bone blood flow after spinal paralysis in the rat

Journal of Orthopaedic Research 8393-400 Raven Press, Ltd., New York 0 1990 Orthopaedic Research Society

Bone Blood Flow After Spinal Paralysis in the Rat

Hiroshi Takahashi, Takao Yamamuro, Hideo Okumura, Ryuichi Kasai, and Kenji Tada

Department of Orthopaedic Surgery, Faculty of Medicine, Kyoto University, Kyoto, Japan

Summary: The goal of this study was to investigate the acute and chronic effects of paralysis induced by spinal cord section or sciatic neurotomy on bone blood flow in the rat. Regional bone blood flow was measured in the early stage with the hydrogen washout technique and the change of whole bone blood flow was measured in the early and the late stages with the radioactive microsphere technique. Four to 6 h after cordotomy at the level of the 13th thoracic vertebra, the regional bone blood flow in the denervated tibia increased significantly (p < 0.01). After hemicordotomy with rhizotomy at the same level, the regional bone blood flow in the denervated tibia increased significantly (p < 0.05) 6 h postoperatively. The whole bone blood flow in the denervated tibia had also increased significantly (p < 0.05) at 6 h and at 4 and 12 weeks postoperatively. After sciatic neurotomy, the regional and the whole bone blood flow in the paralytic tibia did not change significantly. The present study demonstrated that monoplegic paralysis caused an increase in bone blood flow in the denervated hind limb from a very early stage. It was suggested that the spinal nervous system contributed to the control of bone blood flow. Key Words: Bone blood flow-Paralysis-Hydrogen washout technique-Radioactive microsphere technique-Rat.

Paralysis induced by sciatic nerve section or spinal cord injury was reported to affect fracture healing of the denervated bone (5,6). Though the relation between bone metabolism and bone circulation remains uncertain, it is interesting to investigate changes of bone circulation after paralysis. The effect of denervation on bone blood flow in sciatic neurotomy, sympathectomy, and spinal cord section with paraplegia is documented (2,3,8,11, 12,15,16). Several authors have reported an increase in blood flow in the denervated bone after paralysis induced by sciatic neurotomy or sympathectomy (3,11,12,15), and the existence of an autonomic nerve supply to bone (4). The early change of blood flow in the denervated bone is controversial

Received January 30, 1989; accepted September 7, 1989. Address correspondence and reprint requests to Hiroshi Ta-

kahashi, M.D., Department of Orthopaedic Surgery, Faculty of Medicine, Kyoto University, 54 Shogoin Kawara-cho, Sakyo- ku, Kyoto 606, Japan.

(2,8), however, possibly because investigators have used different methods of measuring blood flow and have studied the blood flow in various phases after paralysis. If paralysis directly causes an alteration of bone blood flow, the effect should be immediate and proportionate to the extent of denervation.

The purpose of this study was to investigate the early changes of bone blood flow after spinal paralysis and the late changes of bone blood flow in as- sociation with the immobilization after paralysis. In particular, the effect of monoplegic paralysis induced by hemicordotomy with rhizotomy on bone blood flow was studied, allowing a comparison to be made between the denervated and nondenervated sides. In order to measure bone blood flow in the denervated limb, the hydrogen washout technique and *%--labeled microsphere technique were applied; the former is useful for measuring regional bone blood flow continuously and the latter reflects change of whole bone blood flow.

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H . TAKAHASHI ET AL.

MATERIALS AND METHODS

Seventy-two female Wistar rats, 7-8 months old, weighing 240-300 g were used in this study. The rats were divided into four groups (Table 1): (a) sham operation group as a control, (b) sciatic neurotomy group, (c) hemicordotomy with rhizotomy group (monoplegic paralysis group), and (d) cordotomy group (paraplegic paralysis group). In the sham operation group, the spinous process of the 1st lumbar vertebra was resected. In the sciatic neurotomy group, the right sciatic nerve was resected for 10 mm from the back of the hip joint. In the hemicordotomy with rhizotomy group, the right hemisec- tion of the spinal cord and the rhizotomy of the right nerve roots at the level of the 13th thoracic vertebra (Th,,) were performed after the laminectomy. In the cordotomy group, the spinal cord transection at Th,, level was performed after the laminectomy. We measured the regional bone blood flow continuously with the hydrogen washout technique for 6 h after the sham, sciatic neurotomy , hemicordotomy with rhizotomy, and cordotomy operations. We also measured the whole bone blood flow by using the 85Sr-labeled microsphere technique at 6 h and, at 4 and 12 weeks after the sham, sciatic neurotomy, and hemicordotomy with rhizotomy operations.

Before the measurement of the bone blood flow, the rats were anesthetized with urethane 1000 mgl kg i.p. and atropine sulfate 0.05 mglkg i.m.; the body temperature was maintained at 363°C using a heat lamp and was monitored by a rectal thermom- eter. No transfusion was performed. Prior to the sham, sciatic neurotomy , and hemicordotomy with rhizotomy operations for the 4- and 12-week groups, the rats were anesthetized with ethyl ether and sodium pentobarbital. After the operation the rats were kept in cages, the bottoms of which were

covered with wood shavings. Rats were fed a stan- dard laboratory diet containing 1.20% calcium, 0.96% phosphorus, and an adequate amount of vi- tamin D. Food and water were given ad libitum to all rats during the experimental period.

Regional bone blood flow in the proximal tibial metaphysis was measured with the hydrogen washout technique (1). The proximal metaphyses of the right and left tibias were exposed surgically, with minimal muscle dissection, and 0.3-mm diameter drill holes, 1.5-2.0 mm in depth were made. Plati- num electrodes (0.3 mm diameter) insulated except for the tip (Unique Medical Co., Ltd., Tokyo, Ja- pan) were inserted into the drill holes. An Ag-AgC1 reference electrode was inserted into subcutaneous tissue of the rat's back. A mixture of 90% air and 10% hydrogen gas was administered through a face mask and discontinued after a suitable level of hydrogen concentration was reached. The washout curves were recorded with a pen recorder (PHG type 201; Unique Medical Co., Ltd., Tokyo, Japan) and plotted on a semilogarithmic scale. The half-life obtained from the decay pattern was used to determine the bone blood flow rate, using the following equation (1,17):

F = 0.693X/ti/2

where

F = bone blood flow in mVmid100 ml X = partition coefficient of hydrogen between

blood and bone t,,, = half-life of decay

The partition coefficient value for the metaphysial bone of the rat has not been reported, and the rabbit metaphysial bone value (X = 0.74) reported by Whiteside and colleagues was utilized instead (17). The hydrogen washout curve in the rat showed

TABLE 1. Experimental design

Group"

Experiment l b Experiment 2'

For 6 h after operation At 6 h At 4 wks At 12 wks

Sham operation 8 rats I rats 5 rats 5 rats Sciatic neurotomy 5 5 5 5 Hemicordotomy with rhizotomy 5 5 I 5 Cordotomy 5 - - -

" n = 72 rats. Total 72 rats. Measurement of regional bone blood flow of right and left proximal tibial metaphyses by hydrogen washout

Measurement of whole bone blood flow of tibia by radioactive microsphere technique (ratio of right to left technique.

tibial whole bone blood flow).

J Orthop Res, Vol. 8 , No. 3, 1990

BONE BLOOD FLOW AFTER PARALYSIS 395

a monoexponential decay pattern when plotted on a semilogarithmic scale, indicating homogeneous perfusion around the drill holes (Figs. 1 and 2) with the respiration and systemic circulation unchanged. The bone blood flow rate was measured at least twice preoperatively, and hourly for 6 h after the operation.

To investigate whole bone blood flow in the tibia, we used the radioactive microsphere technique (7,13,14,16). A polyethylene catheter (PE 10; Clay Adams Co., Inc., Parsippany, NJ, U.S.A.) was in- troduced into the left ventricle of the heart through the right carotid artery. Approximately 220,000 microspheres (carbonized '%r-labeled microspheres, 15 pm diameter; 3M Co., Inc., St. Paul, MN, U.S.A.) each having a specific gravity of 1.3, sus- pended in a 10% dextran solution, were injected into the left ventricle in a 0.05-ml volume within 10 s, followed by 0.10-ml saline in order to flush microspheres in the tube into the left ventricle. Suffi- cient shaking by a vortex shaker and Tween-80 were used to prevent microsphere aggregation. The rat was sacrificed with a rapid injection of saturated KCl into the heart. Right and left tibias were dis- sected free from soft tissue and radioactivity was measured in a well-type gamma scintillation counter (Minaxi 5550; Packard Co., Inc., Packard Co., Inc., Downers Grove, IL, U.S.A.). The ratio of right to left tibial bone radioactivity per wet weight was cal- culated to determine the ratio of right to left tibial bone blood flow.

The volume of the tibias was determined by loss of weight of the hydrated tibias when weighed in water. Ash weight of the tibias was measured after heating the bone in an electric furnace at 680°C for 20 h. In order to investigate the demineralization of the denervated bone in each group at 4 and 12 weeks, ash weight per volume of tibia was calcu- lated.

_____ L e f t tibial metaphysis

- Right tibial metaphysis

0 1 2 3 4 5 6 7 8 9 10 11 12 13 Minutes 1- Administration of a mixture o f 90% air and 10% hydrogen gas

FIG. 1. Hydrogen build-up and washout curves of proximal metaphyses of right and left tibias in a nonoperated rat.

[:;;I o---e Left tibial rnetaphysis

1 1 1 1 1 1 1 1 1 1

0 1 2 3 4 5 6 7 8 910 M i n u t e s

FIG. 2. Hydrogen washout curves on a semilogarithmic scale showing a monoexponential decay pattern (same data used as in Fig. 1).

The analysis of variance and Scheffe's method were used for statistical analysis to compare the results.

RESULTS

The pre- and postoperative mean bone blood flow rates measured with the hydrogen washout technique in the early stage are shown in Table 2. In the sham operation and sciatic neurotomy groups, no changes were observed in the regional bone blood flow for 6 h after the operation. In the hemicordotomy with rhizotomy group, the regional bone blood flow rate in the right monoplegic side increased significantly (p < 0.05) at 6 h after the operation compared with control group, but not in the nondenervated side. In the cordotomy group, the regional bone blood flow rates increased significantly (p < 0.01) from 4 to 6 h postoperatively compared with control group. The ratios of right to left whole tibial bone blood flow in each group are shown in Fig. 3. In the sham operation group, the ratios were 1.005 t 0.047 at 6 h, 0.999 2 0.074 at 4 weeks, and 1.004 2 0.089 at 12 weeks, which indi- cated that whole tibial bone blood flows in the right side were nearly equal to those in the left side. In the sciatic neurotomy group, the ratios were 1.083 t 0.095 at 6 h, 1.058 ? 0.064 at 4 weeks, and 1.168 t 0.058 at 12 weeks, which showed a slight increase

J Orthop Res, Vol . 8, No . 3, 1990

396 H. TAKAHASHI ET AL.

a 1.8 - 1.7 - 1.6 - 1.5 - 1.4 - 1.3 - 1.2 - 1.1 - 1.0 - 0.9 - 0.8 - 0.7 -

TABLE 2. Pre- and postoperative regional bone bloodflow rates of right and left proximal tibial metaphyses, measured by hydrogen washout technique

b

I

Hemicordotomy with rhizotomy (n = 5 ) Sham operation (n = 8) Sciatic neurotomy (n = 5) Cordotomy (n = 5)

Group Right Left Right Left Right Left Right Left

Control 13.82 f 2.06 13.30 f 2.03 12.71 i 2.08 12.98 ? 2.21 12.67 f 1.84 12.30 i 2.11 13.18 f 2.33 14.95 f 1.15 l h 14.02 f 2.29 13.90 f 2.27 13.22 f 1.80 12.10f 2.09 11.07 f 2.44 9.51 f 2.78 12.91 ? 2.35 15.18 2 0.37 2 h 13.72 f 2.00 13.50 i 2.06 13.28 f 2.03 12.66 f 2.62 12.90 * 1.81 10.51 f 2.87 14.99 f 1.90 17.18 t 1.42 3 h 13.85 i 2.22 13.72 f 1.93 13.34 f 1.86 12.86 i 2.46 14.28 i 1.45 11.29 f 2.33 16.22 5 2.08 18.29” f 1.57

5 h 13.51 f 1.79 13.17 f 2.09 13.17 f 2.01 12.96 t- 2.14 16.95 f 1.33 13.13 2 2.89 19.84’ f 3.07 22.21’ f 2.88 6 h 13.81 f 2.22 13.08 f 2.10 13.03 f 1.92 12.56f 1.82 17.53O f 1.39 12.95 f 2.90 19.67’ f 2.37 22.216 f 2.88

4 h 14.01 f 2.40 13.83 f 2.12 13.18 f 1.74 12.82 f 2.92 15.53 f 1.87 12.57 * 3.06 18.40n i 2.75 20.58’f 2.53

Values expressed as mean i SD (mVmini100 ml). p < 0.05. Significant differences from the same side of the sham operation group. p < 0.01. Signifcant differences from the same side of the sham operation group.

of the right whole tibial bone blood flow. In the hemicordotomy with rhizotomy group, the ratios were 1.358 k 0.167 at 6 h, 1.399 k 0.266 at 4 weeks, and 1.223 k 0.128 at 12 weeks. Each value was statistically significant at all time points (p < 0.05).

The ash weight per volume of the tibias in each group is shown in Table 3. No change was observed in the sham operation group. In the sciatic neurotomy group, a significant decrease was observed in the denervated tibia after 12 weeks (p < 0.05). In the hemicordotomy with rhizotomy group, significant decreases were observed in the denervated tibias after 4 and 12 weeks (p < 0.01).

r

c

a

b

C

z € f

* p< 0.05 ( Mean * S.D.)

FIG. 3. Whole bone blood flow of tibia, expressed by the ratio of right to left tibial whole bone blood flow, measured with the ”Sr-labeled microsphere technique. a: Six hours after operation. b: Four weeks after operation. c: Twelve weeks after operation. 0, sham operation group; A, sciatic neurot- orny group; *, p < 0.05 (Mean * S.D.). 0, hernicordotorny with rhizotomy group. N = 5 in every group, except sham operation group after 6 h (N = 7) and hernicordotorny with rhizotomy group after 4 weeks (N = 7).

DISCUSSION

Several investigators have reported an increase of blood flow in the denervated bone after sciatic neurotomy or sympathectomy , and most authors have agreed that the sympathetic nervous system exerts control over bone blood flow (3,11,12,15). But the effect of paralysis on bone blood flow in the early stages has been controversial. McElfresh and Kelly showed there was no immediate increase of bone blood flow following a midthigh sciatic nerve section (8). Davies and colleagues also showed there was no immediate increase of bone blood flow after sympathectomy (2). These negative findings may be due mainly to the difficulty in measuring blood flow at various phases of paralysis. In order to elucidate the relation between bone circulation and paralysis, we studied the effect of paraplegic and monoplegic paralysis on bone blood flow. Spi- nal cord section induces rapid and complete denervation below the lesion. in our previous study, a rat with monoplegia induced by hemicordotomy with rhizotomy was established as a model of immobilization osteopenia (9,lO). A monoplegic animal is experimentally superior to a paraplegic animal because the denervated and nondenervated side can be compared; in addition, the animal does not lose total bladder function and is easily kept in cages.

We used the hydrogen washout technique and the radioactive microsphere technique to measure bone blood flow, because the former is very suitable for continuous measurement of the regional bone blood flow and the latter reflects whole bone blood flow. The practical application of the hydrogen washout technique to the bone in the rat has not been reported. Therefore, we examined the applicability of this technique in a preliminary experiment and we

J Orthop Res, Vol. 8, No. 3 , 1990


TABLE 3. Ash weight per volume of tibias in experimental groups 4 and 12 weeks after operation

Group Period after operation

(wks) n Right tibia Left tibia

Sham operation

Sciatic neurotomy

Hemicordotomy with rhizotomy

4 5 822 2 30 (mg/ml) 826 2 35 (mg/ml) 829 2 18 12 5 827 4 14

830 2 25 4 5 792 2 36 818 2 41 12 5 767 2 38"

4 7 757 2 26' 814 2 24 8 2 0 2 13 12 5 712 4 43'

Values expressed as mean 2 SD. " p < 0.05. Significant differences from the same side of the sham operation group. ' p < 0.01. Significant differences from the same side of the sham operation group.

confirmed homogeneous perfusion around the drill holes (Figs. 1 and 2). There are several reports of the application of the radioactive microsphere technique in the rat (13,14,16). In the preliminary studies, we confirmed a sufficient mixing of microspheres in the bloodstream by examining the equal distribution of microspheres in the right and left kid- neys. It was also confirmed that microspheres were completely entrapped in the vascular bed, which was demonstrated by a negligible radioactivity in the venous blood aspirated from the femoral vein immediately after injection of microspheres into the left ventricle. Finally, histological examination re- vealed that the injected microspheres were entrapped properly in the vascular beds of bone cor- tex, bone marrow, and kidney (Figs. 4-6).

The preoperative mean bone blood flow rate mea-

sured with the hydrogen washout technique in the sham operation group was 13.82 rt 2.06 mVmid100 ml in the right and 13.30 f 2.03 ml/min/100 ml in the left tibial proximal metaphysis. The values of the present study approximate those of Shoutens and colleagues who reported that plasma flow in the tibia of the rat was 8.27 rt 3.15 ml/min/100 g wet weight using the "Sr-labeled microsphere technique (13). Whiteside and colleagues reported the tibial proximal metaphyseal bone blood flow value in the rabbit to be 17.0 ? 1.4 ml/min/100 ml, which is higher than the value in our study (17). This may be due to the difference in animal species.

In the paraplegic rat, the regional bone blood flow rate in the denervated tibia increased significantly at 6 h postoperatively (approximately 50% above the preoperative control values). The slope of wash-

FIG. 4. Photomicrograph of a tibia after injection of microspheres. A microsphere (arrow) is lodged in the proximal metaphysis. (H & E x 260)

J Orthop Res, Vol. 8 , No. 3 , 1990

398

FIG. 6. Photomicrograph of a kidney after injection of microspheres. Microspheres (arrow) are lodged in capillaries in the glomerulus. (H & E x130)

H . TAKAHASHI ET AL.

FIG. 5. Photomicrograph of a tibia after injection of microspheres. A microsphere (arrow) is lodged in the cortical bone. (H & E x154)

J Orthop Res, Vol. 8, No. 3, 1990


a

--__- L e f t tibial metaphysis

b 0 10 Minutes

----- L e f t tibial metaphsis

Right tibial metaphysis

0 10 Minutes

FIG. 7. Hydrogen buildup and washout curves of proximal metaphyses of right and left tibias in a paraplegic rat caused by cordotomy. a: Preoperative control. b: Six hours after operation.

out curves at 6 h in both the right and left sides became steep compared with the washout curves of the control (Fig. 7). This change of slope means an increase of bone blood flow. In the monoplegic rat 6 h postoperatively, the regional bone blood flow in the monoplegic side showed a significant increase (38.4% above the preoperative control value). The slope of the washout curve in the monoplegic side also became steep (Fig. 8). The whole bone blood

a W2lj IH,ll ----- L e f t tibial metaphysis

0 10 Minutes b

----- L e f t tibial metaphysis

%---..- - Right tibial metahysis

0 10 Minutes

FIG. 8. Hydrogen buildup and washout curves of proximal metaphyses of right and left tibias in a monoplegic rat caused by right hemicordotomy with rhizotomy. a: Preoper- ative control. b: Six hours after operation.

flow in the monoplegic side increased significantly (35.8% above the left). Therefore, it is clear that from a very early stage of monoplegic or paraplegic paralysis, the blood flow in the denervated limb increased. The present results also showed that less extensive paralysis after sciatic neurotomy could not affect bone blood flow so much as in monoplegic paralysis. In the sciatic neurotomized rat, the increase of bone blood flow at 6 h was less extensive than after hemicordotomy and was not statistically significant from the control. The difference in bone blood flow observed between the monoplegic and the sciatic neurotomized rats in the acute stage may be due to a difference in the adrenergic innervation of bone.

At 4 weeks and 12 weeks in the sciatic neurotomized rat, whole bone blood flow in the denervated tibia increased slightly but not significantly, while in the monoplegic rat whole bone blood flow in the denervated tibia increased significantly: 33% higher than bone blood flow in the nondenervated tibia at 4 weeks and 24% higher than that at 12 weeks (Fig. 3). From 4 weeks to 12 weeks the blood flow value in the denervated tibia in the monoplegic rat had a tendency to return to normal level. Our results showed that monoplegic paralysis caused a rapid and marked increase in bone blood flow in the denervated area, but in the sciatic neurotomized rat a significant change of bone blood flow was not rec- ognized. The monoplegic rat lost all voluntary motion in the denervated hind limb, while the sciatic neurotomized rat retained some motion in the proximal part of the hind limb. The difference in bone blood flow observed between the monoplegic and the sciatic neurotomized rats in the late stage might be due to differences in both the adrenergic innervation of bone and the motion. It was suggested that the spinal cord section caused complete denervation of the intraspinal nervous system which caused severe vasodilatation and increase of blood flow in the bone from the very early stages.

A significant bone loss was observed in the denervated tibia. In the sham operated rat, ash weight per volume of tibia showed no change at 4 and 12 weeks after operation. In the sciatic neurotomized rat, ash weight per volume of denervated tibia decreased 4.6% at 4 weeks and 6.3% at 12 weeks after operation compared with the nondenervated tibia (Table 3). In the monoplegic rat, ash weight per volume of denervated tibia decreased 7.1% at 4 weeks and 13.2% at 12 weeks after operation compared with the nondenervated tibia (Table 3). It was clear that the bone loss in the monoplegic rat was


400 H . TAKAHASHI ET AL.

more severe than in the sciatic neurotomized rat. Verhas and colleagues, using microdensitometry and the *?3r-labeled microsphere technique, reported demineralization and an increase in bone blood flow in the hind limbs 4 and 12 weeks postoperatively in the rat with paraplegia induced by cordotomy at the level of Th,, (16). In our experiment, the later result for the monoplegic rat was in accordance with Verhas’ result. The decrease of bone mass in the paralytic hind limb may be due to the change of bone blood flow and other factors such as a change in activity after spinal paralysis, though there is no proof of a close relation between bone resorption and increased bone blood flow.

In conclusion, the spinal nervous system contrib- utes to the control of bone blood flow, which was demonstrated by a rapid and marked increase of bone blood flow at an early stage after spinal cord section.

Acknowledgment: This study was supported by Grant No. 58570615 from the Ministry of Education, Science and Culture of Japan.

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bone blood flow after spinal paralysis in the rat

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