregation of activated blood platelets.
Hageman factor is activated by exposure to negatively charged surfaces, such as basement
membranes, proteolytic enzymes, bacterial lipopolysaccharide and foreign materials such as nitrogen gas bubbles. The activated form of Hageman factor, namely XIIa, is a protease that initiates subsequent interactions among the other factors involved in the intrinsic pathway. These
include prekallikrein, factor IX, and factor VIII. The activation of Hageman factor, on the otherhand, results in the activation of several additional plasma proteases, which lead to the following:1) conversion of plasminogen to plasmin, 2) conversion of prekallikrein to kallikrein, and 3) activation of the alternative complement pathway.
Plasmin generated by activated Hageman factor induces fibrinolysis. In turn, the productsof fibrin degradation (FDPs) augment vascular permeability in both veins and arteries including
blood capillaries. Plasmin also cleaves to components of the complement system, thus generating
biologically active products, including the anaphylatoxins C3a and C5a. C3a and C5a increasevascular permeability both directly and indirectly. In addition, anaphylatoxins recruit and activate polymorphonuclear leukocytes (PMN) through their chemotactic action. Activated PMN
amplifies tissue damage by releasing cytotoxic substances such as granule-bound enzymes which
may degrade the extracellular matrix, active oxygen metabolites and arachindonic acid products(prostaglandins, thromboxane A2, leukotrienes). They all cause endothelial damage resulting in
18
increase of microvascular permeability. WARD et al. (1990) demonstrated in rabbits that depletion of complement proteins reduced the probability of complement activation and the incidenceof DCS. S TEVENS et al. (1993) 19 reported that an increased plasma level of C3a and C5a corre20
lated with the occurrence of DCS after a saturation dive. H UANG and LIN (1997) investigated
the involvement of the complement system and PMN activation in the air bubble-induced injuryof the rat lung.
21Plasma kallikrein, generated by activated Hageman factor, cleaves to high-molecularweight kininogen, thereby producing several vasoactive peptides of low molecular weight, collectively referrred to as kinins. Bradykinin is the best characterized of these vasoactive kinins.
When injected into skin, bradykinin elicits reversible changes of the endothelium that lead toedema. Many kinins are under the tight regularory control of specific inactivating enzymes. Forinstance, plasma carboxypeptidase N (kininase I) selectively cleaves to the carboxyterminal peptide of bradykinin. A dipeptidase known as kininase II cleaves to the dipeptides of bradykinin.
Kininase II is also termed angiotensin-converting enzyme (ACE), because it converst angiotensin
I to angiotensin II. The action of both kininases renders bradykinin biologically inactive.
On the other hand, the circulating cellular element most intimately involved with confusion

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17of both the blood itself and the vascular endothelium is the blood platelets. Activation of platelets, platelet adherence, aggregation, and degranulation, occur when platelets come in contact
with such foreign surfaces as injured blood vessel walls, gas bubbles, etc. The platelets are important sources of inflammatory mediators, including potent vasoactive substances and growth
factors that modulate mesenchymal cell proliferation. Degranulation is associated with the release of serotonin (5-hydroxytryptamine), which directly induces changes in vascular permeability. In addition, the arachidonic acid metabolite thromboxane A 2 (TxA 2) is produced by platelets.
TxA2 not only plays a key role in the second wave of platelet aggregation but also mediatessmooth muscle constriction. On activation, platelets, as well as phagocytic cells, secrete cationicproteins that neutralize the negative charges on endothelium and promote increased permeability.
Thus, it is appropriate to conclude that activation of Hageman factor and blood platelets
may generate a range of biologically active products resulting in widespread vascular and/orvasculogenic lesions, such as vasodilatation, increase of permeability with severe edema, andeven inflammatory cascades. Although this is only speculation, the femoral changes of the present dog case may have been caused through the activation of both Hagen factor and blood platelets without a detectable thrombosis in the vascular channels at the time of autopsy. For
confirmation of the mentioned hypothesis, further pathophysiological studies on the developmentand progression of DON are necessary.

Acknowledgment
We are grateful to Prof. Peter King for his critical reading of this manuscript.

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