Journal of Orthopaedic Research 9104-112 Raven Press, Ltd., New York 0 1991 Orthopaedic Research Society
Variables Affecting Disc Size in the Lumbar Spine of Rabbits: Anesthesia, Paralysis, and Disc Injury J. Hulse Neufeld, *T. Machado, and tL. Margolin V A Medical Center, *Department of Radiology and ?Animal Research Facility, San Diego, California, U.S.A.
Summary: Methods have been developed that permit repetitive radiographic measurement of the lumbar intervertebral disc space in a rostral-caudal direction (width) in the anesthetized laboratory rabbit. Using isolated control discs and injured discs in which narrowing has been induced for chronic and acute periods, the widths of the lumbar intervertebral disc spaces determined radiographically correlate with widths determined histologically (p < 0.000, r = 0.75). Both an increase (widening) and a decrease (narrowing) in disc width were observed using radiography after different experimental treatments. Anesthesia and lower-body paralysis (an experimentally induced inability to bear weight on and to perceive a pinch stimulus in hind limbs) caused widening of the discs: anesthesia causing a general widening throughout the lumbar spine and lower-body paralysis causing a specific widening low in the lumbar spine. Both disc injection and piercing the disc with needles to recover nucleus pulposus material caused narrowing of the discs. Acridine-orange injection induced a narrowing accompanied by osteophytosis. Experimentally induced narrowing at L4-5 (the result of injury to the disc) resulted in narrowing also at L2-3. These findings are consistent with the hypothesis that in vivo disc-width size in the young rabbit depends on both the quantity of nucleus pulposus material and the force-generating activities of the adjacent spinal muscles, and that disc injury at one level stimulates narrowing at other levels. Key Words: Intervertebral disc size-Rabbit-Radiography-Histology-Induced widening-Induced narrowing.
The purpose of this study was to determine if an induced change in one disc of a rabbit could be observed with radiography and confirmed by histology. In dogs (3,7) and monkeys (19), radiography has been used to document the narrowing of disc width after the injection of nucleolytic substances. In rabbits radiography has been used to document osteophyte formation after lumbar discs had been injured by incising the annular fibers of two or more discs, with a resulting prolapse of the nucleus pulposus (13). If disc width depends on the integrity of annular fibers and/or nucleus pulposus, incising the annular fibers and subsequent anterior prolapse of the nucleus should result in disc narrowing. A discinjury method that minimizes annular-fiber disrup-
It has often been stated that, as an intervertebral disc narrows, the articulating facets telescope or subluxate (2,5,14,16). This study describes how disc widths (rostral-caudal dimension) of abnormal size were produced and measured in rabbits. As a result, researchers can have rabbit populations in which to measure facet position in vitro as a function of disc size. Received November 13, 1989; accepted July 1 1 , 1990. Address correspondence and reprint requests to Dr. J. Hulse Neufeld, Research Associate, at VA Medical Center (151), 3350 La Jolla Village Drive, San Diego, CA 92161, U.S.A. Presented in part at the 35th Annual Meeting, Orthopaedic Research Society, February 6 9 , 1989, Las Vegas, Nevada, U.S.A.
104
105
DISC SIZE IN RABBIT LUMBAR SPINE
tion (needle puncture rather than scalpel incision) was used so that, if disc narrowing were induced, it could be attributed to loss of nucleus pulposus and not to disruption of the annular fibers. To determine the relationship between radiographically and histologically measured disc width, rabbit lumbar intervertebral discs from control and injured specimens were prepared for histological examination after radiography of posttreatment lumbar spines in anesthetized rabbits. Also the importance to disc width of (a) the quantity of nucleus pulposus material and (b) the force-generating activities of the spinal muscles was determined from radiography of lumbar spines before and after experimental treatments. The relationship of disc width to muscle activity was studied indirectly by measuring disc width at different planes of anesthesia and before and after lower-body paralysis. MATERIALS AND METHODS Animals and Animal Care Rabbits of a New Zealand strain were housed at an ambient temperature and a humidity standard for rabbits. Food and tap water were available ad libitum. Experimental and control rabbits were picked at random from 49 rabbits aged 3 to 5 months and weighing 2.5 to 3.5 kg. Lumbar disc width was studied with radiography under ketamine-rompun-acepromazine (KRA) anesthesia (26 normal rabbits) or under 2% halothane anesthesia (23 normal rabbits). (See Experimental Design below for explanation.) Rabbits initially studied under KRA anesthesia either were studied again later (results, Table I), or they were anesthetized to surgical levels with KRA TABLE 1. Mean disc width and average coefficient of variation in measuring disc width from radiographs of anesthetized rabbits
and placed into one of six experimental, sham, or control groups: (a) surgical exposure of the L3-4 or L4-5 disc and acridine-orange injection (5 rabbits); (b) surgical exposure of the L4-5 disc and diluent injection (2 rabbits serving as shams to acridineorange injection); (c) surgical exposure of the L4-5 disc and closure (1 rabbit of control group); (d) fluoroscopic guided-needle puncture of the L4-5 disc with nucleus pulposus recovery (6 rabbits); (e) fluoroscopic guided-needle puncture of the L4-5 disc without nucleus pulposus recovery (2 rabbits serving as shams to needle puncture with recovery); or (f) fluoroscopic guided-needle puncture up to but not into the L4-5 disc (1 rabbit of control group). The other nine rabbits were used in experiments not reported here or they died. Four of the 26 rabbits studied under KRA anesthesia were measured repeatedly over 42 to 76 days before the experimental treatments. Rabbits initially studied with 2% halothane either were studied again later or were placed into one of two experimental groups: (a) aortic occlusion (of 19 attempts, 12 rabbits were paralyzed); or (b) a sham procedure for aortic occlusion (2 rabbits). The other two rabbits were used in an experiment not reported here. Four of the 23 rabbits studied under 2% halothane were measured repeatedly at a 1-day interval before experimental treatments. Paraplegic rabbits were cared for twice daily by a nursing routine of expressing the urinary bladder, handwashing the lower body, and placing the rabbit on disposable liners to cushion the rabbit’s contact with the cage. Parasthesias were evident 1 week postparalysis as chewing on hind limbs. Such selfmutilation was prevented by putting wide collars around the rabbits’ necks and gauze booties on their hind paws. Methods of Quantifying Disc Space Size Radiographic Procedure
Anesthetic agent KRAa Disc width
(mm) One occasion‘ More than one occasiond
-
2% Halothane CVb (%)
Disc width (mm)
CVb (%)
1.36 5 0.08
4.3
2
0.6
1.05 ? 0.12
6.2 2 1.8
1.34 2 0.05
5.4
?
0.9
1.02 ? 0.11
5.2
2
2.8
Values are 2 S D (disc width) and 5SEM (coefficient of variation). Ketamine-rompun-acepromazinemixture. Coefficient of variation. Within rabbits on one occasion of anesthesia and radiography. Within rabbits on more than one occasion of anesthesia and radiography.
Details of the radiographic and histologic procedures have been described elsewhere for use with rats (9,ll). In this study a rabbit was positioned correctly by placing foam-rubber supports beneath the lumbar area of the back and between the knees. Magnified views of the lumbar discs were obtained by magnification radiography (6). Magnification was 2.5 to 3.5 0.25 and was achieved by having a focal spot-film distance of 120 ? 5 cm and an objectfilm distance of 80 ? 5 cm; the microfocal spot x-
*
J Orthop Res, Vol. 9, No. 1 , 1991
J . H . NEUFELD ET AL.
106
ray tube (0.4 mm magnification tube; Eureka, Chicago, IL, U.S.A.) was employed at 85 peak kilovoltage (kVp) and 160 milliamperes (mA) for 1.6 s. The degree of magnification was determined with a magnification control (MC) consisting of a metal clip with a hole 1.O k 0.1 mm in diameter as determined with a X 6 hand lens calibrated to 0.1 mm (Leitz Wetzlar, Germany). Repetitive measurements of a rabbit’s lumbar intervertebral disc spaces were made by repositioning the rabbit and the MC between determinations. The disc space width (mm) was determined by direct measurement on radiographs (Fig. 1). In summary, the average of the three middle lines between endplates was corrected for magnification and used to measure the disc width (1 1). Histological Technique
Histologic samples embedded consisted of two halves of two adjacent vertebral bodies and associated intervertebral disc and articulating facets (motion segments). Of the eight rabbits euthanized to obtain the motion segments used for histologicradiographic correlation, six had radiographic evidence of disc narrowing for more than 3 months and two had no radiographic evidence of disc injury. In this study, 27 motion segments from eight euthanized rabbits were used for histologic-radiographic correlation. The samples had a wide range of disc widths in order to increase the ease of obtaining a significant relationship (18). The spines from an-
other four rabbits were fixed in extension, flexion, or lateral flexion to obtain 12 motion segments to be used to determine the effect on disc width of the spine’s position during fixation. Discs that had lesions with osteophytes were measured after the outer marks approximating the disc were drawn between the presumed original edges of the vertebral epiphyses (Fig. 2B, line c). The depths of the indentations on the superior and inferior vertebral endplates in eight specimens were measured to compare disc width determined radiographically with that determined histologically before and after the indentation depths were subtracted. An indentation was measured as the perpendicular distance from the deepest part of the indentation in an endplate to a line between the edges of the indentation. Experimental Treatments Evidence for both disc widening (Figs. 3 and 4) and disc narrowing (Figs. 5-7) was observed using radiography after different experimental treatments. Widening of lumbar discs was induced by anesthesia or by lower-body paralysis; narrowing of lumbar discs was induced by disc injury (injection of acridine orange in diluent, injection of diluent alone, or puncturing the disc with needles). Induced Widening of Lumbar Discs
Anesthesia method. Rabbits were anesthetized with KRA or 2% halothane so that disc width at
FIG. 1. A radiograph of the lumbar spine of a rabbit showing induced narrowing of disc-width space at the L3-4 disc level. The L2-3, L3-4, and L4-5 discs are marked for width determinations (original magnification x3.25).
J Orthop Res, Vol. 9, No. I , 1991
DISC SIZE IN RABBIT LUMBAR SPINE
107
FIG. 2. A: The midsagittal section of a L4-5 disc injected with diluent 4 months earlier and marked for disc-width measurement. Average b-line length = 1.43 mm. B:The midsagittal section of an L4-5 disc injured 4 months earlier by acridine-orange injection. The edges of the vertebral endplates before injury are used to determine the outer marks on line a. Average b-line length = 0.94 mm. a = line bisecting the disc longitudinally, b = lines between vertebral endplates perpendicular to line a, c = approximate ventral edge of disc before osteophyte formation (original magnification x7).
high and low planes of anesthesia could be compared. (See Experimental Design below for explanation.) Unanesthetized rabbits could not be radiographed due to motion artifacts. 1.5
02% Halothone B88 KRA ,--.
anesthesia disc level interaction
p p p
( (
>
Paralyzed
group disc level interaction
p p p
( ( (
.02
,005 .02
1.4
1.4 h
-Z:2k
1.3
1.3
a .2
2e
g .D
%
17 Normals
.04 .3
Z b 3
1.5
.001
E x
5:
Paralysis method. Lower-body paralysis was induced by aortic occlusion (12). Rabbits were sedated for surgery with 4% halothane at a flow rate of 4 L oxygedmin, and the left femoral artery in the
1.2
L!?z n U L
1.1
1.1 1 .o
1.2
L!?g n
L2-3
L4-5
FIG. 3. The effect of anesthesia on disc-width size. The L2-3 and L4-5 disc widths in rabbits sedated with 2% halothane and KRA were analyzed for specificity of effect of anesthesia on disc size.
1 .o
L2-3 L4-5 FIG. 4. Comparison of disc size in normal and paralyzed rabbits. The L2-3 and L4-5 disc widths in normal and paralyzed rabbits were analyzed for specificity of effect of the paralysis procedure.
J Orthop Res, Vol. 9, No. 1. 1991
J . H . NEUFELD ET AL.
108
-
0.4
L
v
o.2
I
1
L? Controls IAcridine orange Diluent
> .4 b ( .05
QTOUD
D
isc'level interaction
p ) .25
h
E E v
0.4 , -
0.3
1
W
N
6
-0.4
-
n x Z "-0.6
1
W 0
Z
5 I 0
-0.8
1
I
I
[
-1.0
0
12-3 L4-5 FIG. 5. Effects on the L2-3 and L4-5 disc-width sizes at more than 80 days after injecting the L4-5 disc with acridine orange or diluent compared with control rabbits. The disc size change was after-treatment values minus before-treatment values.
inguinal region was surgically exposed. A balloon tip, pressure-sensitive catheter (Berman angiographic balloon, AI-07134; Arrow International, Inc., Reading, PA, U.S.A.) was inserted into the femoral artery, positioned just below the renal arteries, and inflated for 30 k 5 min. In the sham rabbits for experimental paralysis the catheter was positioned, but the balloon was not inflated. Induced Narrowing of Lumbar Discs
The lateral aspect of a rabbit's lower back was shaved, and a lumbar disc was injured by one of the three methods described below. All invasive procedures were performed after the rabbit was sedated and maintained at surgical planes with KRA. Radiographic measurements, obtained shortly before euthanasia and ranging from immediately to 9 months after disc injury, were used to correlate disc size that had been determined radiographically and histologically. Acridine-orange injection. The L3-4 (1 rabbit) or
v
o,2
17 Controls
group
p ( ,005
D Diluent
interoction
p
)
2
W
N v)
0
0.1
-0.6
L2-3
L4-5
FIG. 6. As in Figure 5, 1 to 2 weeks after treatment.
J Orthop Res, Vol. 9, No. 1 , 1991
post treatment period disc level interaction
p ( .05 p ( .02 p ) .6
L
/
-
3 :
8
-L7
0.0
: : g e-0.2
E7d L2-3 a~ 4 - 5
-0.4
'
1
Immediately 1-3 Weeks AFTER INJURY WITH NEEDLE P U N C T U R E
FIG. 7. Effects on L2-3 and L4-5 disc-width sizes immediately and 1 to 3 weeks after the L4-5 disc was pierced with needles. The disc size change was after-treatment values minus before-treatment values.
L4-5 (4 rabbits) discs were exposed surgically (15) and injected with a 2% suspension of acridine orange (1) (Eastman Kodak Co., Rochester, NY, U.S.A.) in sterile phosphate-buffered saline (Tisu-Sol Solution; Travenol Laboratories Inc., Deerfield, IL, U.S.A.) using a 25-gauge needle. The ventrolateral aspect of the disc was exposed by sequential incisions of skin and fascia. Blunt tissue separation was used to separate muscles. Apparently, high pressure in the disc was responsible for difficulty in injecting a known volume. Verification of dye injection was obtained by euthanizing a rabbit 2 weeks after injection and observing an orange stain in the disc. In three of the other four rabbits, the discs injected with acridine orange developed disc space narrowing and severe osteophytosis over a period of 4 months or longer. Diluent injection. In two rabbits the L4-5 disc was surgically exposed and injected with diluent. Shaminjected discs were narrowed but did not develop osteophytes. One rabbit that was surgically opened to expose the L4-5 disc and closed showed no narrowing nor osteophytes. Needle puncture. The L4-5 disc of six rabbits sedated with KRA was pierced with 22-gauge needles guided by fluoroscopy. The procedure required an additional 30 min under anesthesia after preradiographs had been obtained. Two needles were used to obtain nucleus pulposus material. Discs suffered partial enucleation when air pressure was applied with a syringe attached to one of the needles. Two of the six rabbits were radiographed immediately after the procedure, and the other four were radiographed 1 to 2 weeks after needle puncture. When only one 22-gauge needle with the stylet held in place was used, there was little or no recovery of
DISC SIZE IN RABBIT LUMBAR SPINE nucleus pulposus material. One rabbit that received a needle up to but not into the disc showed no narrowing nor osteophytosis. Histological characterization showed that the material recovered from the needles consisted of multinucleated cells in a metachromatic matrix, i.e., rabbit nucleus pulposus material (8), and showed that the injured disc retained nucleus pulposus material. Experimental Design
Because radiography was used for repetitive measurements on each rabbit over time, it was necessary to demonstrate that the radiographically determined disc measurements in normal rabbits were stable over time. This was accomplished by comparing the average coefficient of variation (CV) in measuring the L4-5 disc width of rabbits in repetitive measurements on one occasion of radiography to measurements repeated over time (more than one occasion of radiography). One source of the experimental error in the radiographic procedure was estimated by analyses of copies of a film marked for measurement. Comparison of the precision of histologic and radiographic techniques was investigated by determining the CV between radiographs of all intervertebral discs and histological sections of the same discs. The variation in repeat readings of histological sections by two independent observers was determined. Because disc width was measured in living rabbits, changes over time could be expected. Therefore, trends in width change were looked for in rabbits undergoing experimental treatments designed to alter the disc width by measuring both soon after treatment and later. The study compared an experimentally altered disc (L4-5) to a control disc (L2-3) in the same spine, i.e., a repeated-measures design. Normal rabbits were used to establish the mean width and the CV in the radiographic measurements of anesthetized rabbits, in comparison to paraplegic rabbits, and to compare the effects of two different planes of anesthesia. High and low levels of anesthesia were used for disc width measurement with radiography. Since no electromyographic activity could be obtained from an errector spinae muscle when rabbits were anesthetized with KRA or 4% halothane, but could be observed with 2% halothane, spinal muscle activity was suppressed by anesthesia, and a high level of anesthesia suppressed spinal muscle activity more than a low level of anesthesia (preliminary study to results reported in 10). A high level of anesthesia
109
was obtained with KRA; it was used for histologicradiographic correlation because it was assumed that, after muscles relaxed during anesthesia, disc width would more closely resemble the width after death. The same high level of anesthesia was used for studies on histologic-radiographic correlation and on the effects of disc injury. The anesthetic KRA consisted of 50 mg/kg ketamine, 10 mg/kg of xylazine (rompun), and 1 mg/kg of acepromazine and was administered subcutaneously or intramuscularly. Radiographs were obtained within 30 min after injection of KRA. Radiographs obtained immediately after needle puncture were obtained under conditions of prolonged anesthesia, approximately 1 h after injection. In studies on the effects of lower-body paralysis on disc width, where some force-generating activity in normal spinal muscles was desired, a low level of anesthesia was used. For paralysis studies, halothane was delivered in controlled gas-flow anesthesia of 2% at a flow rate of 3 L oxygedmin. Statistical Methods
Linear regression analysis was used to correlate the disc widths determined radiographically and histologically (radiographic values as dependent variable and histologic values as independent variable). Two-way analysis of variance (ANOVA) with a repeated-measures design was used to determine significance of difference in means between treatment groups and disc levels. The specificity of the anesthesia and paralysis treatments was determined using disc width data obtained high (L2-3) and low (L4-5) in the lumbar spine. The results of comparing control rabbits to rabbits injected with diluent or with acridine orange and the results of comparing needle-puncture injury immediately to 1 to 3 weeks after injury were presented as significance of difference of change in width of L2-3 and L4-5 (aftertreatment values minus before-treatment values). Figure results are given as disc width in millimeters (mean & SEM). Table results include disc width in millimeters (mean _t SD) and the average CV in repetitive measurements in percent (mean k SEM). The significance level chosen was p < 0.05. RESULTS Radiography and Histology
The experimental error in repeat determinations of the L4-5 disc width on one occasion of radiogra-
J Orthop Res, Vol. 9, No. I , 1991
J . H . NEUFELD ET AL.
110
phy was approximately 5%, and no experimental variation, beyond the error in the procedure itself, occurred between occasions of radiography (Table 1). As the variation in reading marked films was approximately 1%, the main cause of experimental error in the radiographic analyses was the marking of the radiographs before measuring the marked films. Because the variation between all discs, analyzed both by radiography and histology, was similar (16.4%vs. 17%), the radiographic and histologic determinations were equally precise. The error in repeat readings of widths in histologic sections was minimal (
Variables Affecting Disc Size in the Lumbar Spine of Rabbits: Anesthesia, Paralysis, and Disc Injury J. Hulse Neufeld, *T. Machado, and tL. Margolin V A Medical Center, *Department of Radiology and ?Animal Research Facility, San Diego, California, U.S.A.
Summary: Methods have been developed that permit repetitive radiographic measurement of the lumbar intervertebral disc space in a rostral-caudal direction (width) in the anesthetized laboratory rabbit. Using isolated control discs and injured discs in which narrowing has been induced for chronic and acute periods, the widths of the lumbar intervertebral disc spaces determined radiographically correlate with widths determined histologically (p < 0.000, r = 0.75). Both an increase (widening) and a decrease (narrowing) in disc width were observed using radiography after different experimental treatments. Anesthesia and lower-body paralysis (an experimentally induced inability to bear weight on and to perceive a pinch stimulus in hind limbs) caused widening of the discs: anesthesia causing a general widening throughout the lumbar spine and lower-body paralysis causing a specific widening low in the lumbar spine. Both disc injection and piercing the disc with needles to recover nucleus pulposus material caused narrowing of the discs. Acridine-orange injection induced a narrowing accompanied by osteophytosis. Experimentally induced narrowing at L4-5 (the result of injury to the disc) resulted in narrowing also at L2-3. These findings are consistent with the hypothesis that in vivo disc-width size in the young rabbit depends on both the quantity of nucleus pulposus material and the force-generating activities of the adjacent spinal muscles, and that disc injury at one level stimulates narrowing at other levels. Key Words: Intervertebral disc size-Rabbit-Radiography-Histology-Induced widening-Induced narrowing.
The purpose of this study was to determine if an induced change in one disc of a rabbit could be observed with radiography and confirmed by histology. In dogs (3,7) and monkeys (19), radiography has been used to document the narrowing of disc width after the injection of nucleolytic substances. In rabbits radiography has been used to document osteophyte formation after lumbar discs had been injured by incising the annular fibers of two or more discs, with a resulting prolapse of the nucleus pulposus (13). If disc width depends on the integrity of annular fibers and/or nucleus pulposus, incising the annular fibers and subsequent anterior prolapse of the nucleus should result in disc narrowing. A discinjury method that minimizes annular-fiber disrup-
It has often been stated that, as an intervertebral disc narrows, the articulating facets telescope or subluxate (2,5,14,16). This study describes how disc widths (rostral-caudal dimension) of abnormal size were produced and measured in rabbits. As a result, researchers can have rabbit populations in which to measure facet position in vitro as a function of disc size. Received November 13, 1989; accepted July 1 1 , 1990. Address correspondence and reprint requests to Dr. J. Hulse Neufeld, Research Associate, at VA Medical Center (151), 3350 La Jolla Village Drive, San Diego, CA 92161, U.S.A. Presented in part at the 35th Annual Meeting, Orthopaedic Research Society, February 6 9 , 1989, Las Vegas, Nevada, U.S.A.
104
105
DISC SIZE IN RABBIT LUMBAR SPINE
tion (needle puncture rather than scalpel incision) was used so that, if disc narrowing were induced, it could be attributed to loss of nucleus pulposus and not to disruption of the annular fibers. To determine the relationship between radiographically and histologically measured disc width, rabbit lumbar intervertebral discs from control and injured specimens were prepared for histological examination after radiography of posttreatment lumbar spines in anesthetized rabbits. Also the importance to disc width of (a) the quantity of nucleus pulposus material and (b) the force-generating activities of the spinal muscles was determined from radiography of lumbar spines before and after experimental treatments. The relationship of disc width to muscle activity was studied indirectly by measuring disc width at different planes of anesthesia and before and after lower-body paralysis. MATERIALS AND METHODS Animals and Animal Care Rabbits of a New Zealand strain were housed at an ambient temperature and a humidity standard for rabbits. Food and tap water were available ad libitum. Experimental and control rabbits were picked at random from 49 rabbits aged 3 to 5 months and weighing 2.5 to 3.5 kg. Lumbar disc width was studied with radiography under ketamine-rompun-acepromazine (KRA) anesthesia (26 normal rabbits) or under 2% halothane anesthesia (23 normal rabbits). (See Experimental Design below for explanation.) Rabbits initially studied under KRA anesthesia either were studied again later (results, Table I), or they were anesthetized to surgical levels with KRA TABLE 1. Mean disc width and average coefficient of variation in measuring disc width from radiographs of anesthetized rabbits
and placed into one of six experimental, sham, or control groups: (a) surgical exposure of the L3-4 or L4-5 disc and acridine-orange injection (5 rabbits); (b) surgical exposure of the L4-5 disc and diluent injection (2 rabbits serving as shams to acridineorange injection); (c) surgical exposure of the L4-5 disc and closure (1 rabbit of control group); (d) fluoroscopic guided-needle puncture of the L4-5 disc with nucleus pulposus recovery (6 rabbits); (e) fluoroscopic guided-needle puncture of the L4-5 disc without nucleus pulposus recovery (2 rabbits serving as shams to needle puncture with recovery); or (f) fluoroscopic guided-needle puncture up to but not into the L4-5 disc (1 rabbit of control group). The other nine rabbits were used in experiments not reported here or they died. Four of the 26 rabbits studied under KRA anesthesia were measured repeatedly over 42 to 76 days before the experimental treatments. Rabbits initially studied with 2% halothane either were studied again later or were placed into one of two experimental groups: (a) aortic occlusion (of 19 attempts, 12 rabbits were paralyzed); or (b) a sham procedure for aortic occlusion (2 rabbits). The other two rabbits were used in an experiment not reported here. Four of the 23 rabbits studied under 2% halothane were measured repeatedly at a 1-day interval before experimental treatments. Paraplegic rabbits were cared for twice daily by a nursing routine of expressing the urinary bladder, handwashing the lower body, and placing the rabbit on disposable liners to cushion the rabbit’s contact with the cage. Parasthesias were evident 1 week postparalysis as chewing on hind limbs. Such selfmutilation was prevented by putting wide collars around the rabbits’ necks and gauze booties on their hind paws. Methods of Quantifying Disc Space Size Radiographic Procedure
Anesthetic agent KRAa Disc width
(mm) One occasion‘ More than one occasiond
-
2% Halothane CVb (%)
Disc width (mm)
CVb (%)
1.36 5 0.08
4.3
2
0.6
1.05 ? 0.12
6.2 2 1.8
1.34 2 0.05
5.4
?
0.9
1.02 ? 0.11
5.2
2
2.8
Values are 2 S D (disc width) and 5SEM (coefficient of variation). Ketamine-rompun-acepromazinemixture. Coefficient of variation. Within rabbits on one occasion of anesthesia and radiography. Within rabbits on more than one occasion of anesthesia and radiography.
Details of the radiographic and histologic procedures have been described elsewhere for use with rats (9,ll). In this study a rabbit was positioned correctly by placing foam-rubber supports beneath the lumbar area of the back and between the knees. Magnified views of the lumbar discs were obtained by magnification radiography (6). Magnification was 2.5 to 3.5 0.25 and was achieved by having a focal spot-film distance of 120 ? 5 cm and an objectfilm distance of 80 ? 5 cm; the microfocal spot x-
*
J Orthop Res, Vol. 9, No. 1 , 1991
J . H . NEUFELD ET AL.
106
ray tube (0.4 mm magnification tube; Eureka, Chicago, IL, U.S.A.) was employed at 85 peak kilovoltage (kVp) and 160 milliamperes (mA) for 1.6 s. The degree of magnification was determined with a magnification control (MC) consisting of a metal clip with a hole 1.O k 0.1 mm in diameter as determined with a X 6 hand lens calibrated to 0.1 mm (Leitz Wetzlar, Germany). Repetitive measurements of a rabbit’s lumbar intervertebral disc spaces were made by repositioning the rabbit and the MC between determinations. The disc space width (mm) was determined by direct measurement on radiographs (Fig. 1). In summary, the average of the three middle lines between endplates was corrected for magnification and used to measure the disc width (1 1). Histological Technique
Histologic samples embedded consisted of two halves of two adjacent vertebral bodies and associated intervertebral disc and articulating facets (motion segments). Of the eight rabbits euthanized to obtain the motion segments used for histologicradiographic correlation, six had radiographic evidence of disc narrowing for more than 3 months and two had no radiographic evidence of disc injury. In this study, 27 motion segments from eight euthanized rabbits were used for histologic-radiographic correlation. The samples had a wide range of disc widths in order to increase the ease of obtaining a significant relationship (18). The spines from an-
other four rabbits were fixed in extension, flexion, or lateral flexion to obtain 12 motion segments to be used to determine the effect on disc width of the spine’s position during fixation. Discs that had lesions with osteophytes were measured after the outer marks approximating the disc were drawn between the presumed original edges of the vertebral epiphyses (Fig. 2B, line c). The depths of the indentations on the superior and inferior vertebral endplates in eight specimens were measured to compare disc width determined radiographically with that determined histologically before and after the indentation depths were subtracted. An indentation was measured as the perpendicular distance from the deepest part of the indentation in an endplate to a line between the edges of the indentation. Experimental Treatments Evidence for both disc widening (Figs. 3 and 4) and disc narrowing (Figs. 5-7) was observed using radiography after different experimental treatments. Widening of lumbar discs was induced by anesthesia or by lower-body paralysis; narrowing of lumbar discs was induced by disc injury (injection of acridine orange in diluent, injection of diluent alone, or puncturing the disc with needles). Induced Widening of Lumbar Discs
Anesthesia method. Rabbits were anesthetized with KRA or 2% halothane so that disc width at
FIG. 1. A radiograph of the lumbar spine of a rabbit showing induced narrowing of disc-width space at the L3-4 disc level. The L2-3, L3-4, and L4-5 discs are marked for width determinations (original magnification x3.25).
J Orthop Res, Vol. 9, No. I , 1991
DISC SIZE IN RABBIT LUMBAR SPINE
107
FIG. 2. A: The midsagittal section of a L4-5 disc injected with diluent 4 months earlier and marked for disc-width measurement. Average b-line length = 1.43 mm. B:The midsagittal section of an L4-5 disc injured 4 months earlier by acridine-orange injection. The edges of the vertebral endplates before injury are used to determine the outer marks on line a. Average b-line length = 0.94 mm. a = line bisecting the disc longitudinally, b = lines between vertebral endplates perpendicular to line a, c = approximate ventral edge of disc before osteophyte formation (original magnification x7).
high and low planes of anesthesia could be compared. (See Experimental Design below for explanation.) Unanesthetized rabbits could not be radiographed due to motion artifacts. 1.5
02% Halothone B88 KRA ,--.
anesthesia disc level interaction
p p p
( (
>
Paralyzed
group disc level interaction
p p p
( ( (
.02
,005 .02
1.4
1.4 h
-Z:2k
1.3
1.3
a .2
2e
g .D
%
17 Normals
.04 .3
Z b 3
1.5
.001
E x
5:
Paralysis method. Lower-body paralysis was induced by aortic occlusion (12). Rabbits were sedated for surgery with 4% halothane at a flow rate of 4 L oxygedmin, and the left femoral artery in the
1.2
L!?z n U L
1.1
1.1 1 .o
1.2
L!?g n
L2-3
L4-5
FIG. 3. The effect of anesthesia on disc-width size. The L2-3 and L4-5 disc widths in rabbits sedated with 2% halothane and KRA were analyzed for specificity of effect of anesthesia on disc size.
1 .o
L2-3 L4-5 FIG. 4. Comparison of disc size in normal and paralyzed rabbits. The L2-3 and L4-5 disc widths in normal and paralyzed rabbits were analyzed for specificity of effect of the paralysis procedure.
J Orthop Res, Vol. 9, No. 1. 1991
J . H . NEUFELD ET AL.
108
-
0.4
L
v
o.2
I
1
L? Controls IAcridine orange Diluent
> .4 b ( .05
QTOUD
D
isc'level interaction
p ) .25
h
E E v
0.4 , -
0.3
1
W
N
6
-0.4
-
n x Z "-0.6
1
W 0
Z
5 I 0
-0.8
1
I
I
[
-1.0
0
12-3 L4-5 FIG. 5. Effects on the L2-3 and L4-5 disc-width sizes at more than 80 days after injecting the L4-5 disc with acridine orange or diluent compared with control rabbits. The disc size change was after-treatment values minus before-treatment values.
inguinal region was surgically exposed. A balloon tip, pressure-sensitive catheter (Berman angiographic balloon, AI-07134; Arrow International, Inc., Reading, PA, U.S.A.) was inserted into the femoral artery, positioned just below the renal arteries, and inflated for 30 k 5 min. In the sham rabbits for experimental paralysis the catheter was positioned, but the balloon was not inflated. Induced Narrowing of Lumbar Discs
The lateral aspect of a rabbit's lower back was shaved, and a lumbar disc was injured by one of the three methods described below. All invasive procedures were performed after the rabbit was sedated and maintained at surgical planes with KRA. Radiographic measurements, obtained shortly before euthanasia and ranging from immediately to 9 months after disc injury, were used to correlate disc size that had been determined radiographically and histologically. Acridine-orange injection. The L3-4 (1 rabbit) or
v
o,2
17 Controls
group
p ( ,005
D Diluent
interoction
p
)
2
W
N v)
0
0.1
-0.6
L2-3
L4-5
FIG. 6. As in Figure 5, 1 to 2 weeks after treatment.
J Orthop Res, Vol. 9, No. 1 , 1991
post treatment period disc level interaction
p ( .05 p ( .02 p ) .6
L
/
-
3 :
8
-L7
0.0
: : g e-0.2
E7d L2-3 a~ 4 - 5
-0.4
'
1
Immediately 1-3 Weeks AFTER INJURY WITH NEEDLE P U N C T U R E
FIG. 7. Effects on L2-3 and L4-5 disc-width sizes immediately and 1 to 3 weeks after the L4-5 disc was pierced with needles. The disc size change was after-treatment values minus before-treatment values.
L4-5 (4 rabbits) discs were exposed surgically (15) and injected with a 2% suspension of acridine orange (1) (Eastman Kodak Co., Rochester, NY, U.S.A.) in sterile phosphate-buffered saline (Tisu-Sol Solution; Travenol Laboratories Inc., Deerfield, IL, U.S.A.) using a 25-gauge needle. The ventrolateral aspect of the disc was exposed by sequential incisions of skin and fascia. Blunt tissue separation was used to separate muscles. Apparently, high pressure in the disc was responsible for difficulty in injecting a known volume. Verification of dye injection was obtained by euthanizing a rabbit 2 weeks after injection and observing an orange stain in the disc. In three of the other four rabbits, the discs injected with acridine orange developed disc space narrowing and severe osteophytosis over a period of 4 months or longer. Diluent injection. In two rabbits the L4-5 disc was surgically exposed and injected with diluent. Shaminjected discs were narrowed but did not develop osteophytes. One rabbit that was surgically opened to expose the L4-5 disc and closed showed no narrowing nor osteophytes. Needle puncture. The L4-5 disc of six rabbits sedated with KRA was pierced with 22-gauge needles guided by fluoroscopy. The procedure required an additional 30 min under anesthesia after preradiographs had been obtained. Two needles were used to obtain nucleus pulposus material. Discs suffered partial enucleation when air pressure was applied with a syringe attached to one of the needles. Two of the six rabbits were radiographed immediately after the procedure, and the other four were radiographed 1 to 2 weeks after needle puncture. When only one 22-gauge needle with the stylet held in place was used, there was little or no recovery of
DISC SIZE IN RABBIT LUMBAR SPINE nucleus pulposus material. One rabbit that received a needle up to but not into the disc showed no narrowing nor osteophytosis. Histological characterization showed that the material recovered from the needles consisted of multinucleated cells in a metachromatic matrix, i.e., rabbit nucleus pulposus material (8), and showed that the injured disc retained nucleus pulposus material. Experimental Design
Because radiography was used for repetitive measurements on each rabbit over time, it was necessary to demonstrate that the radiographically determined disc measurements in normal rabbits were stable over time. This was accomplished by comparing the average coefficient of variation (CV) in measuring the L4-5 disc width of rabbits in repetitive measurements on one occasion of radiography to measurements repeated over time (more than one occasion of radiography). One source of the experimental error in the radiographic procedure was estimated by analyses of copies of a film marked for measurement. Comparison of the precision of histologic and radiographic techniques was investigated by determining the CV between radiographs of all intervertebral discs and histological sections of the same discs. The variation in repeat readings of histological sections by two independent observers was determined. Because disc width was measured in living rabbits, changes over time could be expected. Therefore, trends in width change were looked for in rabbits undergoing experimental treatments designed to alter the disc width by measuring both soon after treatment and later. The study compared an experimentally altered disc (L4-5) to a control disc (L2-3) in the same spine, i.e., a repeated-measures design. Normal rabbits were used to establish the mean width and the CV in the radiographic measurements of anesthetized rabbits, in comparison to paraplegic rabbits, and to compare the effects of two different planes of anesthesia. High and low levels of anesthesia were used for disc width measurement with radiography. Since no electromyographic activity could be obtained from an errector spinae muscle when rabbits were anesthetized with KRA or 4% halothane, but could be observed with 2% halothane, spinal muscle activity was suppressed by anesthesia, and a high level of anesthesia suppressed spinal muscle activity more than a low level of anesthesia (preliminary study to results reported in 10). A high level of anesthesia
109
was obtained with KRA; it was used for histologicradiographic correlation because it was assumed that, after muscles relaxed during anesthesia, disc width would more closely resemble the width after death. The same high level of anesthesia was used for studies on histologic-radiographic correlation and on the effects of disc injury. The anesthetic KRA consisted of 50 mg/kg ketamine, 10 mg/kg of xylazine (rompun), and 1 mg/kg of acepromazine and was administered subcutaneously or intramuscularly. Radiographs were obtained within 30 min after injection of KRA. Radiographs obtained immediately after needle puncture were obtained under conditions of prolonged anesthesia, approximately 1 h after injection. In studies on the effects of lower-body paralysis on disc width, where some force-generating activity in normal spinal muscles was desired, a low level of anesthesia was used. For paralysis studies, halothane was delivered in controlled gas-flow anesthesia of 2% at a flow rate of 3 L oxygedmin. Statistical Methods
Linear regression analysis was used to correlate the disc widths determined radiographically and histologically (radiographic values as dependent variable and histologic values as independent variable). Two-way analysis of variance (ANOVA) with a repeated-measures design was used to determine significance of difference in means between treatment groups and disc levels. The specificity of the anesthesia and paralysis treatments was determined using disc width data obtained high (L2-3) and low (L4-5) in the lumbar spine. The results of comparing control rabbits to rabbits injected with diluent or with acridine orange and the results of comparing needle-puncture injury immediately to 1 to 3 weeks after injury were presented as significance of difference of change in width of L2-3 and L4-5 (aftertreatment values minus before-treatment values). Figure results are given as disc width in millimeters (mean & SEM). Table results include disc width in millimeters (mean _t SD) and the average CV in repetitive measurements in percent (mean k SEM). The significance level chosen was p < 0.05. RESULTS Radiography and Histology
The experimental error in repeat determinations of the L4-5 disc width on one occasion of radiogra-
J Orthop Res, Vol. 9, No. I , 1991
J . H . NEUFELD ET AL.
110
phy was approximately 5%, and no experimental variation, beyond the error in the procedure itself, occurred between occasions of radiography (Table 1). As the variation in reading marked films was approximately 1%, the main cause of experimental error in the radiographic analyses was the marking of the radiographs before measuring the marked films. Because the variation between all discs, analyzed both by radiography and histology, was similar (16.4%vs. 17%), the radiographic and histologic determinations were equally precise. The error in repeat readings of widths in histologic sections was minimal (
