The effect of a 5-day space flight on the immature rat spine

The Spine Journal 2 (2002) 239–243

Clinical Studies

The effect of a 5-day space flight on the immature rat spine Raj K. Sinha, MD, PhDa,*, Suken A. Shah, MDb, Eric L. Hume, MDc, Rocky S. Tuan, PhDc a Department of Orthopaedic Surgery, Division of Adult Reconstruction, University of Pittsburgh Medical Center, 5200 Center Avenue, Suite 415Pittsburgh, PA 15232, USA b DuPont Institute, 1600 Rockland Drive, Wilmington, DE, 19899 USA c Department of Orthopedic Surgery, Thomas Jefferson University, Philadelphia, PA d Cartilage Biology and Orthopaedics Branch, NIAMS, National Institutes of Health, Bethesda, MD, USA Received 11 October 2001; accepted 30 April 2002

Abstract

Background context: Spaceflight has many reported effects upon the musculoskeletal system structure and function. This study was designed to determine the effect of a 5-day flight on the rat spine. Methods: In September 1991, 8 neonatal rats were flown aboard the Space Shuttle Columbia flight STS-48 during a 5-day mission. Upon return to earth, the spines were dissected, frozen and shipped to our laboratory. Matched ground-based rats were used as controls. The spines were radiographed and then slowly thawed. Individual vertebrae were subjected to compressive biomechanical testing using an Instron tester (Instron Corp, Canton, MA, USA) and then processed for determination of calcium and phosphorus content. The intervertebral discs were placed in physiological saline and the stress-relaxation characteristics measured. The discs were then lyophilized and assayed for collagen and proteoglycan content. Disc height on radiographs was measured by image analysis. Results: After space flight, the heights of the discs were found to be 150 to 200 microns greater, although the values were not statistically significant. There was no difference in the resiliency of the thoracic discs as determined by stress-relaxation. However, in the lumbar discs, space flight increased the resiliency (p.01). There was no difference in water content. In both the thoracic and lumbar discs there was a 3.3-fold increase in hydroxyproline–proteoglycan ratio after space flight. However, because of the small sample size, these values were not statistically significant. In the vertebrae, there was no difference in calcium–phosphate ratio or compressive strength. Conclusions: These data suggest that even after a short 5-day flight, the spine begins to undergo biomechanical and biochemical changes. In addition, the weightless environment in space may provide a good model to study the effects of immobilization on earth. © 2002 Elsevier Science Inc. All rights reserved.

Keywords:

Disc, Vertebrae, Biomechanics; Biochemistry; Spaceflight

Introduction With the advent of the space age, the effect of weightlessness on human physiology became of great concern. Initial studies demonstrated that humans readily adapted to weightlessness through a variety of physiological changes [1]. All organ systems are affected. For example, cardiovascular changes include decreased blood volume, cardiac ar-

FDA device/drug status: not applicable. Nothing of value received from a commercial entity related to this research. * Corresponding author. Department of Orthopaedic Surgery, Division of Adult Reconstruction, University of Pittsburgh Medical Center, 5200 Center Avenue, Suite 415, Pittsburgh, PA 15232, USA. Tel.: (412) 8024100; fax: (412) 802-4120. E-mail address: [email protected] (R.K. Sinha)

rhythmias, fluid and electrolyte imbalances and decreased hemoglobin [2–4]. Endocrine abnormalities include altered liver enzyme activity [5] and suppressed pituitary function [6]. In addition, the immune function is compromised by weightlessness, resulting in sluggish granulocyte migration and decreased numbers of lymphocytes [7]. Perhaps the most striking changes have been found with regard to the musculoskeletal system [8]. These include decreased bone formation and loss of bone mass [1,8–12] and muscular atrophy [4,13]. The spine also undergoes dramatic changes when exposed to microgravity. These include increased length of the spinal column [4] and loss of vertebral bone volume [14–16]. The effect on the intervertebral discs, however, has not been well studied. Fluid imbibition has been implicated as the cause for increased spinal length in astronauts [4]. However, there was no difference in swelling

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pressure observed in rat lumbar discs after space flight [1]. Recent studies have described histologic [17] and biochemical changes in intervertebral discs [18,19], although no biomechanical data are currently available. The purpose of this study was to determine the effects of a short 5-day space flight on the biochemistry and biomechanics of spinal components.

Materials and methods In September 1991, 8 neonatal female Sprague-Dawley rats were flown aboard the Space Shuttle Discovery flight STS-48 during a 5-day mission. Upon return to earth, the animals were sacrificed by cervical dislocation. The spines were dissected and frozen in liquid nitrogen at the NASA Ames Research Center and then shipped to our laboratory. Matched ground-based rats were used as controls and were processed in a manner similar to that of the flight animals. These rats had been maintained in the same type of enclosures with similar conditions regarding food and water availability, exercise, light/dark cycles and temperature and humidity, as the space rats. Initially, the intact spines were radiographed. The radiographs were used to measure intervertebral disc height using an image analysis program (CUE-2; Olympus Corporation, Melville, NY, USA). Each disc space was traced with the image analysis software, and nine random measurements of disc height for each disc space were taken. The data were divided into thoracic versus lumbar discs for both control and flight animals. The spines were then slowly thawed and individual vertebrae and intervertebral discs were dissected for biomechanical and biochemical testing. The vertebrae were subjected to compressive biomechanical testing using an Instron tester (Model 1000), and compression load to fracture of the vertebrae was measured. The vertebrae were then lyophilized and processed for determination of calcium content by atomic absorption spectroscopy (Thermo Jarrel Ash Corporation, Franklin, MA, USA) and phosphorus content [20]. The intervertebral discs were allowed to reach an air stable weight, which was recorded. The discs were then placed in phosphate-buffered saline, pH 7.4. While maintained in the phosphate-buffered saline, the discs were subjected to a static, uniaxial load. A load of 15 to 20 N was applied by the Instron tester at a loading rate of 6 mm/min. Once the load reached the targeted range, the crosshead was stopped, and the resultant dissipation of stress in the discs was measured on a polygraph chart recorder. The curves were analyzed on a GBStat statistics program (Dynamic Microsystems, Inc., Silver Spring, MD, USA) on an IBM personal computer. For chemical analysis, the discs were then lyophilized, dry weights were measured and assays to determine collagen [21] and proteoglycan [20,22] content were performed. To estimate the collagen content, hydroxy-proline was quantified. The lyophilized discs were hydrolyzed in 6

N HCl at 130° C for 3 hours, treated sequentially with 0.05 M chloramine T for 20 minutes at room temperature, 3.15 M perchloric acid for 5 minutes at room temperature and then with p-dimethylaminobenzaldehyde for 20 minutes at 60° C, and then A560 was measured [21]. To determine proteoglycan content, the lyophilized discs were separated into two fragments and weighed. One fragment was used to measure hexosamine and hexuronic acid content [20]. The sum of these two measurements was used to estimate proteoglycan content per disc [22]. For hexosamine, the sample was added to 10 l saturated NaHCO3, 10 l reagent A (0.5 g acetic anhydride dissolved in 10 ml water at 4° C), incubated in a boiling water bath for 3 minutes and then cooled to room temperature. Then, 50 l reagent B (1 g K2B4O7.4H2O in 20 ml water) was added, the sample was placed again in a boiling water bath for 7 minutes and cooled to room temperature. Then 500 l glacial acetic acid and 200 l Ehrlich reagent C (16 g p-dimethylaminobenzaldehyde in 95 ml glacial acetic acid and 5 ml concentrated HCl) was added and the sample was incubated at 38 C for 20 minutes. A585 was measured, using 2 g/l glucosamine.HCl as the standard. For hexuronic acid, the lyophilized disc sample was placed in 1,200 l conc. H2SO4, heated for 20 minutes in a boiling water bath and cooled to room temperature. Then 40 l of carbazole reagent (10 mg carbazole diluted to 10 ml with 95% ethanol) was added, and the sample was incubated at room temperature for 2 hours. A535 was measured, using 1 g/l uronic acid in water as the standard.

Results Vertebral bodies The results of the chemical analysis of the vertebral bodies are summarized in Table 1. The vertebrae were grouped into thoracic and lumbar specimens, and flight versus groundbased animals were compared. After a 5-day flight, there was no significant difference in Ca, P or Ca/P ratio. Also, as shown in Table 2, there was a 1.3-fold increase in compressive strength between flight and ground-based groups, although this difference was not statistically significant.

Table 1 Chemical composition of rat vertebral bodies

Thoracic Flight (n13) Control (n10) Lumbar Flight (n9) Control (n14) *p .05.

Ca (g/mg dry weight)

P (g/mg dry weight)

Ca/P

1.47  0.57 2.42  1.58

0.24  0.05 0.25  0.11

6.13  0.62 9.68  1.69

1.47  0.58 1.10  0.51

0.24  0.04 0.27  0.06

4.07  0.57 6.13  0.62

R.K. Sinha et al. / The Spine Journal 2 (2002) 239–243 Table 4 Stress-relaxation measurements of intervertebral discs*

Table 2 Compressive strength of rat vertebral bodies (N/m2) Flight Control

241

Thoracic

Lumbar

3.24  1.3 (n13) 2.44  1.3 (n13)

3.76  1.25 (n13) 2.77  1.73 (n13)

Flight Control

Thoracic

Lumbar

1.95  0.15 (n11) 2.18 0.12 (n11)

1.60  0.85† (n11) 1.27  0.34 (n7)

*The stress-relaxation curve was defined by the equation y1/(abx), where ystress, xtime and bslope of the curve. The slope at the steepest portion of the curve was determined, and these values ( 10 3 are shown. † p  .01.

*p  .05.

Intervertebral discs Using the CUE-2 image analysis program, the height of individual intervertebral discs was measured from a radiograph of the intact spinal columns. These data are shown in Table 3. For both the thoracic and lumbar discs, space flight resulted in an increase of up to 150 to 200 m, or 20% to 30% of the control disc height. Although these differences were quite large, the data were not statistically significant, probably as a result of the small sample size. Because there was a demonstrable change in disc height, it was reasonable to expect differences in mechanical and chemical properties after space flight. Fig. 1, A and B, demonstrate examples of stress-relaxation curves for flight and a ground-based intervertebral disc, with the equation used to describe the curve also shown. The value B represents the slope of the curve at the steepest portion and is used to estimate resiliency of the discs [2], that is, the larger the value for B, the more resilient the disc. Table 4 shows the mean values of B for each group. In the thoracic group, there was no statistical difference. In the lumbar group, space flight resulted in a 1.3-fold increase in resiliency, which was statistically significant (p.01). In order to explain the biomechanical changes, the chemical composition of the discs was studied. The results of the chemical analysis are summarized in Table 5. There was no difference in water content for either the thoracic or the lumbar discs as a result of space flight. However, there was a 25% mean decrease in dry weight of the thoracic discs after flight, although this was not statistically different. There was no difference in dry weight for the lumbar discs. Hydroxyproline content has previously been used to estimate collagen content [21]. After space flight, there was a 3.0fold increase in hydroxy-proline in the thoracic discs and a 1.4-fold increase in the lumbar discs. However, these differences were not statistically significant. Similarly, hexuronic

acid plus hexosamine content has been used to estimate proteoglycan content [22]. In both thoracic and lumbar groups, there was a slight, albeit statistically insignificant, decrease in proteoglycan content after flight. The hydroxyproline–proteoglycan ratio showed a 3.3-fold increase (p.05) in the thoracic discs and a 3.3-fold increase (p.05) in the lumbar discs. These differences in chemical composition correlate with the changes in mechanical properties that were observed.

Table 3 Intervertebral disk height (m)* Flight Control

Thoracic

Lumbar

632  125 (n24) 557  110 (n20)

1,201  146 (n14) 1,025  154 (n16)

*Values were determined by image analysis measurements of disc spaces on plain radiographs of intact spinal columns. p  .05.

Fig. 1. Examples of stress-relaxation curves for flight (A) and control (B) L4-5 intervertebral disks. The X-axis is time in minutes over which stressrelaxation was measured during uniaxial, unconfined fixed stress-loading. The Y-axis is stress-relaxation as recorded by the polygraph chart recorder. The equations in each panel describe the respective curves. The value B represents the slope of the curve at the steepest portion, and is used to estimate resiliency of the disks14, i.e. the larger the value for B, the more resilient the disk. Thus, this figure demonstrates that the flight sample disk appears to be more resilient.

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Table 5 Chemical analysis of intervertebral discs

Thoracic flight Control Lumbar flight Control

Dry weight* (mg)

% Water† (%ASW‡)

Proteoglycan§ (g)

Collagen (g)

Collagen/ proteoglycan¶

1.8  0.9 (n11) 2.5  1.5 (n11) 2.3  0.9 (n11) 2.6  1.2 (n8)

71.5  8.3 (n11) 60.5  10.0 (n11) 74.2  9.0 (n11) 76.4  8.1 (n8)

0.66  0.27 (n11) 0.81  0.38 (n10) 0.83  0.35 (n11) 01.91  0.49 (n8)

5.04  4.23 (n10) 1.66  1.21 (n10) 3.15  1.98 (n10) 2.22  2.56 (n6)

6.11  3.75 (n9) 1.88  1.12 (n10) 4.57  3.62 (n10) 1.36  0.81 (n6)

*Dry weight was measured after lyophilization of intervertebral discs. ASW  air stable weight. ‡ Percent water content was determined by the formula [(ASW dry weight)/ASW] 100. § Proteoglycan content was estimated by the sum of the hexuronic acid and hexosamine contents (for additional details, see Materials and Methods).  Collagen content was estimated by hydroxyproline content (for additional details, see Materials and Methods). ¶ The values for collagen/PG ratio were calculated for each intervertebral disc sample for which both values were available. Values shown in the table reflect the mean of these samples. p.05. †

Discussion The effect of prolonged microgravity is not fully understood. Despite nearly 30 years of space flight and experimentation, many of the data obtained have been inconclusive [23]. The use of human subjects has severe limitations, such as small sample size and poor accessibility to astronauts because of preflight preparations and postflight precaution [23]. The large body of data that exists is difficult to interpret, because missions have been of varying duration, tissues were harvested and processed in different manners and experimental techniques were not uniform. In addition, because of cost and the relatively small number of flights, very few studies are reproducible. Nevertheless, attempts must be made to use the existing data to predict the effect of long-term weightlessness on humans in space, especially with long missions planned for the near future. In order to allow the safe return of space travelers, the effects of microgravity must be minimized or reversed. Although most adaptations are merely minor disturbances for astronauts, evidence clearly indicates that changes steadily progress with continued exposure to microgravity [1]. During short space flights, minor changes are readily reversed upon return to earth. However, with prolonged exposure to zero gravity, some changes, such as severely osteopenic skeletons, could make return to earth functionally impossible. However, the precise relationship between length of exposure and the reversibility of physiologic effects must be clearly delineated. Previous studies have shown that a 14-day space flight resulted in the inhibition of bone formation and increased bone turnover in the thoracic vertebrae [16]. Furthermore, these parameters did not return to normal after a 14-day recovery period on earth. Another study identified no difference in vertebral hardness or bone mineral density in rat vertebrae after a 14-day flight [15]. Histology revealed a decrease in lamellar bone after space flight. Other reports showed that bone formation in vertebrae was reduced and

that osteoclastic activity was possibly increased [11,12,14] after flights of 12 to 13 days. In the current study, a relatively short 5-day flight had no significant effect on compressive strength or chemical composition, either in thoracic or lumbar vertebrae. With regard to the discs, Pedrini-Mille et al. [18] have recently described significant biochemical changes, including decreased wet and dry weights and increased collagen–proteoglycan ratio, after 14 days in space. Maynard [19] also found lower wet and dry weights of annuli, and an increased collagen–proteoglycan ratio after a 14-day flight. We showed similar results after only 5 days, suggesting that alterations in the spine clearly progress with continued exposure to weightlessness. In addition, we showed changes in resiliency of the intervertebral discs, possibly explained by the concomitant alterations in water, collagen and proteoglycan content. For example, degenerative discs have been shown to have an altered collagen–proteoglycan ratio and have an associated increase in stiffness and less ability to absorb shock [24]. In addition, previous studies also have shown increases in length of the spinal column of up to 2.7 inches [4] and backaches in astronauts after space travel [4,8]. Thus, there is a need to understand more thoroughly the effect of space flight on the discs. Future studies on the spine should focus on whether biochemical and biomechanical changes brought about by prolonged weightlessness or immobilization predispose discs or other components of the spinal column to injury.

Acknowledgments The authors thank the NASA recovery and dissection teams and Dr. Thora Birch and her colleagues at the NASA Ames Research Center for providing tissues for this study, as well as for the opportunity to participate in the PARE.01 experiments.

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