Model of tail suspension and its effect in some mechanical properties of the rat bone

Abstracts

A manutenção do metabolismo mineral normal dos ossos é um resultado de vários fatores inclusive das solicitações mecânicas que são aplicadas aos ossos pelas contrações musculares e pela força da gravidade. O propósito desta investigação foi estudar um modelo de suspensão de rato pela cauda que simulasse assim as alterações esqueléticas que podem acontecer em um ambiente de microgravidade. O modelo foi analisado em termos de tolerância do animal e dos efeitos sobre a resistência mecânica do complexo tíbia-fíbula. Após a realização do ensaio de flexão em três pontos, foram obtidos os principais parâmetros mecânicos (carga e deflexão no limite máximo, carga e deflexão no limite elástico, rigidez e resiliência). Foram utilizadas cinqüenta e três ratas fêmeas, distribuídas em quatro grupos conforme o período de suspensão (controle, 7, 14 e 21 dias). O modelo de suspensão mostrou-se eficaz com boa adaptação dos animais e promoveu um enfraquecimento significativo nos ossos principalmente no período de 21 dias.

Simulação de microgravidade; Osso; Ratos

ORIGINAL ARTICLE

Model of tail suspension and its effect in some mechanical properties of the rat bone

Adriana Valadares da Silva^I; José Batista Volpon^II

^IMaster in Bioengineering and Professor - Universidade de Franca - Physiotherapist

^IIAssociate Professor - Faculdade de Medicina de Ribeirão Preto

^{Correspondence}

SUMMARY

The maintenance of the normal metabolism of minerals in the bone is a result of several factors including the mechanical demands that are applied to the bones by muscle contractions and gravity force. The proposal of this investigation was to study a model of tail suspension of rats thus simulating the skeletal alterations that may occur in a microgravity environment. The model was analyzed in terms of animal tolerance and the ensuing effects on the mechanical resistance of the tibiofibular complex. After a three-point bending test in flexion the main mechanical parameters were obtained, (load and deflection at the ultimate limit, load and deflection at the yielding point, stiffness and resilience). 53 adult female rats were used and distributed in four groups according to the length of time in suspension (control, 7, 14 and 21 days). The model of suspension was efficient with good animals adaptation and it caused a significant weakness of bones mainly in the 21-day period.

Keywords: Weightlessness simulation; Bone; Rats.

INTRODUCTION

There are significant evidences that an environment with no mechanical stimulus may produce direct effects in the bone structure and function⁽⁶⁾.

According to Johnson⁽⁷⁾, periods of seven or fourteen days without any ground contact are enough to promote bone alterations, specially in those ones that support the body weight such as the femur and the tibia.

Therefore, the bone remodeling is extremely sensitive to alterations of the mechanical tension generated in the bone tissue.

The use of mechanical forces during a normal physical activity can be defined as a mechanical stimulus that causes small malformations in the bone architecture, stimulating the osteogenesis. The physical activity may directly act in the bone formation. But the immobilization leads to a bone reabsorption process^(5,15).

Studies on the effect of the long resting-on-the-bed periods on the calcium metabolism showed that the calcium increase in the urine excretion during this long period of confinement doesn't occur due to the lack of activity. It occurs because of the longitudinal compression on the long bones caused by the type of decubitus⁽¹⁾.

In order to live or explore the environment with its different climates and temperatures, men need a physiological adaptation. Innumerous investigations have been performed aiming to discover the influence of the gravity in our evolution and its role in physiology⁽¹⁰⁾.

The experiments performed in orbital flights are very limited. Therefore NASA (National Aeronautics and Space Administration) has performed several studies trying to determine the mechanisms of the muscular atrophy and the bone attrition that occur in individuals exposed to microgravity, simulating the space flight conditions⁽¹³⁾.

Several models were applied to simulate the alterations that occur in a microgravity environment. The system of elevation by the body and the model of tail suspension of the animal are the most used. Kasper et al.⁽⁸⁾ and Musacchia et al.⁽¹²⁾ assured that the ideal model would be the one which enabled free mobility of the animal but with a certain restriction of posterior members movements, i.e., the model of tail suspension of the animal.

In this situation, the posterior members of the animal remain with free movements but without ground contact. According to the research performed by Morey-Holton and Globus⁽¹¹⁾, the animals that remain in simulated microgravity situations present physiological alterations similar to the animals that were in space flights. Therefore, the model of tail suspension of the rat is considered an effective model to evaluate the physiological and cellular mechanisms of the musculoskeletal system that change responding to a new stimulus.

The bone mechanical behavior can be studied by the performance of mechanical tests of the bone tissue that are important because they provide the fundamental parameters on the resistance of these structures⁽⁴⁾.

The general aim of this study was to evaluate a model of tail suspension of the rat that could simulate the alterations in skeletal in a microgravity environment analyzing its effectiveness referring to the animal tolerance and the suspension repercussion on the mechanical resistance of the bone. The particular aim was to perform mechanical tests by flexing the tibia-fibula bone complex after different intervals of time of tail suspension in order to determine the variation of some of its mechanical properties.

Material and Methods

The paper was performed according to the rules of the research committee of the Medical School of Ribeirão Preto – USP. The experiment used female adult young rats, from the race Rattus Norvegicus albinus, variety Wistar and mean weight of 250 gr. At first, the animals were maintained in common cages, in number of five in each one. When they reached the ideal weight, they were transferred to special individual cages. In these cages, they were maintained with the posterior members suspended in order to enable the support using the anterior members only for the locomotion and water and ration access.

The animals were divided into four experimental groups, according to the type of treatment. The Group 1 (control) was formed by animals that didn't receive any specific treatment. In the group 2, the animals remained in suspension for 7 days; in the group 3 the suspension period was of 14 days and in the group 4 the suspension was of 21 days. All the animals were killed after this period and the tibias associated with the fibulas underwent the three-point bending mechanical test in flexion.

Suspension technique

The animals were anesthetized with etilic ether inhalation in order to install the suspension process. At first, the animal tail was washed with water and detergent and after drying, Benjoim tinck was applied in the whole skin. After that, the tail was enfolded by an adhesive spuma (Reston®) from its origin until the two proximal thirds, approximately, aiming to protect the skin and avoid cutaneous injuries.

Adhesive elastic strip was applied on the adhesive spuma. The strip was tensioned homogeneously enfolding all the spuma. A narrow elastic ribbon was placed on the elastic strip, fixed by bandages of the adhesive elastic strip and additional bandages of court plaster and twine in order to form a handle used to connect the animal to the suspension system.

After that, with the animal still anesthetized, the distal border of its tail that wasn't bandaged was amputated by a shear, aiming to avoid the local necrosis during the period the animal was suspended.

Applied cage

The restraint system of the animal was constituted by a two-part cage. The lower part was formed by a box with 35 cm of width, 35 cm of length and 21,5 cm of height, open in the upper extremity. It was made in transparent acrylic to enable a better visualization and animal control. It was made a lateral perforation to introduce the water source point.

Another box with metallic bars with 35,5 of width, 35,5 cm of length and 24 cm of height was placed in an inverted position on this acrylic box. This superior box was made with lateral sockets to assure a perfect fixation of the acrylic box. The superior component was used as a support for the fixation system of the animal. The two cages were connected to fit perfectly by the opening, forming a unique compartment.

The size of the cage enabled the animals to perform free movements using the anterior members but maintaining the posterior members suspended without a support on the cage floor or on the sides.

After assembling the cage, it was made a suspension axle. The fixation system was basically composed by a straight steel twisted shaft, with 40 cm of length and 4,5 mm of diameter. In the central part of this shaft, in the other extremity, a clasp was fit with a socket to the elastic handle which was part of the connection system of the animal (Figure 1B).

A small aluminum plate was placed in each extremity of the shaft, fixed by two "nuts" delimiting the movement space of the animal in the cage (Figure 1C). The course limit to the animal movement was of 25 cm. This shaft was fixed transversally in the superior cage, in the larger diameter, and presented several attachments such as nuts, washers and butterfly nuts (Figure 1 A) with the proposal of fixing it to the cage bars enabling the adjustment of the lateral excursion and height according to the animal size.

After the preparation and complete assembly of the cage, the animals were weighted and placed in suspension. The clasp placed on the suspension bar was united to the elastic fixed to the animal tail (Figure 2).

Sawdust for the hygiene and also the animal ration were placed on the cage floor and replaced diary. The provision of water to the animal, as previously described, was made by the fixation of a bottle on the lateral wall. During the animal movements, there was a displacement of the clasp that remained connected to the tail and fixed on the steel shaft. The animals could move to a 360º angle with free access to water and ration and without a support on the lateral walls.

Mechanical Tests

The rats were weighted and killed by excessive inhalation of sulfuric ether in order to collect the material. After that the posterior members were dissected and weighted; the right and left tibio-fibular units were cleaned and removed from the soft parts around. The tibia and fibula bones remained connected and were distally merged in the rat. Therefore, the periosteum and the articular cartilage of the borders were preserved and the dissection of the muscles was performed carefully in order to avoid bone damages.

After the dissection, the bones were enfolded in a bandage humidified by physiological serum and stocked in "freezer" in an approximate temperature of -20º C.

Three-point bending mechanical test in flexion

In these mechanical test, it was used a universal test machine of the Bioengineering Laboratory of the Medical School of Ribeirão Preto of the Universidade de São Paulo. 24 hours before, the bones were removed from the "freezer" and kept in a common refrigerator. Some hours before being tested, they were removed and kept under ambient temperature in order to get the thermal equilibrium. They were still enfolded in a bandage humidified by physiological serum.

The tibia-fibula set of each side underwent three-point bending in simple flexion. The load application point was the posterior side of the tibia and the anterior side was turned down. The distance between the support points was 2,5 cm. It was applied a pre-load of 200 g, with an accommodation time of 1 minute, vertically, from top to bottom.

The load was applied in a speed of 0,25 mm/min and registered by a load cell of 200 kgf (Kratos®) connected to the CAE 201 Sodmex^® amplifier, until the tibia fracture. The tibia wasn't directly submitted to the load. All the tests were performed in similar conditions and during all of them the bones were kept humidified by physiological serum in order to avoid drying up.

Mechanical parameters analysis

To each test, a load x deflection graphic was built and the following mechanical parameters were obtained: elastic limit, maximum limit, rigidity and the energy absorbed by the bone in the elastic phase (resilience) (Figure 3).

Statistical analysis

Animals weight

The animals weight of the groups 2, 3, and 4, individually, was analyzed before and after the suspension, using the t test of Student paired.

The statistical comparison of the animals' weight before the suspension was performed by the ANOVA test to simultaneous comparison among the groups and the Student-Newman-Keuls method to compare the pairs of groups. The same tests were applied in comparison to the groups 2, 3 and 4 after the suspension.

Parts weight

The statistical comparison of the parts weight was performed applying the ANOVA test to simultaneous comparison among the groups and the Student-Newman-Keuls method to compare the pairs of groups.

Mechanical properties

For the statistical analysis of the load and deflection of the elastic limit, deflection of the maximum limit and resilience, ANOVA test was used to simultaneous comparison among the groups and the Student-Newman-Keuls method to compare the pairs of groups.

The load of the maximum limit and rigidity presented a non-parametrical distribution, therefore it was applied the Kruskal-Wallis test to simultaneous comparison among the groups and the Dunn's method to compare the pairs of groups. The level of significance was settled in 5%.

RESULTS

45 animals were placed in suspension, and two died during the experimental phase. These animals were the first to be placed in suspension. The tail border wasn't amputated resulting in necrosis due to the excessive compression caused by the fixation system of the animal connected to the suspension axle. Therefore, the death of these animals may be related to the tail necrosis. After that, all the animals had their tails extremities amputated and there weren't more complications.

The right and left tibia and fibula bones of 43 animals that underwent suspension were tested, in a total of 86 tests. Of these animals, 14 were from the groups 2 and 3 and 15 from group 4. Ten animals of the control group were also analyzed and only the right side was tested in these ones.

To obtain the final results, the graphics presenting atypical behaviors were discharged what generally reflects the technical problems occurred during the test. Therefore, of the 96 tests 87 were used for the final analysis.

Animals weight

By analyzing the animals weight values, it wasn't observed any statistical difference among the groups at the beginning of the experiment (p = 0,054). The mean weight of the animals from the group 1 (control) was 257,3 g; in the group 2, it was 245,1 g; in the group 3, it was 246,8 g and in the group 4 it was 260,4 g.

At the end of the experiment, the overall weight of the group 2 (7 days of suspension) presented a significant statistical difference comparing to group 4 (21 days of suspension). Therefore, between group 2 and group 3 there wasn't any statistical difference as well as between groups 3 and 4. After the suspension, the mean weight of the animals in group 2 was 234,3 g, in group 3 was 241,1 g and in group 4 was 256,0 g.

The animals' weight in each group was also analyzed, before and after the suspension period. The group 2 presented significant difference (p=0.006). In the group 3, there wasn't any significant difference (p=0.061) and in the group 4 there was a significant difference (p=0.024).

Parts weight

By analyzing the parts weight values (tibia-fibula), it was observed a difference among all groups (p=0,0140). The mean parts weight of the animals in the group 1 (control) was 0,86 g, in the group 2 was 0,77 g, in the group 3 was 0,79 g and in the group 4 was 0,76g. The group 1 presented difference when compared to all the other groups: Group 1 and Group 2 (p=0.035), Group 1 and Group 3 ( p=0,020) and group 1 and group 4 (p=0.0015). When the groups 2, 3 and 4 are compared it isn't observed any difference: groups 2 and 3 (p=0,332), groups 2 and 4 (p=0,948) and groups 3 and 4 (p=0,275)

MECHANICAL ESSAY

From the load x deflection graphic, the mechanical property values of the flexion tests were obtained in three points of the tibia and fibula bones of the control group and of the females rats that underwent different suspension periods (7, 14 and 21 days)

Figure 4 shows the mean load value in the elastic limit (EL) obtained in the tests with tibia and fibula bones of the rats of the group 1 (control) that was (46,75±5,00)N. In the group 2 it was (41,39±5,59)N, in the group 3 it was (37,58±7,25)N and in the group 4 it was (32,17±5,27)N. The simultaneous comparison among all the groups was statiscally different (p=0,009). After analyzing the load values in EL, it was verified that there was a significant difference between the group 1 (control) and the group 2 (p=0,0064), between the group 1 (control) and the group 3 (p=0,0085) and between the group 1 (control) and the group 4 (p=0,0096). It was also observed a statistically significant difference between the group 2 and 3 (p=0,029) , between the groups 2 and 4 (p<0,0011) and between the groups 3 and 4 (p=0,002).

The deflections in EL presented mean values of (0,478±0,046)x10^-3m in the group 1 (control), (0,484±0,075)x10^-3m in the group 2, (0,457±0,099)x10^-3m in the group 3 and (0,455±0,094)x10^-3m in the group 4. The simultaneous comparison among all the groups wasn't statiscally different (p=0,580). By comparing the deflection values in EL, it was verified that there wasn't a statiscally significant difference among the groups. The values were: (p=0,816) between the groups 1 and 2, (p=0,529) between the groups 1 and 3, (p=0,464) between the groups 1 and 4, (p=0,291) between the groups 2 and 3, (p=0,485) between the groups 2 and 4 and (p=0,928) between the groups 3 and 4 (figure 5).

The load mean value in the maximum limit (ML) was (60,01±7,84) in the group 1 (control), (51,29±.4,37)N in the group 2, (51,05±.8,78)N in the group 3 and (43,77±.4,49)N in the group 4. The simultaneous comparison among all the groups was statiscally different (p=<0,001). It could be observed that there was a statiscally significant difference between the group 1 and 2 (p=<0,001), 1 and 3 (p=0,008) and 1 and 4 (p=<0,001). There was also a statiscally significant difference between the groups 2 and 4 and 3 and 4, but there wasn't difference between the groups 2 and 3. (Figure 6).

The deflections in ML in the tests of the rats presented a mean value of (0,697±0,071)x10^-3m in the group 1 (control), (0,729±0,088)x10^-3m in the group 2, (0,711±0,075)x10^-3m in the group 3 and the (0,765±0,114)x10 in the group 4. The simultaneous comparison among all the groups showed that there wasn't statiscally significant difference among the groups. The obtained value between the group 1 and 2 was (p=0,306), (p=0,615) between 1 and 3 and (p=0,086) between 1 and 4. There wasn't statistic difference between the groups 2 and 3 (p=0,481), 2 and 4 (p=0,176) and 3 and 4 (p=0,098) (Figure 7).

The mean value of rigidity in the tests was (102,99±17,88)x10³N/m in the group 1, (87,36±12,04)x10³N/m in the group 2, (82,89±19,78)x10³N/m in the group 3 and (71,18±11,47)x10³N/m in the group 4. The simultaneous comparison among all the groups was statiscally different (p<0,001). By comparing the rigidity values, it could be observed that there was a statistically significant difference between the groups 1 and 2 (p=0,004), 1 and 3 (p=0,008) and 1 and 4 (p<0,001). There was a statistic difference between the groups 2 and 4 but there wasn't difference between the groups 2 and 3 or 3 and 4 (Figure 8).

The calculated resilience presented a mean value of (1,041±0,116)x10^-2J in the tests of the group 1 (control), (0,933±0,229)x10^-2J in the group 2, (0,803±0,270)x10^-2J in the group 3 and (0,648±0,335 )x10^-2J in the group 4. The simultaneous comparison among all the groups was statiscally different (p=0,002). There wasn't a statistic difference between the group 1 and 2 (p= 0,168). However there was a difference between the group 1 and the group 3 (p=0,011) and between the groups 1 and 4 (p=0,001). There was a statistically significant difference between the groups 2 and 4, but there wasn't difference between the groups 2 and 3 or 3 and 4. The obtained values between the groups 2 and 3 were (p=0,105), (p=0,002) between the groups 2 and 4 and (p=0,052) between the groups 3 and 4. (Figure 9).

DISCUSSION

The research related to the astronauts' stay in the space shows that a series of musculoskeletal alterations may really occur, even after returning to the Earth. This research can be useful in order to determine prevention measures or a treatment to osteopenia, not only for astronauts but also to patients presenting osteoporosis or needing to rest in bed for long periods^(2,14).

The most important in this research is related to the necessity of understanding and managing the forces that act on the human body such as the muscular tone, the external resistance, the friction. The aim is to identify the risk factors settling prevention and therapeutical measures of rehabilitation adequate to the bone weakness caused by inactivity.

Martin et al.⁽¹⁰⁾ compared the data obtained in animals that underwent suspension. The similarities found in the tests suggest that the suspension model is valid, mainly to the analysis of musculoskeletal alterations.

Our suspension model was developed from Kasper et al.⁽⁸⁾ papers with some changes. An example was the shaft used in the suspension axle. In our pilot study, performed according to the original description, it was smooth, what caused difficulty in controlling the animals' movements since they slid unintentionally even while sleeping. This shaft was then substituted by a twisted one, what enabled a greater stability of the animal during its movements making it easier the suspension axle fixation. The cage size was projected in order to obstruct the animals support in any part of the cage. The system of tail suspension was meticulously developed in order to avoid any lesion in the animal tail.

During the bibliographical research, there were only few papers performing mechanical tests in animals maintained in microgravity simulation environment.

The aim of this paper came from the interest in analyzing the bone alterations occurred during stays in microgravity environment or in periods of physical activity restriction, by the essay of flexion in three points. This essay was chosen because it appears effective in the purposed analysis and because it is simple.

The animals selected for this paper were adult-and young since they present a reasonable size of the tibia and fibula, facilitating the management and fixation of the samples to a better view of the events occurred during the mechanical essay.

The mean weight of each animal was 250 gr., This size was considered adequate for the cage size and for the suspension system developed in this paper.

In Desplanches et al.⁽³⁾ results, the animals presented a higher loss of weight during the first days of suspension but, after fourteen days, most of them restored the lost weight.

In our paper, the analysis of the animals weight variation among the groups and in different periods inter-groups showed that, at first, there was a significant weight loss which is natural and expected because of the stressing condition of the animal. However, after that it occurred a partial recovery showed by the significant weight increase in the group 4, when the mean weight is analyzed at the beginning and at the end of the experiment.

This is clear when we compare the present increase between the final mean weight in the groups 2 and 3. The analysis of the weight in the group 3 (14 days) shows that it is a transitional period.

Summarizing, regarding the weight analysis, our results showed that at first the animal was resentful with the new imposed condition but after that it underwent adaptation and recovered almost totally at the end of 21 days. The same result was observed by Morey-Holton e Globus⁽¹¹⁾ that report maintenance of the animals' weight after the adaptation to the suspension.

The question that follows is if it would be convenient or not to maintain these animals in suspension for longer periods aiming to verify the probable complete recovery of weight. By this measure, probably, it would present complications related to the tail fixation system, that would have to be remade or maybe other complications such as respiratory ones.

The mean weight of the parts (tibia and fibula) obtained for the essay were analyses and the group 1 (control) presented a statistical difference compared to other groups. This can be explained by the probable bone hypertrophy caused by the immobilization.

Analyzing the mechanical properties as a whole, it was verified that, except to the deflection in the elastic system and the maximum limit, there was a decrease in all of them from the first to the fourth group, indicating an overall decrease of the bone resistance to the applied force. This shows the effectiveness of the suspension system in causing the bone weakness, more stressed for the longer period. Therefore the animals finish the experiment present bone weakness. This is an important factor in order to determine the following step in case of evaluating human beings undergoing a rehabilitation process due to a real microgravity condition exposure or due to a long period of rest in bed or immobilization.

This way, the data provide grants to test several programs or rehabilitation techniques in experimental conditions in order to optimize the concepts "shorter time and shorter risk". So, other papers can be developed from this experiment including the drug essay that can act in the bone metabolism.

By detailing the data obtained with the mechanical essay, it was verified that the earlier alteration was the one of the load in the elasticity limit, while the other alterations were later and well characterized in 21 days. The fourteen-day group really represented a transition phase even regarding the body weight and therefore it should not be useful for the definitive analysis.

As the deflection didn't present alterations in 21 days, it can suggest that a longer time of suspension would be more adequate to modify it. From the same reasoning used in the weight analysis, there would be a necessity to test other intervals in order to optimize this period but maybe a limiting factor is the animal tolerance.

However, twenty-one days appear to be a good experimentation period since at the end of this time, important properties such as the maximum load and rigidity will be significantly altered. These properties strictly show the bone weakness and the higher risk of fractures and, in a rude way, are equivalent to the finds of other authors in other circumstances⁽⁹⁾.

The detailed relationship between the type of mechanical property and the microstructure of the bone is still to be determined but overall it depends on the type/quality of the collagen and the mineral part, mainly the calcium. Therefore, it is difficult to explain, regarding the microstructure, what happens to the bone with the suspension that can significantly weaken it but doesn't interfere in its deflection capacity. We think this should be a reason for a new investigation.

In the literature, Morey-Holton and Globus⁽¹¹⁾ mention that microgravity environment or those ones that simulate its effects promote a decrease in the formation and in the reabsorption of the bone. These mechanisms should directly act in the collagen quantity. Other factor that may interfere in the bone resistance is the spacial distribution of the collagen and the formation of the bone gills that, according to Wolff's Law, depends on the mechanical requests.

We only need to find if 21 days are enough to observe these alterations.

Other studies can be performed from the model developed in this paper. As an example, testing longer exposure intervals in suspension, better characterizing the alterations found in calcium and phosphorus metabolism by a metabolic point of view, studying the bone microstructure alterations after the suspension, testing different rehabilitation methods using this studied model and still testing the substances influence that positively act in the bone neoformation such as the dysphosphonates

CONCLUSION

The suspension model developed in this paper showed effectiveness, simplicity and is well tolerated by the animals.

The provoked hypoactivity caused significant decreases in the values of most of the mechanical properties of the bone leading to its weakness.

REFERÊNCIAS BIBLIOGRÁFICAS

Work performed in the Bioengineering Laboratory – USP – Ribeirão Preto

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Publication Dates

• Publication in this collection
  24 June 2004
• Date of issue
  Mar 2004

History

• Accepted
  19 Feb 2004
• Received
  18 Dec 2003