Editorial of volume 8, n°2, December 1996 "Basic needs for a relevant orthopaedic practice in less-developed countries" by S. Hughes, led to comments from F. Burny (Brussels), which grew to a full text that we insert here.
F. Burny
Department of Orthopedics and Traumatology - Cliniques Universitaires de Bruxelles - Hôpital Erasme - Brussels - Belgium.
Major changes in African health productivity creates increasing concern about the viability of the local medical system and education program. The last years witnessed the general medical decline of health care, mostly due to political instability and malpractice. To afford health care represents a major problem in most African countries, where care availability is in a worse situation than 40 years ago: should we still speak of orthopaedic surgery in countries as Zaire? What does this editorial mean to refugees around Kisangani? In January 1997, there was only, to our best knowledge, five fully trained ortho-paedic surgeons in Zaire; in some African countries, not the less developed, there is no one orthopedic surgeon !
A major problem is to recruit and retain qualified health personal in unattractive areas; specialists do prefer to work in towns, even with minimal infrastructure. Some populations are unique in the extend of physical barriers they encounter to obtain health care. In areas with high poverty and (or) very low population density, a single facility may be the only provider of communities vital services, including basic emergency, primary, acute, and long term care. It is important to assess how travel distances and transportation limitations affect access to hospital care: long distances for the patients and no means of transportation has enormous impact, and explain the long delay before the patients reach the first aids: some open fractures reach the hospital after more than 1 month. The same situation exists in Kinshasa where 90% of the trauma patients reach only an hospital after 24 hours of delay; most of the several injured die before, the few who reach it do not all survive, and a majority of the survivors will not be able to return to their previous activities.
The medical help to developing countries is selective and mostly oriented towards parasi-tology, infectious or nutritional diseases. Orthopedics and traumatology represent less morbidity and mortality, growing up however, from year to year, with an increasing economic impact, as the majority of the victims are young and active men. Road accidents are an endemic problem, most of the time involving pedestrians and cyclists.
It is possible to perform orthopedic surgery in Africa, when adapted techniques are used with, however, special skill, as X-ray facilities are most of the time non-existent, or unusable. Precarious hygiene, a source of contamination, constitutes a major risk of sepsis in orthopedic surgery (over 50% of septic complication after internal fixation of open fractures). Another major cause of infection is sickle cell anemia, in Sub-Saharian Africa.
The treatment must be adapted to the situation, and surgery performed for major pathologies only (open or infected fractures, impairment of function). In many surgical fields, basic instrumentation is required; orthopaedic surgery often relies upon sophisticated ancillary and implants. Any missing element may result in catastrophic condition, even worse than in some traditional practices. Most fractures should be treated conservatively, complications only should indicate surgery. Internal osteosynthesis is the open door to infection when all the adequate conditions of operation are not entirely fulfilled. External fixation of fracture, when surgery is required, should be preferred to internal osteosynthesis.
The population has no, or very restricted, access to governmental health care which, when existing, is often under-equipped, and over-crowded. The orthopaedic surgeon faces cultural and economic constraints and must adapt treatments where, despite modern concepts of medicine and surgery are introduced, traditional medicine remains very strong. Due to the lack of medical doctors and of other health practitioners, people turns to traditional medicine (the two thirds of the population still remain under the influence of traditional medicine). Should the hospital fail, for any reason, the patient will return to the witch doctor who offers the advantage of availability, and is close to his cultural environment.
The level of medical care in the most demanding countries relies on medical training and skill. Patients and providers are often physically and professionally isolated. Economic and political barriers prevent specialists from receiving adequate education; some happy few are trained in Europe. The enormous diversity in health problems suggests that it is appropriate to maintain a strong support to the demanding countries. Orthopaedics and traumatology represent a major aspect of medicine. The training of orthopaedic surgeons, as of most specialists, must remain, up to now, supervised by western specialists bearing in mind the local medical facilities and pathology. It is important to assess the availability of educational programs and the remedial strategies we have to adopt, to influence the educational politics. Private initiative will help, but must be transferred at a wider level; African people should not only rely on foreign support, but also manage their own resources and adapt it to specific problems.
No single strategy is appropriate for health care delivery, but to let people untrained is the worst decision. The success of strategies varies from country to country; some strategies vital to a community may offer little to others; multiple approaches are more likely to obtain results. One option should be:
* to identify essential health care facilities which could be implemented with assistance from developed countries (criteria should include level of medical services and indigence of the area population, evidence of plans or actions, as well as the techniques of evaluation of the results);
* to establish a short-term (1-2 years) advisory tasks force to provide guidance on the implementation of new structures.
A problem is to identify areas that require special protection. This should be the origin of pilot projects on the development of ortho-trauma centers, with some help of national or international authority as the European Union. There is a situation of over-production of physicians in most countries of the European Union. A specialization should be organized, directed towards developing countries, to adapt specific problems, as orthopedics and traumatology.
Belgian universities recognize the need for a complete, but adapted training, equivalent to the European one. Belgian Government Agencies will allow 3 years of training, in the fear that a full training could incite African surgeons to stay in Europe, and to not return to their own country (even if the authorization to practice depends on the same Agency). Some European governments claim that teaching of the future specialist is possible in the country of origin which, in fact, is impossible, in most African countries we had the opportunity to visit. To refuse such a training is equivalent to maintaining the country in a state of dependance and permanent under development. No local solution can be found in an emergency crisis, only international help could solve the major problems of which orthopaedics and trauma-tology are only a small part.
The wish of the WHO "medicine for everybody in the year 2000" does not seem to be of application for under-developing countries. This overview of a very few of the existing challenges is far from exhaustive. A special concerned meeting should be organized for an in depth analysis of the problems with African and European countries. To bring such projects to success requires local political willingness, together with the support of the International comrnunity. It should be expected that some help could come from European countries in fields as medical care and education.
We acknowledge very useful discussion with Prof. F. Panda, Drs M. Elanga, E. Pay Pay, G. Tsiagadigui and F. Yila.
BONE BLOOD FLOW IN THE RATS - EFFECTS OF SEX HORMONES
The circulation of blood in the bones is a relatively narrow problem, about which little is known and on which little has been written, but on the other hand, is an important problem from the physiological, pathological, and clinical point of view. The basic physiological data are known, but clinicians would like to know much more, for example: Are there changes in bone blood flow during bone diseases? What kind of changes? Which diseases are they attendant to? Do changes in bone blood flow contribute to the pathogenesis of bone diseases? Can they be influenced by treatment or prevention? We can provide only very partial answers and we cannot foresee an improvement of the situation in future: given the technical difficulties, we cannot expect the discovery of an accessible method of bone blood flow measurement in humans in the near future. Thus the experimental approach still forms the basis for research, namely, experiment on a small and available animal, which would also be suitable for the purpose (such as laboratory rat), together with a technically feasible method (such as uptake of labelled microspheres). By definition, the results of experiments can be applied with limitations: a model always corresponds only partially to human pathology, and the interpretation of results is difficult and uncertain.
In the 1980s we moved from endocrinological blood flow studies to bone blood flow and demineralization influences in our laboratory. At that very time, Schoutens et al. (1984) published an abstract showing that bone blood flow increased after castration and other demineralization effects; the result was interpreted as "an initial step of bone demineralization in the rat". These interesting results confirmed the objective of our own programme: to investigate the relations between bone blood flow and bone mineralization in various experimental models simulating clinical pathological conditions with demineralization. We focused mainly on experimental situations induced by castration of female and male rats, administration of sex hormones, and application of some potentially regulatory effects. In this article, I provide an overview of the main results of these experimental studies.
I shall provide only a brief overview of the materials and methods used in the experiments necessary for the scope of this article. For more details, see the cited publications.
All experiments were conducted on female or male Wistar rats (Research Institute for Pharmacy and Biochemistry, Prague; Velaz, Prague, Czech Republic). Castrations were performed usually four, exceptionally five weeks before the experiment: oophorectomy (OOX) by dorsal approach, and orchidectomy (ORX) by scrotal approach. In the following overview, I shall list the hormonal preparations and other substances administered to rats after castration, that is, usually four weeks before the experiment:
- Estradiol benzoate (EB, long-acting preparation Agofollin Depot inj., Biotika, Slovak Republic), 1 mg per rat or 5 mg/kg BW s.c. once a week or once every 5 days.
- Estradiol dipropionate (Agofollin inj., Biotika, Slovak Republic), 1 mg/kg BW s. c. every day.
- Testosterone isobutyrate (T, long-acting preparation Agovirin Depot inj., Biotika, Slovak Republic), 25 mg/kg BW s. c. once every 5 days.
- Acetylsalicylic acid (ASA, Acylpyrin tbl., Slovakofarma, Slovak Republic), 0.13% in the food.
- Hydrocortisone (Hydrocortison tbl., Léciva, Czech Republic), 0.04% in the food.
- Methylene blue (Sigma, USA), 0.5% or 0.1% or 0.04% in the food.
We used the method of tissue uptake of microspheres labelled with radioactive strontium 85Sr (NEN, USA) (Rudolph and Heymann,1967; Kapitola et al., 1987) for determining the circulatory indicators. We express the microsphere uptake value in % of the dose in 1 g of tissue as a relative indicator of local tissue blood flow changes. The advantage of this non-physiological value is that it is not influenced by current changes of the overall circulation, that is, by changes in the cardiac output. If we multiply the 85Sr-microsphere uptake value by the cardiac output, we obtain the blood flow in ml per min. The cardiac output is estimated by an artificial organ method, that is, by a calculation from the radioactivity of a blood sample withdrawn from a. femoralis dx. after the administration of 85Sr-microspheres, and from the known velocity of pumping. Simultaneously, mean blood pressure and heart rate are measured in each rat.
In each experiment, the density of tibia is established on the basis of Archimedes principle, and the ashes of the tibia are weighed after incineration in a muffle furnace (expressed in g per ml of bone volume).
In parallel, 24 hours incorporation of 45Ca (Poland) and 3H-proline (Amersham, England) into the tibia are established as indicators of the mineralization process and of production of organic matrix, which are to be related to the local blood flow results (they are expressed in dpm per mg of tissue) (Globus et al., 1986). The uptake of radioactive strontium 85Sr in bone tissue was measured in some earlier experiments.
We mention only these two aspects in the area of basic physiology of bone blood flow contained in our materials: they are interesting and important, as they must be taken into consideration when judging the impact of experimental influences.
Local differentiation is a particularity of the skeleton. There are anatomical, metabolical, and functional differences; the cortical bone is different from the trabecular bone, etc. Local differences in rats manifest themselves significantly in the blood flow in various bones (Fig. 1), as well as in the sections of long bones (Fig. 2): the distal end of the tibia has 15 times lower blood flow than the proximal metaphyseal area (Kapitola et al., 1989).
Local bone blood flow differences can be of some importance in clinical medicine in some pathological processes, for example, in fractures and their healing.
Another interesting phenomenon is the decrease of bone blood flow in rats with age. We confirmed our earlier observations with radioactive rubidium by an experiment with 85Sr-microspheres in the age range 35 to 135 days in one experiment, and 45 to 110 days in another. The latter results are shown on Fig. 3 (Kapitola et al., 1989). A comparison with other organs and tissues showed that blood flow decrease concerned almost exclusively bones: a significant decrease was found only in the skin of older rats (not in heart, kidneys, liver, intestine, or skeletal muscle). It is related probably to the metabolism of the bone tissue. This is suggested by the decrease in the retention of 99m Tc-methylenediphosphonate, as well as the uptake of 85Sr in the bones of older rats. In our experiments, we were not dealing with changes observed at an advanced old age, such as the loss of bone marrow. The animals ranged in age from young to adult ones, but not very old. A similar unique observation in old humans has also been made: lower blood flow in the great trochanter of femur was recorded, using the method of 133Xe (Lahtinen et al., 1981).
Over the last few years we have carried out a number of experiments investigating the effects of the deficit (castration) and the supply of sex hormones (estradiol and testosterone) on local bone blood flow in rats. The aim was to contribute to the sofar thin knowledge of physiological and/or pathological role of local blood flow through investigation of the changes of the bone blood flow in the above mentioned situations. The experiments concerned mainly the relations between the responses of the local blood flow indicators of the bone formation, and the mineral content in the bone. The castration of female rats is a particularly suitable experimental model: it induces metabolical changes in the bone similar in nature and their time course to the changes in the bones of women after oophorectomy and, to an extent, also after menopause (Wronski et al., 1988).
In the initial experiments, we verified the effects of oophorectomy in the conditions similar to those described by Schoutens et al. (1984) (Kapitola et al., 1989; 1991).
The main results are shown in Fig. 4 and Tab. 1. Increase in blood flow was found in bones, but no similar change was observed in soft tissues. Increased uptake of 85Sr, increased incorporation of 45Ca and 3H-proline, and decrease of mineral content were found in the tibia.
| 1 | 2 | 3 | |
| Controls | Sham-operation | OOX | |
| Number of rats | 10 | 10 | 10 |
| Body weight (g) | 270±4 | 290±5a) | 341±6a)b) |
24h incorporation (dpm per mg): 45Ca |
15.69±0.27 |
14.17±0.32a) |
17.81±0.55a)b) |
| 3H-proline | 4.30±0.13 | 3.63±0.14a) | 4.98±0.24a)b) |
Also, we wanted to find out whether the substitution with estradiol would have any effect in this situation. Already the first results were surprising ( Fig. 5 ): administration of estradiol benzoate to OOX females entirely inhibited the increase in bone blood flow; furthermore, it decreased significantly the blood flow also in non-castrated, sham-operated females compared with sham-operated controls (Kapitola and Kubickova, 1990). These results were unexpected and encouraged a number of further experiments aimed at more detailed investigation of the observed effects.
The following results were obtained with respect to the effect of estradiol (Kapitola et al., 1991; 1992): as to the effect in various bones and their parts, it seems that a more significant decrease occurs in the predominantly trabecular bone (Fig. 6); the decrease in blood flow after the administration of estradiol was recorded only in kidneys from among all the other organs and soft tissue samples under examination (Fig. 7); apart from the blood flow values, the 24 hours incorporation of 45Ca and 3H-proline and the 3 hours uptake of 85Sr also decreased significantly in the tibia after the administration of estradiol; the indicators of the mineral content, that is, the bone density and weight of the ashes of the incinerated tibia, increased non-constantly after the administration of estradiol (Fig. 8). The time course of the effect of estradiol was investigated. The effect appeared rather quickly: the decrease in blood flow was observed already in the first day after the daily s. c. administration of estradiol dipropionate and became significant on the 9th day (Fig. 9). A dose-response relationship was established for doses ranging from 0.5 to 2.0 mg estradiol benzoate per animal (administered once a week for four weeks). A comparison showed that estradiol had inhibiting effects on bone blood flow in both female and male rats.
The effects of estradiol and testosterone on local blood flow and mineral content in female and male rats - castrated and non-castrated - were compared in the following series of four identical experiments. The results are summarized in Fig. 10. These experiments showed that OOX and ORX increase the uptake of 85Sr-microspheres and bone blood flow, and decrease the mineral content in the tibia, while estradiol constantly and testosterone non-constantly (with the administered dosage) decrease the uptake of 85Sr-microspheres and blood flow, and increase bone mineral content. Thus bone blood flow responses to sexual hormones were not sex-specific in rats (Kapitola et al., 1995).
The results suggest that under the described experimental conditions a relation probably exists between local blood flow and its changes, and bone metabolism and bone mineral content. Further, it is very likely that these changes and relations are related to the process of bone resorption. These regulations and relations in humans are not known, neither is the effect of the lack or administration of estrogen on bone blood flow. However, there are sporadic observations of estrogen effects in humans: decrease in the 24 hours retention of 99mTc-methylenediphosphonate in the body (Fogelman et al., 1980), decrease in hydroxyproline excretion, and decreased concentration of blood osteocalcin (Civitelli et al., 1988; Stock et al., 1985).
In all experiments so far, the results were obtained in rats with artificially changed level of sex hormones through castration and/or administration of hormonal preparations. In the next series of experiments, we measured bone blood flow and incorporation of 45Ca and 3H-proline in female rats in an advanced stage of pregnancy, that is, with naturally increased estrogen level (Kapitola et al., 1995). The results are shown in Fig. 11 and Tab. 2. In two blood-flow experiments, the uptake of 85Sr-microspheres and blood flow were significantly lower in tibia and distal femur in pregnant female rats; blood flow was lower in humerus and calvaria. No difference in the uptake of 85Sr-microspheres was recorded in liver, muscle, and skin; blood flow was lower in kidneys. The uptake of 85Sr-microspheres and blood flow were several times higher in the perirenal fat of the pregnant females than in the control group. 24 hours incorporation of 45Ca and 3H-proline was significantly lower in the pregnant females; bone density and weight of the ashes of the tibia were higher in the pregnant females in the first experiment. Plasma estradiol, measured in the pregnant females in the second experiment, was significantly although not much increased (+24.8%). Lower bone blood flow and decreased incorporation of 45Ca and 3H-proline with normal or higher mineral content in the tibia were found in pregnant females, i. e. in the situation when increased bone resorption and bone formation were described. It is not yet possible to explain these changes and relations and, particularly, the lower bone blood flow and its causes in pregnant females; the results neither prove nor reject the role of estrogens (possibility of involvement of other estrogen than estradiol may be considered; most likely, it could be estrone, not measured in these experiments).
| Controls | Pregnant | |
| Body weight (g) | 263±8 | 360±1 |
| Cardiac output (ml/min) | 62.1±3.4 | 67.7±3.5 |
| (ml/min/100g) | 24.3±1.3 | 18.1±0.8** |
| Heart rate (beats per min) | 411±8 | 435±10 |
| Blood pressure (kPa) | 15.4±0.5 | 10.9±0.6** |
| Density of tibia | 1.54±0.01 | 1.54±0.01 |
| Ash weight (g/ml) | 0.629±0.011 | 0.638±0.009 |
| Estradiol (nmol/l) | 1.01±0.05 | 1.26±0.10* |
We gathered a great amount of results on the effects of sex hormones on bone blood flow. After the collection of experimental data, however, a question arose as to the mechanisms of the observed responses.
It does not seem likely that, in our experiments, the changes observed resulted from a direct action of sex hormones on blood vessels. Literature records only very few - and usually older - data on the effects of estradiol on blood flow (but not in bones): these studies report on the increase in the cardiac output, increase in the muscle or entire hind extremity blood flow, and decrease in the blood flow fraction to the brain and kidneys (Hart et al.,1985; Solti et al., 1965; 1968). In the cited literature however the effects were immediate, possibly direct, and they cannot be compared to our results of long-term hormonal action.
Prostaglandins, and especially PGE2, may be considered as the possible mediators of the effects of withdrawal or excess of sex hormones on bone blood flow. They are produced in bone tissue and have important roles in bone metabolism including bone resorption (Watrous and Andrews, 1989), and they are usually vaso-active. With regard to our interests, the observations made by Feyen and Raisz (1987) are important: the production of PGE2 by rat osteoblasts in vitro was increased after OOX, and inhibited after the administration of estradiol to both intact and OOX females (and also by administering hydrocortisone in vivo to females or by supplying hydrocortisone in vitro). The experimental situation and the results are to be put in balance with our observation of an increase in bone blood flow after OOX, and its inhibition after administration of estradiol.
In a series of experiments, we obtained results supporting the hypothesis of the possible participation of prostaglandin (PG) in blood flow changes induced by the deficit and/or supply of estradiol. We chose the acetylsalicylic acid (ASA) as the known inhibitor of cyclo-oxygenase and eikosanoid synthesis. The working hypothesis was as follows: estrogen should inhibit the production of PG (we do not know how); the estrogen deficit after OOX would result in the increase of PG production (probably PGE2), which should act as a mediator for bone blood flow increase; the blockade of the PGE2 induced by the administration of ASA should prevent the increase in bone blood flow after OOX.
The results of the first experiment are described in Fig. 12. They confirmed the presuppositions mentioned above: the uptake of 85Sr-microspheres and the blood flow were increased in the tibia and distal end of the femur in OOX females, while the concurrent administration of ASA inhibited the increase significantly compared with the OOX group (Kapitola et al., 1994). In the following experiment on females, local circulatory values were increased significantly in the tibia and in the distal femur after OOX, insignificantly in the diaphysis of the femur and in the calvariae ; the increase was inhibited significantly in the tibia by the concurrent administration of ASA, and insignificantly in the distal end and the diaphysis of the femur, this effect of ASA was not proved in the calvaria. Local blood flow was decreased by the effect of ASA in the kidneys of the OOX females; blood-flow changes were not proved in the liver, muscle, and skin. The mineral content in the tibia was decreased significantly in both groups of OOX females. A similar experiment on males (Tab. 3) showed an increase in the local circulatory values four weeks after ORX in the tibia, distal femur, and diaphysis of the femur, and the inhibition of the increase with the concurrent administration of ASA. The changes were not proved in the calvaria and in the soft tissues which were subjected to the measurements. In the experiment with 45Ca and 3H-proline (Fig. 13), the incorporation of both substances into the tibia was inhibited significantly by the administration of ASA in both the control and OOX females. (Kapitola et al.,1996).
| Controls | ORX | ASA | ORX + ASA | |
| Number of rats | 10 | 10 | 9 | 10 |
| Body weight (g) | 338±8 | 357±4 | 322±11 | 332±8 |
| Cardiac output (ml/min) | 55.5±3.4 | 58.6±3.5 | 53.1±2.1 | 55.9±3.4 |
| (ml/min/100g) | 16.7±1.1 | 16.4±3.1 | 16.6±0.9 | 16.7±0.7 |
| Blood pressure (kPa) | 15.0±0.8 | 15.7±0.5 | 15.6±0.7 | 14.5±0.3 |
| Heart rate (beats/min) | 391±7 | 429±12 | 393±15 | 391±14 |
| Blood flow (ml/min/g): | ||||
| Tibia | 0.19±0.01 | 0.25±0.02a) | 0.18±0.02 | 0.20±0.01 |
| Distal femur | 0.25±0.018 | 0.36±0.038a) | 0.27±0.026 | 0.27±0.020b) |
| Diaphysis of femur | 0.081±0.008 | 0.108±0.012a) | 0.077±0.083 | 0.065±0.059b) |
| Calvaria | 0.11±0.007 | 0.12±0.010 | 0.11±0.009 | 0.13±0.011 |
| Kidneys | 4.33±0.36 | 4.28±0.45 | 3.70±0.48 | 3.82±0.68 |
| Liver | 0.11±0.01 | 0.10±0.01 | 0.11±0.02 | 0.13±0.02 |
| M. gastrocnemius | 0.092±0.019 | 0.068±0.006 | 0.082±0.010 | 0.078±0.09 |
| Skin | 0.073±0.010 | 0.089±0.009 | 0.075±0.005 | 0.067±0.00 |
The results of the experiments confirm that ASA inhibits the increase in bone blood flow after castration, and thus suggest the probability of the prostaglandin - most likely PGE2 - participation. The blood flow changes described above are induced by a local response of the blood vessels, and occur in both male and female rats. The inhibition of the 45Ca and 3H-proline incorporation by the administration of ASA supports the blood-flow results and indicates important relations between metabolic processes and local blood flow. We conclude that prostaglandin is probably a factor regulating bone blood flow.
Considering the known important effects of corticoids on the bone, we wanted to investigate their effects on local bone blood flow.
Significant changes appeared already in the first experiment (Fig. 14; Kapitola et al., 1992). They were probably at least partially related to the effects on the production of prostaglandins. That was the reason for focusing on this issue in several subsequent experiments (Kapitola et al., 1995). The significant decrease in the uptake of 85Sr-microspheres and in blood flow was proved again in the tibia, distal femur, and lumbar vertebra in the treated sham-operated females compared with the control group. The blood flow values decreased in a similarly significant way also in the bone of treated OOX females compared with the OOX group. The samples of some soft tissues were examined simultaneously and did not show any similar differences. Hydrocortisone reduced the 24 hours incorporation values of 45Ca and 3H-proline into the tibia in the sham-operated females as well as in the OOX females (Tab. 4).
Glucocorticoids are known to cause a blockade of the eikosanoid synthesis by the inhibition of the arachidonic acid production. There is a probability that the above described effects of hydrocortisone on local bone blood flow in rats are mediated by this mechanism, and that the results support the presupposition of the prostaglandin participation in the bone blood flow regulation. The very significant effects of hydrocortisone on bone blood flow, however, could also play a part in the occurence of other effect of corticoids on bone and in the pathogenesis of clinical bone disorders induced by corticoids.
| 1 | 2 | 3 | 4 | |
| Controls | OOX | Hydro-cortisone | OOX + hydro-cortisone | |
| Number of rats | 11 | 10 | 10 | 9 |
| Body weight (g) | 231 ± 3 | 261 ± 4a) | 211 ± 2a) | 236 ± 7a)b) |
24-h incorporation (dpm per mq) |
||||
| 45Ca | 4.18 ± 0.16 | 4.32 ± 0.19 | 3.20 ± 0.14a) | 3.38 ± 0.25b) |
| 3H-proline | 3.88 ± 0.18 | 4.53 ± 0.17 | 3.00 ± 0.14 | 3.22 ± 0.26b) |
In our investigation of mechanisms of effects of sex hormones on bone blood flow in rats, we had to consider the influence of the recently discovered and powerful "endo-thelium-derived relaxing factor" (EDRF), which was analysed as the simple chemical compound nitric oxide (NO) (Kapitola et al., in press). It is again a vasodilating factor and, therefore, the preliminar hypothesis was similar as in the case of prostaglandin: the inhibition of the increase in the bone blood flow following OOX by the EDRF-NO blockade would suggest the probable participation of EDRF-NO in the local blood flow response in the bones of female rats after OOX. Methylene blue (MB) was used as the blocking agent in the first series of experiments. MB, among other effects, blocks the EDRF-NO production and its action on blood vessels (Wang et al., 1995).
In the first experiment, MB was administered four weeks prior to the experiment in the high concentration of 0.5% in the food. The uptake of 85Sr-microspheres and the blood flow in the tibia were reduced significantly in the sham-operated female rats as well as after OOX; further, the cardiac output and the blood pressure were reduced. The latter changes occured proportionately to the former so that the total peripheral resistance was not significantly changed (Table 5). The density of the tibia and the weight of the ashes was lower in both OOX groups.
In the following experiment, MB was administered in the concentration of 0.1% in the food: the uptake of 85Sr-microspheres was reduced in all bone samples (tibia, distal femur, diaphysis of the femur, calvaria), but significantly only in the tibia of OOX females; no similar change was noted in soft tissues.
Blood flow values were not significantly changed, neither were cardiac output and blood pressure. In this experiment, bone density and the weight of the ashes were reduced in the sham-operated females following the administration of MB.
In the last experiment, the 24 hours incorporation values of 45Ca and 3H-proline into the tibia decreased significantly in OOX females following administration of the low concentration of 0,04% in the food (Table 6).
| Controls | OOX | Methylene blue | OOX+methylene | |
| Number of rats | 11 | 11 | blue | |
| Body weight (g) | 234±3 | 12 | 193±5a) | 12 |
| Cardiac output (ml/min) | 50.0±4.2 | 252±2a) | 33.5±3.6a) | 215±5a)b) |
| (ml/min/100g) | 21.4±1.8 | 49.2±4.7 | 17.2±1.8a) | 35.1±3.2a)b) |
| Heart rate (beats/min) | 424±12 | 19.5±1.8 | 330±9a) | 16.2±1.3a) |
| Blood pressure (kPa) | 15.1±0.6 | 413±9 | 12.1±0.3a) | 337±9a)b) |
| Tibia: | 15.3±0.3 | 11.8±0.6a)b) | ||
| 85Sr-microspheres (%/g) | 0.46±0.03 | 0.33±0.02a) | ||
| Blood flow (ml/min/g) | 0.20±0.02 | 0.54±0.04a) | 0.ll±0.0la) | 0.41±0.03b) |
| Density | 1.56±0.01 | 0.21±0.02 | 1.56±0.01 | 0.13±0.01a)b) |
| Ash weight (g/ml) | 0.65±0.01 | 1.51±0.01a) | 0.65±0.01 | 1.51±0.01a) |
| Distal femur: | 0.59±0.01a) | 0.60±0.01a) | ||
| 85Sr-microspheres (%/g) | 0.72±0.06 | 0.56±0.04a) | ||
| Blood flow (ml/min/g) | 0.32±0.03 | 0.86±0.08 | 0.19±0.02a) | 0.66±0.06a)b) |
| 1 | 2 | 3 | 4 | |
| Controls | OOX | Methylene blue | OOX + methylene blue | |
| Number of rats | 10 | 8 | 13 | 12 |
| Body weight (g) | 241±4 | 309±10 | 229±2a) | 262±6a)b) |
24h incorporation (dpm per mg): |
||||
| 45Ca | 1.89±0.05 | 2.22±0.09a) | 1.75±0.08 | 1.86±0.09b) |
| 3H-proline | 1.30±0.05 | 1.85±0.07a) | 1.27±0.05 | 1.61±0.06a)b) |
l) High MB dosage reduced bone blood flow in both OOX and non-castrated females, which suggests the probability of EDRF-NO participation in the regulation of local blood flow.
2) Methylene blue did not increase the cardiac output or blood pressure. High concentration even reduced both values.
3) Methylene blue reduced the incorporation values of 45Ca and 3H-proline into the tibia in OOX females, in proportion to the blood-flow effects.
4) Methylene blue reduced - in one experiment - bone density and the weight of the bone ashes in non-castrated, sham-operated female rats - that is an interesting, but so far unexplained result.
5) The methylene blue effects on cardiac output and blood pressure, which were observed in our experimental conditions, differ from the reported effects of arginine-derived blocking agents, which induce a significant increase of blood pressure.
The effects of methylene blue are complex, which makes the interpretation of the results difficult (therefore, we plan further experiments with L-NAME). Despite this fact, we consider the results a very likely evidence of the EDRF-NO participation in bone blood flow regulation.
I will supply a brief commentary to the results presented here and, further, I will point out some more general problems and relations.
In correspondence with the objectives explained in the Introduction, we attempted to contribute to the issues related to the role of bone blood flow in demineralizing processes, by means of methods available to us. The main focus was to investigate bone blood flow in rats in experimental situations with artificial deficit of sex hormones following castration. However, we modified the thematic plan of our experiments already after the first experiment considering the role of excess estradiol administration, and focused also on the effects of sex hormones on bone blood flow. Castration continued to be included in almost all experiments. The basic effects, that is, the increase in bone blood flow after castration and its decrease following the administration of sex hormones, were proved repeatedly and further investigated. The decrease in bone blood flow was proved following the external supply of pharmacological doses of estradiol, and also in the physiological increase of endogenic estrogens in pregnant female rats.
We explored the mechanism of the estradiol effects on bone blood flow. We presupposed a mediated, rather than direct effect. The experiments with the acetylsalicylic acid showed a probability of prostaglandin participation; the participation of EDRF-NO is also likely (study not yet completed). The results obtained in the experimental conditions and presented here proved the existence of interesting relations between sex hormones and bone blood flow. However, they also induce questions, so far unanswerable. It is not known whether and how the relations we observed work physiologically in rats, and whether sex hormones participate in physiological regulations of bone blood flow in rats. It is also unknown whether similar effects of sex hormones on bone blood flow occur in humans too. If they do, the regulatory effects of sex hormones would constitute an important factor in the physiology, and probably also in the pathology, of the skeleton.
Some more general facts and broader problems emerge from the results of a large number of experiments in various model situations.
At the beginning of this study, we pointed out some anatomical and functional differences of various parts of the skeleton, that is, of various bones and also parts of the same bone, and we showed the extent of the differences in blood flow in basal physiological conditions. The results of the following experiments show that the responses to experimental influences (castration, hormones) also differ: the changes are the most frequent and the most significant in the tibia and distal femur, they are less frequently significant in the diaphysis of the femur, and the least significant in the calvaria.
The concrete causes and mechanisms of these differences in bone blood flow responses are not clear. Nevertheless, they are not surprising, considering that also other estrogen and castration effects differ with location and the type of bone. For example, the resorption of bone after OOX does not rise in the calvaria as it does at other locations (Turner et al., 1992).
An interesting phenomenon, which concerns all experimental situations that were used, is also local tissue specificity of the observed blood-flow responses: local blood-flow changes induced by withdrawal or excess of sex hormones were proved only in bones and kidneys (not quite constantly in the latter) (Kapitola et al. 1994). Similar changes were not proved in other organs and tissue under examination, apart from insignificant exceptions. It has been long known that sex hormones influence kidneys, especially their growth and weight, and that receptors for sex hormones are present in the kidney tissue (Kochakian 1976; Shukla et al., 1992; Wing 1990). However, it is also known that estrogen receptors are present diffusely in the cardiovascular system (Shan et al., 1994). And finally, if we suppose prostaglandin, and possibly also EDRF-NO participation in the effects of sex hormones on bone blood flow, it is difficult to understand why the effects occur only in bones.
The important findings, at which we repeatedly arrived, are:
a) the relation between bone blood flow and the incorporation of 45Ca and 3H-proline, and particularly,
b) the relation of local blood flow and metabolic incorporation, to the current bone density and the weight of the ashes of the incinerated bone.
The changes in blood flow and incorporation correspond (although they do not show constant statistical significance) in the majority of the experimental conditions. That suggests significant participation of local blood flow in these processes and vice versa, the credibility of blood-flow results on the basis of the corresponding changes in the incorporation.
The relations of local blood flow and the incorporation of 45Ca and 3H-proline to bone density and the weight of the ashes are particularly interesting. The increase in the bone density and the mineral content, with the concurrent decrease in the indicators of the organic matrix production and mineral deposition, and the decrease of the local bone blood flow occurred following estradiol administration, in pregnant females, and following hydrocortisone administration. The situation is probably similar to the one described by Wronski et al. (1986) following the chronic administration of 1,25-Dihydroxyvitamin D3, and which he reported as "increased bone but impaired minera-lization". Our observation suggests that a similar situation probably occurs more frequently at least in experimental conditions.
Finally, one more particular and complex aspect of the relations between local blood flow and metabolism occurs only in bone. The increase of local blood flow generally corresponds to metabolical and functional demands of the tissue and, therefore, is a positive reaction with respect to the tissue.
Decreased local blood flow has often unfavourable consequences for the tissue. It applies to soft tissue as well as to bone. However there is also another and reversed relation in bone: it is the relation between bone blood flow and bone resorption. In this regard, increased local blood flow seems to be a component of unfavourable metabolical changes, associated with bone resorption and demineralization. Decreased blood flow can, as shown above, be related to decreased bone resorption and the reduction of loss of the bone mineral.
Nevertheless, we do not know the functional importance and the role of blood flow changes in these and other situations: local blood flow may represent an active regulatory factor, it may be merely one element in the process of metabolical changes, or it may be only a secondary consequence of metabolical changes.
In our study, these problems are associated with the observed hormonal effects on bone blood flow. A comprehensive understanding of these issues could bring concrete explanation of the role of local bone blood flow, and of its physiological or pathological importance.
We realize that, although our results contributed some partial findings, we have not managed to find solutions to many problems, and we have not yet found answer concerning the importance of bone blood flow. We hope, however, that our studies at least drew attention to the importance of an area which has been so far little investigated.
Acknowledgement: This work was supported by grant No. 306/93/0851 from the Grant Agency of the Czech Republic Government.
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