J Manipulative Physiol Ther 2007 (May); 30 (4): 279–294 ~ FULL TEXT
Charles N.R. Henderson, DC, PhD, Gregory D. Cramer, DC, PhD, Qiang Zhang, MD, DC,
James W. DeVocht, DC, PhD, and Jaeson T. Fournier, DC, MPH
Palmer Center for Chiropractic Research,
Davenport, Iowa, USA.
OBJECTIVES: The purpose of this study was to characterize intervertebral stiffness and alignment changes in the external link model and evaluate it as an experimental mimic for studying the chiropractic subluxation.
METHOD: A controlled test-retest design was used to evaluate rats with spine segments linked in 3 alignment configurations and controls that were never linked. Dorsal-to-ventral spine stiffness was measured with a load platform, and flexion/extension misalignment was assessed on lateral radiographs obtained with a spine extension jig. Descriptive statistics were computed for study groups, and multiple linear regression models were used to examine all potential explanatory variables for the response variables "stiffness" and "joint position."
RESULTS: Rats tested with links in place had significantly higher dorsal-to-ventral stiffness in the neutral configuration than rats in the flexed configuration. This difference remained after the links were removed. Stiffness after link removal was greater for longer linked periods. Surprisingly, stiffness after link removal was also greater with longer unlinked periods. Longer linked periods also produced greater misalignments during forced spine extension testing. Although link configuration was not a statistically significant predictor of misalignments, longer times after link removal did produce greater misalignments.
CONCLUSIONS: This study suggests that the external link model can be a valuable tool for studying the effects of spine fixation and misalignment, 2 cardinal features of what has been historically described as the chiropractic subluxation. Significant residual stiffness and misalignment remained after the links were removed. The progressive course of this lesion is consistent with subluxation theory and clinical chiropractic experience.
From the FULL TEXT Article
This article introduces a new animal model, the external link model (ELM), that allows researchers to produce and study spine lesions with the cardinal biomechanical features of what has been historically described as a chiropractic subluxation (ie, fixation, hypomobility/stiffness, and misalignment).  As the name suggests, spine fixation and misalignment are produced in the ELM by means of an external link system attached to spine segments (Fig 1, Fig 2). The external links may be removed after a variable link period (weeks linked) without stressing the animal but allowing normal forces to be reintroduced to the motion segments.
The critical biomechanical features of vertebral subluxation are joint fixation (stiffness) and misalignment. [2, 3] Therefore, these features were assessed to evaluate the ELM as an experimental subluxation mimic. Segmentally localized stiffness was obtained by linking the externalized stems of 3 spinous attachment units (SAUs) that were implanted on consecutive lumbar vertebrae in the rat (Fig 1, Fig 2). Although the spine segment can be fixed and misaligned with links in place, it is the residual stiffness and misalignment after the links are removed that offer promise as a subluxation mimic. This residual fixation and malposition, in contrast to that observed while the links are in place, must be due to modification of spinal and paraspinal tissues. This is an outstanding feature of the ELM. The ELM allows us to move beyond the study of acute effects—we may now examine the chronic effects of spine fixation. Therefore, examination of residual stiffness and misalignment was the primary focus of this study. Because the ELM is a long-term survival model, with the rats studied for many months after experimental spine fixation, we believe it may be used to study the putative chronic effects of subluxation as well as the effects of various therapeutic interventions.
The first article in this series explained the need for the model, its biomechanical rationale, and its current application in research related to chiropractic theory.  This article reports the biomechanical characterization of the ELM, particularly as it relates to the chiropractic subluxation concept. Development and evaluation of the ELM was a collaborative effort between the Palmer Center for Chiropractic Research, Davenport, Iowa, and the National University of Health Sciences, Lombard, Ill.
Diagram of SAUs linked
in neutral on the rat spine
This figure shows the ELM
with 3 link configurations
Intervertebral fixation was examined by measuring
lumbar dorsal-to-ventral (D-V) stiffness with a load testing
platform (Fig 3), and joint position (misalignment) was
examined by measuring intervertebral flexion/extension
relationships on lateral lumbar spine stress radiographs
(Fig 4). The D-V stiffness study was a repeated measures
design with 3 arms (Fig 5): (1) link period without a
subsequent unlinked period; (2) link period followed by an
unlinked period; and (3) control (never linked). Rats in
the 2 experimental group arms (E) were linked in 1 of
3 configurations: neutral (EN), flexed (EF), or rotated (ER).
Animals in arm 1 of Figure 5 were linked for 1 of 6 link
periods (1, 2, 4, 8, 12, or 16 weeks) and then unlinked,
with no survival period after unlinking. Rats in arm 2 were
linked for 1 of 3 link periods (1, 4, or 8 weeks) and then
unlinked for 1 of 5 unlinked survival periods (1, 2, 4, 8, or
12 weeks). Control rats (arm 3) all received SAU implants
but were never linked. In all experimental groups, D-V
stiffness testing was performed just before linking (U1),
immediately after linking (L1), immediately before link
removal (L2), immediately after link removal (U2), and at
the conclusion of any unlinked period (U3). Dorsal-toventral
stiffness tests were performed on control rats after
survival periods that were equivalent to experimental group
rats (Fig 5, C-U1, C-L1, C-L2, C-U2, and C-U3). The last
bequivalent periodQ in the control arm, bto 20 weeks,Q
represents the additional unlinked period required for
comparison with the 8–week linked plus 12–week unlinked
rats in arm 2. The animal counts shown in Figure 5 are
explained in the Methods section.
Joint position was examined in a separate comparison
study with 3 arms (Fig 6). Rats in the experimental group
(E) were linked in 1 of 3 configurations: neutral (EN), flexed
(EF), or rotated (ER). Rats in arm 1 were linked for 1 of
6 link periods (1, 2, 4, 8, 12, or 16 weeks) and were not
unlinked. Rats in arm 2 were linked for 1 of 3 link periods
(1, 4, or 8 weeks) and were subsequently unlinked for 1 of
5 unlinked periods (1, 2, 4, 8, or 12 weeks). Control rats had
either sham SAU implant surgeries (CSURG) or were
implanted with SAUs that were never linked (CSAU).
Radiographs were taken at the end of the link period in
arm 1 (R Linked), at the end of the unlinked period in arm 2
(R Unlinked), and at the end of equivalent control periods in
arm 3 (R Control). The animal counts shown in Figure 6 are
explained in the Methods section.
X-ray positioning jig
stiffness study design.
Joint position study design.
Pilot studies generally examine a large number of variables and, therefore, may not have the power for definitive hypothesis testing of all explanatory (predictor) variables. In this report, we present all of our a priori hypotheses because they shaped the study design, and their statistical examination provides at least a first approximation of the relationship between study variables that may then be examined in future, adequately powered, studies.
We expected that the D-V stiffness of control rats (and not-yet-linked experimental rats) would not substantially increase over the age range covered by this study (13–48 weeks). We also thought that increased segmental stiffness during the link period (linked stiffness) would be so large that the differential effect of weeks linked or link configuration would not be detectable while the links were in place. By contrast, we expected a much smaller increased segmental stiffness immediately after link removal (immediate residual stiffness), which would be demonstrably effected by both the length of the link period and the link configuration (neutral, flexed, or rotated). Furthermore, we anticipated that this residual stiffness would progressively decrease after the links were removed—approaching, but probably not attaining, each animal's baseline (before linking) stiffness. Any long-term residual stiffness was expected to be greater for longer link periods.
Therefore, 4 hypotheses were identified for the stiffness testing component of this pilot study:
Control rat stiffness (CSAU rats) will be the same as the baseline stiffness of experimental rats (EN, EF, ER) for the L4–L6 spine region, and this stiffness will not significantly increase with increasing age.
Linked stiffness of the L4–L6 spine region during the link period will be significantly greater than baseline stiffness, and the magnitude of the difference will not be influenced by the length of the fixation period or the link configuration.
Immediate residual stiffness of the L4–L6 spine region will be significantly greater than baseline stiffness, and the magnitude of the difference will be influenced by both the length of the fixation period and the link configuration.
Long-term residual stiffness of the L4–L6 spine region will be significantly greater for longer link periods but decrease with longer unlinked periods (weeks unlinked), approaching but not attaining baseline stiffness.
A separate population of rats were radiographed in an extension jig for intersegmental misalignment associated with SAU linking. As in the stiffness studies, joint position was examined during the link period, immediately after the link period, and after a variable unlinked period. We expected that rats with the L4–L6 segment linked in a flexed configuration would show segmental flexion when radiographed in the extension jig (positive L4–L6 posterior body angle [PBA]). We also expected that radiographic examination in the extension jig immediately after link removal would reveal either a small residual flexion of L4–L6 (a small positive PBA) or, compared with control rat radiographs, a smaller extension of the L4–L6 segment (a larger, less negative PBA).
By contrast, rats linked in a neutral or rotated configuration would be expected to have a much smaller flexion effect across the L4–L6 region (associated with the slightly flexed, normal posture of the rat). As in the stiffness tests, we anticipated that longer link periods would be associated with greater positional effects after link removal, but it would have no demonstrable effect on rats radiographed with the links still in place. Finally, we anticipated that rats radiographed after having their links removed for variable periods of time would have little or no residual effects—depending on the length of the linked and unlinked periods, and any link configuration interaction.
Therefore, additional hypotheses were evaluated for the radiologic component of the study:
Linked position of the L4–L6 spine region will be flexed (positive PBA), and the magnitude of this flexion will be related to the link configuration (EN, EF, and ER), but not the length of the link period (weeks linked).
Immediate residual position of the L4–L6 spine region will be less extended immediately after the link period than that of control rats (>PBA), and the magnitude of the difference will be related to both the link configuration (EN, EF, and ER) and the length of the link period (weeks linked).
Long-term residual position of the L4–L6 spine region will be less extended than or the same as that of control rats (?control PBA), depending on the interaction between the link configuration (EN, EF, and ER), the length of the link period (weeks linked), and the length of the unlinked period (weeks unlinked).
Before study implementation, the Institutional Animal Care and Use Committees of both institutions reviewed all experiments for compliance with the National Research Council Guide for the Care and Use of Laboratory Animals.  Three hundred twelve male Sprague-Dawley rats (350–450 g) were obtained for the D-V stiffness and joint position studies (Harlan Laboratories, Indianapolis, Ind). All animals were housed in individual plastic cages at the vivariums of either the Palmer Center for Chiropractic Research or the National University of Health Sciences. These animal housing facilities are operated in compliance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals. Water and Purina Lab Block (Ralston Purina, St Louis, Mo) were available to the rats at all times.
Biomechanical characterization of the ELM was performed as part of a larger project to develop the model and gather preliminary data on the anatomical, biomechanical, and neurologic consequences of spine fixation and misalignment. Feasibility constraints on that developmental project required that animal counts for the various outcome studies be kept at the minimum necessary to establish trend plots. For the D-V stiffness study (Fig 5), it was decided that 4 rats would be evaluated for each of the unique variable combinations in arm 1 of the experimental group (n = 72), which provided data on residual stiffness immediately after link removal. Three rats would be examined for each of the numerous variable combinations in experimental arm 2 (n = 135), which evaluated long-term residual stiffness after link removal.
Finally, 4 rats would be evaluated for each of 7 equivalent periods in arm 3 (1, 2, 4, 8, 12, 16, and 20 weeks; n = 28), the control arm. In the radiographic joint position study (Fig 6), data were collected in a separate rat population to avoid potential treatment effects that might be associated with D-V stiffness testing (see Discussion section). One rat was radiographed for each of the 63 unique variable combinations in the experimental arms (arm 1 and arm 2, n = 63) and equivalent periods for the 2 control configurations (arm 3, n = 14). Therefore, 312 rats were used for the biomechanics and radiographic studies (D-V stiffness, 235; joint position, 77).
Surgical Attachment of the External Fixator System
For the purposes of this article, a brief description of the ELM is needed. All rats in the experimental groups and rats in one control group (CSAU) had external link systems surgically implanted while under halothane anesthesia. Each external link system consisted of 3 SAUs that could be externally interconnected at a later date by 2 small metal links to experimentally fix the 3 vertebrae (Fig 1, Fig 2). All components were made of surgical stainless steel. After surgery, each animal was returned to its cage and placed under a small incandescent lamp to maintain body temperature while recovering from the anesthesia. An analgesic, acetaminophen, was administered for the first 3 postsurgical days (Children's TYLENOL Liquid, grape flavor [Johnson & Johnson, New Brunswick, NJ], approximately 300 mg/kg a day via water bottle). Rats were included in the link or control groups only after a 1–week recovery period. All animals were closely observed for 2 days after discontinuance of the analgesics for possible pain behavior (decreased eating or drinking, increased resting, immobility, or scratching). Pain behavior was very rare after this procedure (<2%). Neither baseline outcome measures nor initiation of experimental fixation procedures were performed on an animal exhibiting postsurgical pain behavior.
Experimental and Control Configurations
Animals having their SAUs linked were first placed under anesthesia using a halothane and oxygen mixture (2.5% halothane at 1 L/min flow rate). The L4, L5, and L6 vertebrae of rats in the experimental group (E) were linked in 1 of 3 configurations to produce fixation without misalignment (neutral) or with misalignment (flexed or rotated). These experimental configurations were the same in both the stiffness (biomechanics) and joint position (radiographic) studies (Fig 5, Fig 6). The “neutral” configuration (EN) was obtained by linking the 3 SAU stems of the external link system in the position they assumed when the anesthetized animal was placed prone on a flat surface (Fig 2B). The flexion configuration (EF) was obtained by linking a rat with its trunk moved into near maximum flexion. To obtain a rotated configuration, we shifted the L5 SAU stem laterally relative to the L4 and L6 SAU stems (Fig 2C). This was done by attaching the links between the proximal ends of horizontal bolts inserted on the L4 and L6 SAU stems and the distal end of the L5 SAU stem's horizontal bolt. This produced approximately a 6° lateral tilt of the SAU stem. Note that for the rotation configuration, the rat's spine was also in a neutral position with regard to flexion/extension when the L5 vertebra was axially rotated. At the end of all linking procedures, a drop of 222 MS thread locker (Loctite Corporation, Rocky Hill, Conn) was applied to the external nuts to prevent subsequent loosening.
A subpopulation of rats in the experimental group of both studies (ie, stiffness and position) were examined for spontaneous recovery by removing the external links and reexamining them at the end of 5 discrete unlinked survival periods: 1, 2, 4, 8, or 12 weeks (Fig 5, Fig 6, arm 2). Rats were divided into these discrete groups, rather than performing tests on each animal at multiple times during an extended unlinked period. This was done to avoid any treatment effect that might otherwise have occurred as a result of repeated testing after the link period. These unlinked rats had all been previously linked for 1, 4, or 8 weeks in 1 of the 3 link configurations (neutral, flexed, or rotated). For example; after being linked in flexion for 4 weeks, the links on a rat were removed for a subsequent 2–week unlinked survival period (EF4-U2).
A never-linked control configuration, CSAU, was examined in both the stiffness and joint position studies. CSAU rats had SAUs surgically attached, but the SAU stems were never linked (Fig 2, Fig 5, Fig 6). An additional control group, CSURG rats, was examined in the joint position study (Fig 6). CSURG rats received a sham implant procedure in which the SAUs were actually placed on the spinous processes and holes were drilled, but the SAUs were removed before wound closure. The CSURG group was not included in the stiffness study because D-V stiffness testing required SAUs to be present (Fig 3A).
Dorsal-Ventral Spine Stiffness Testing
This stiffness testing protocol was adapted from a procedure developed for in vivo testing of the human spine.6, 7 Rats linked in neutral (EN), flexion (EF), and rotation (ER) configurations were evaluated for spine stiffness before and after experimental fixation with the external link system (Fig 5). Spinous attachment unit–bearing control rats (CSAU) were age matched and tested for spine stiffness at times corresponding to the experimental group rats. Spine stiffness was determined by measuring D-V loading forces in halothane anesthetized rats using a PCM-Versa Test load frame (Mecmesin, Santa Rosa, Calif) with a 10–N load cell. Each anesthetized rat was placed prone on a rigid surface and tested using a 3–point load applied to the 3 SAU stems of the external link system (Fig 3A). The 2 outer SAU stems were supported by small brass turnbuckles suspended from rigid supports. This allowed the middle vertebra to undergo D-V displacement, whereas the supported outer vertebrae moved in conjugate sagittal rotation, but not D-V displacement. The load frame's motor-driven indenter was applied to the middle (L5) SAU stem at a constant displacement rate of 0.3 mm/s until a preset 3–N maximum load was obtained. This maximum load was adopted because it was well within the physiologic range of the joints and it was thought that it would produce little or no treatment effect. Preliminary investigations with a larger load cell and euthanized rats revealed that joint failure required D-V loads in excess of 35 N. In addition, examination for stiffness changes across multiple test cycles confirmed that there was no treatment effect with this small load (unpublished data). The split foot pad of the load frame indenter applied essentially symmetrical D-V force on a small pin inserted through a hole in the L5 SAU stem (Fig 2, Fig 3). The force vs displacement curve produced by this test had an initial nonlinear “toe” region below 1–N loads, above which a nearly linear portion of the plot was seen (Fig 3B). The slope of the regression line for this linear region represents the end-range D-V stiffness coefficient (k) of the spine segment.6, 8 We determined the slope using the line segment corresponding to the 2.0– to 3.0–N load region of each stiffness test. Each test cycled 5 times. The first cycle of each test was always discarded. The remaining 4 cycles moved between preset minimum and maximum loads of 0.4 and 3 N, respectively. The mean slope of these 4 cycles was used to determine the stiffness coefficient (k). In addition to measuring intersegmental linked stiffness (Fig 5, mean of stiffness tests L1 and L2), we examined spine stiffness within minutes after the links were removed, that is, immediate residual stiffness (Fig 5, stiffness tests U2), and at the conclusion of each of 5 discrete unlinked survival times (1, 2, 4, 8, and 12 weeks) after the link period, called long-term residual stiffness (Fig 5, stiffness tests U3). Spinous attachment unit–bearing control rats that were never linked (CSAU) were examined at comparable study times (Fig 5: CU2, CU3, and the mean of stiffness tests CL1 and CL2).
Intervertebral joint position was radiographically assessed in an additional group of 77 rats immediately after they were euthanized. Thirteen rats were filmed at the end of the linked period while the links were still in place (4 EN, 5 EF, and 4 ER), and 46 unlinked rats were filmed at the end of an unlinked period (14 EN-U, 16 EF-U, 16 ER-U). Eighteen control group animals (7 CSURG and 11 CSAU) were also evaluated for joint position. All films were obtained using a standardized stress-radiograph procedure in which each euthanized animal's lumbar spine was held in extension with a positioning jig (Fig 4A).
These lateral radiographs were exposed using a single-phase x-ray unit set at 12 mAs at 52 kVp with a 40–in target-film distance. Flexion/extension misalignment was assessed on the radiographs by measuring the L4–6 PBAs. In this procedure, the posterior body margins of L4 and L6 were extended on the radiograph, and the angle formed by their intersection was recorded (Fig 4B). Flexion was assigned a positive value (+) and extension a negative value (?). The stainless steel SAUs obscured much of the spine in dorsal-ventral radiographs; therefore, these views were not taken.
Euthanasia was accomplished by a 2–step procedure. Each rat was first anesthetized in a halothane induction chamber (4% halothane/oxygen at a 1 L/min flow rate for 3 min) and then given a lethal intraperitoneal injection of sodium pentobarbital (1.5 mg/kg). This method was rapid, nontraumatic, and consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.9
Table 1 lists all explanatory and response variables in the study. Differences in spine stiffness among control rats and before-linking stiffness of experimental rats were examined with a 1–way analysis of variance (ANOVA). The effect of age on D-V stiffness was evaluated with simple linear regression. Multiple linear regression models were used to assess the effect of potential explanatory variables (baseline stiffness, age at baseline, link period, unlinked period, and link configuration) for each of the 3 stiffness response variables (linked stiffness, immediate residual stiffness, and long-term residual stiffness) and joint position (PBA). Regression diagnostics were performed, and the estimated regression coefficients, standard errors, and P values were reported for the final models. The level of significance was set at .05.
Animal use is reported in Table 2. Three hundred twelve rats were originally planned for the biomechanical characterization studies (grand total in parentheses). However, it was observed that the axial tilt (left-right) of the L5 SAU stem in rats linked in the rotation configuration, ER, converted much of the D-V loading into a moment about the animal's body axis. This precluded valid D-V loading measurements in ER rats after they were linked, although valid baseline (prelinking) D-V stiffness measurements were obtained in this group. We discovered this problem after 34 rats had been linked in the rotation configuration. Consequently, we obtained baseline data for these 34 ER rats but did not measure linked, immediate residual, or long-term residual stiffness with them. This experimental group was discontinued in the stiffness study.
In Table 2, differences between the actual rat counts (outside parentheses) and the expected counts (inside parentheses) are due to exclusion of the ER group from measurements other than baseline values, and animal loss due to complications occurring during the 8– to 20–week period after the implant procedure (spinous fracture, implant loosening, and developing pathology). The actual number of control rats was greater than expected because additional control animals were added to the larger developmental study from which these biomechanics data were drawn.
Intervertebral fixation was assessed by comparing the D-V stiffness of each rat before linking (baseline stiffness), while linked (linked stiffness), and immediately after the links were removed (immediate residual stiffness). A subpopulation of rats was also evaluated for D-V stiffness after various unlinked periods (long-term residual stiffness). One-way ANOVA showed no statistically significant differences in intersegmental stiffness between controls and not-yet-linked animals (Table 3). Therefore, to examine whether spine stiffening increased with age, stiffness data from the control and not-yet-linked animals were pooled (D-V stiffness = 14.52 ± 4.47 N/mm [mean ± SD], n = 202). As shown in Figure 7, D-V spine stiffness increased very little over the age range examined in this study, 13 to 48 weeks, making only a negligible contribution to overall stiffness (slope = 0.08 and r2 = 0.02).
The D-V stiffness of rats tested while linked was substantially increased (linked stiffness = 44.26 ± 11.06 N/mm, a 237% increase from baseline, n = 130). Spine stiffness assessed immediately after the links were removed, immediate residual stiffness, was much less than linked stiffness but greater than baseline values (immediate residual stiffness = 19.64 ± 6.10 N/mm, a 47% increase from baseline, n = 117). Long-term residual stiffness varied with weeks unlinked but was always greater than baseline stiffness (long-term residual stiffness = 21.29 ± 6.71 N/mm, a 59% increase from baseline, n = 80). These values are grand mean values not reflecting differences because of weeks-linked, weeks-unlinked, and link configuration. Figure 8 presents a graph summary showing 4 representative plots. The control animal (dashed line) was never linked but was tested at periods equivalent to the linked rats. The 3 experimental group rats (solid lines) were all linked in the neutral configuration for 8 weeks and then unlinked for 4, 8, and 12 weeks. The 3 experimental animal plots show the general trend of marked linked stiffness (linked), immediate residual stiffness modestly increased above baseline, and additionally increased long-term residual stiffness. Multiple linear regression analysis (Table 4) reports the effects and interactions of stiffness study variables (Table 1).
Age at baseline (age before linking) was significantly related to linked stiffness. In addition, baseline stiffness was significantly related to both immediate residual stiffness and long-term residual stiffness. Therefore, both baseline stiffness and age were controlled for in each of the multiple linear regression models. Although the link period (weeks-linked) was not significantly related to linked stiffness, it was positively related to both immediate residual stiffness and long-term residual stiffness. Surprisingly, the unlinked period (weeks-unlinked) was also positively related to long-term residual stiffness. Finally, the neutral link configuration (N) had significantly higher stiffness values than the flexed configuration (F) for each of the 3 stiffness variables.
Joint position was examined in a separate population of rats. Figure 9 shows a difference in the PBA measurements from radiographs of never-linked control rats (CSAU and CSURG) and experimental animals linked in neutral, flexed, and rotated configurations (EN, EF, and ER). In addition, modeling of PBA in control rats and linked rats, with weeks-linked and configuration (CSAU, CSURG, EN, EF, and ER) as explanatory variables, showed a statistically significant difference between the control and experimental animal groups (Table 5). The link period (weeks-linked) was not a statistically important influence on PBA in rats while the links were still in place. However, in animals that had the links removed and were then examined weeks after the link period, greater link periods predicted greater PBA values (Table 6 and Fig 10). A squared weeks-linked term (weeks-linked2) was included in the multiple regression model (Table 6) to address the nonlinear portion of the linked time vs PBA plot (Fig 10). In these rats radiographically examined in the extension jig after the links were removed, configuration was not statistically significant, but greater periods of time after the links were removed (weeks unlinked) produced larger PBA values. The magnitude of this weeks-unlinked effect was small compared with the influence of weeks-linked.
Age effect on
for control and linked rats.
for unlinked rats.
The findings from this study suggest that the ELM can be a valuable tool for studying biologically significant effects of spine fixation and misalignment. We showed that the external link system provided a means for producing both experimental spine fixation and misalignment, the 2 cardinal biomechanical features of subluxation. Both were marked while the external links were in place, and significant residual stiffness and misalignment remained after the links were removed. Most interesting was the apparently progressive effect of this experimental biomechanical lesion. After the links were removed, the spine segment continued to become stiffer, and misalignment observed on the stress radiographs was greater over the subsequent 12–week period of the experiments. These observations are consistent with both subluxation theory and clinical chiropractic experience.
Posterior-Anterior and Dorsal-Ventral Stiffness Testing
Manual posterior to anterior (P-A) stiffness testing is often used by chiropractors and physical therapists to identify the site and nature of biomechanical spine lesions. [10–14] Early manual methods showed poor reliability and validity; however, a newer reference-based method developed by Maher et al  showed good reliability as well as criterion-related validity (interrater ICC[2,1] = 0.78, criterion-related validity = 0.74). [10, 11, 15] Chiradejnant et al  reported that therapists could distinguish between stiffness stimuli that vary by only 9% when using this method. The validity of this reference-based manual method was determined by assessing agreement with an instrumented stiffness testing device, the stiffness assessment machine (SAM).  Stiffness testing instruments such as the SAM apply a 3–point load wherein the loading points are widely separated on the spine. The subjects lay prone on a lightly padded table as a small indenter loads the spine P-A. The spine is treated essentially as an elastic beam suspended between the thorax and pelvis. These stiffness testing instruments have become a criterion standard against which manual methods are tested. Three such instruments have been developed and validated in the past decade: (1) the spinal physiotherapy simulator (SPS),  (2) the SAM,  and (3) the spinal posteroanterior mobilizer (SPAM).  Excellent test-retest reliability have been reported for all 3 instruments (ICC[2,1] = 0.88 SPS, 0.96 SAM, and 0.98 SPAM). [7, 16, 17] In vitro accuracy has also been established for all 3 instruments using elastic beams of known stiffness. [7, 16–18]
In our study, we used a proprietary material testing instrument, the PCM-Versa Test load frame (Mecmesin), to evaluate D-V stiffness in the rat. This is comparable with P-A stiffness in humans. In vitro test-retest studies of the load frame in our laboratory demonstrated excellent instrument reliability (ICC[2,1] = 0.998) on 3 silicone rubber pads (McMaster-Carr Elmhurst, IL) that were selected because they had similar stiffness values to what we observed in the rat spine. The PCM-Versa Test load frame has a displacement resolution of 5 µm, and the 10–N load cell is accurate to 10.00 ± 0.02 N (SD). This equipment is calibrated regularly in our facility. We have also determined the intrarater and interrater reliability (ICC[2,1] = 0.815 and 0.692, respectively) for our application of this device in measuring D-V stiffness in the rat (unpublished data).
The SAUs gave direct access to the spine segments of interest (L4–L6). Therefore, we were able to focus the 3–point load to these segments by supporting the 2 outer SAU stems (L4 and L6) with small brass turnbuckles suspended from rigid supports while the indenter loaded L5. We reasoned that focusing the load in this way would minimize the dampening error that must occur when segmental stiffness is measured using a method that actually distributes the applied load over most of the spine, as in the SPS, SAM, and SPAM approaches. Suspending the region of interest with the turnbuckle supports should also have reduced, although not completely eliminated, the confounding influence of abdominal/thoracic compression. Consequently, we believe that our load-localizing approach has face validity for reducing the competing systematic errors contributed by nonfocused loading and abdominal tissue support. Reducing these confounding influences should provide more accurate measures of segmental spine stiffness.
It was interesting that the L5 D-V stiffness observed in our pooled control rats/not-yet-linked rats (D-V stiffness = 14.52 ± 4.47 N/mm [mean ± SD], n = 202) was similar to the L4 P-A stiffness reported in humans. Lee and Liversidge  reported L4 P-A stiffness values in healthy human volunteers to be 17.5 ± 4.8 N/mm and Viner et al  reported 15.5 ± 3.3 N/mm. Because the rat has 6 lumbar vertebrae, we consider the L5 vertebra in the rat to be in a similar “biomechanical position” to the L4 vertebra in humans.
Investigators have commented on the large amount of variation in P-A stiffness across populations of healthy individuals and have suggested that, when looking at treatment effects, a comparison of stiffness changes within each subject may be of greater value than comparison between subjects. [16, 20, 21] We also found great variability in D-V stiffness across rats (see standard deviations in the intervertebral fixation section of “Results”). Consequently, the stiffness assessments in this study compared stiffness changes within rats (change from baseline).
As noted in the “Methods” section, rats linked in the rotation configuration had a substantial axial (left-right) tilt of the L5 SAU stem, which converted much of the D-V load into a moment about the animal's body axis. For this reason, animals linked in the rotation configuration were excluded from D-V stiffness testing. However, preliminary work currently being conducted by our group indicates that this vertebral rotation fixation may produce unique anatomical changes of the Z joints, when evaluated with light microscopy. In future studies, we will continue to assess the features of this configuration, including stiffness using measurement methods that are not effected by SAU stem tilt.
Evaluation of Working Hypotheses
Our working hypotheses for stiffness changes were largely supported in this study. The D-V spine stiffness of control rats was the same as that of not-yet-linked experimental rats, and this did not significantly increase with age (Hypothesis 1). This finding is consistent with a clinical study by Viner et al  of 42 men and women, aged 20 to 45 years, with no history of low back pain. They also found no statistically significant correlation between age and spine stiffness. The observation in Table 4 that age at baseline (age before linking) was negatively related to linked stiffness was because younger rats were generally linked in the longest link periods, and longer link periods produced greater stiffness.
As expected, the spine stiffness of rats tested while linked was markedly greater than SAU bearing control animals (CSAU), and linked stiffness was not significantly greater for longer link periods (Hypothesis 2). However, contrary to our expectation, rats linked in the neutral link configuration had greater D-V stiffness than rats linked in a flexed configuration. We expected that, while the links were in place, the immobilization produced by all 3 link configurations would be essentially complete, with no measurable stiffness differences among them. Our data suggest that the links produced various amounts of reduced segmental mobility but not complete immobilization. This is consistent with the concept of spine fixation used by chiropractors when referring to subluxated segments. Fixation, in chiropractic use, is synonymous with spine hypomobility rather than complete immobilization.
Retention of considerable mobility, even in surgically fused spines, has been reported in both animal and human studies. In an analysis of single-level (L5–L6), bilateral, posterolateral intertransverse process fusion in a New Zealand white rabbit model, Erulkar et al  reported statistically (P = .01) and biologically significant decreases in flexion (81%) and extension (61%) at 5 weeks postsurgery. They noted that, although posterolateral fusion reduced spine motion, it did not eliminate it. Similarly, Lee and Langrana  simulated posterolateral, posterior, and anterior fusion in human cadavers with transfixion wires and polymethylmethacrylate. They reported that posterolateral fusion increased spine stiffness by 48%, whereas posterior fusion and anterior fusion increased stiffness by 92% and 47%, respectively. Cadaver studies have also showed increased stiffness without complete rigidity immediately after implantation of fixator instruments (time 0 studies). [24–26]
We do not know why rats linked in a neutral configuration (EN) were stiffer than those linked in the flexed configuration (EF), but we note that the normal posture of the L4–L6 region in the resting (crouched) rat is moderately flexed.  The neutral link configuration in this study was obtained by yoking the 3 SAU stems of the external linking system in the position they assumed when the anesthetized animal was placed prone on a flat surface. Therefore, it could be argued that our neutral link configuration actually placed the L4–L6 region of the spine in a more extended position than that assumed when the animal is in its normal crouched posture. By contrast, the flexed link position probably placed the lumbar spine in a posture that is much closer to its resting position. Therefore, linking the L4–L6 region in neutral position may actually have placed that spine segment in an abnormal (ie, extended) posture that was closer to its end range of motion. At end range, the bony “stops” of the inferior articular processes meet the pars interarticularis of the vertebra below. 
As anticipated, we found that D-V stiffness immediately after the links were removed (immediate residual stiffness) was much less than while linked (linked stiffness), but greater than baseline (prelinked stiffness). Also, immediate residual stiffness was greater for longer linked periods and was influenced by the link configuration (Hypothesis 3). This effect of link configuration on immediate residual stiffness was similar to, but was less marked than, that observed during the link period. The opportunity for spontaneous recovery was examined by observing stiffness changes within subgroups of rats at 1, 2, 4, 8, or 12 weeks after link removal (long-term residual stiffness). As expected, D-V stiffness measured weeks after link removal was greater for longer link periods (Hypothesis 4). But, surprisingly, long-term residual stiffness also increased with greater weeks unlinked, rather than decreasing toward baseline as we anticipated (Table 4). However, this effect was less than that observed for longer link periods (weeks linked). It appears that a progressive stiffening of the spine was initiated by the experimental fixation. If this phenomenon is also present in human populations with naturally occurring spine fixations, it suggests that such lesions are progressive and have increasing biomechanical consequences.
Spine stiffness is of interest to patients and clinicians because it is widely thought that abnormal stiffness (increased or decreased) is associated with pain, degenerative change, or reduced function. [29–32] Grob et al  reported that external fixation relieved pain in 89% of patients with suspected cervical spine instability, and it has been shown that manual P-A stiffness testing can identify the level of intervertebral joint dysfunction. [34, 35] Latimer et al  reported that, in 25 patients with low back pain, P-A stiffness at the most symptomatic level decreased by a mean 8% as the level of pain decreased to at least an 80% improvement. This suggests that an 8% change in stiffness may be clinically significant. Nine of the 25 low back pain subjects in the Latimer et al  study demonstrated a 14% to 37% reduction in stiffness. Consequently, the immediate residual stiffness increases of linked animals over control or not-yet-linked animals observed in our ELM may well be biologically significant. Immediate residual stiffness increases above baseline differed with the configuration and with number of weeks linked (Table 4). Moreover, long-term residual stiffness also substantially increased with increased number of weeks linked. These observations are consistent with both subluxation theory and clinical chiropractic experience. 
Our recently published work on degenerative spine changes after experimental fixation showed one way this model may be used to explore the biologic effects of spinal fixation (hypomobility, subluxation).  Consistent with the stiffness data reported here, we observed degenerative changes in zygapophysial joints (Z-joints), and these changes were greater for animals experimentally linked for longer periods. In addition, like long-term residual stiffness, we observed progressive degenerative changes after the links were removed. However, it was interesting that spontaneous improvement in Z-joint articular surface degeneration and osteophyte formation appeared to occur in some rats after the external links were removed. When present, this improvement was associated with a link-time threshold. Some rats linked for 1 to 4 weeks, and then observed weeks after the links were removed showed improvement in articular surface degeneration. Similarly, osteophytes were less frequent and/or severe in some rats linked for 4 to 8 weeks and then observed for weeks after link removal. It was noted that rats linked for more than 4 weeks showed only progressive articular surface degeneration, and rats linked for more than 8 weeks developed only more (or larger) osteophytes during the subsequent unlinked period. These observations suggest 2 very interesting questions for future study: (1) How are D-V stiffness and degenerative Z-joint changes correlated? (2) Can therapeutic intervention stop or even reverse increased spine stiffness and/or degenerative spine changes? We are currently examining these questions with the ELM.
Misalignment is a frequently cited component of subluxation theory. [1, 37, 39] Historically, it has often been referred to as “a bone out of place.” The early notion was that subluxation was a static mechanical lesion, often demonstrable on a neutral radiograph wherein 1 vertebra was out of normal alignment with adjacent segments. This misalignment was asserted to be less than a dislocation, hence, the term sub (less than) luxation (dislocation). From its earliest presentation by the founders of chiropractic, this static perspective of the subluxation has been vigorously challenged within the chiropractic profession. [39, 40] The current view of vertebral subluxation describes a dynamic biomechanical lesion wherein an error of movement is present that may, or may not, be associated with static misalignment. [29, 41]
We expected that rats examined radiographically in the extension jig while linked (during the link period) would have decreased extension. We also thought that a stable misalignment in the sagittal plane would be achieved with the links in place and, therefore, anticipated that this decreased extension would be correlated with link configuration, but not with the length of the link period (Hypothesis 5). Our data support this hypothesis (Table 5). We anticipated that some residual of this dynamic misalignment would remain immediately after the links were removed—with weeks linked and link configuration influencing the magnitude of the PBA (Hypothesis 6). Although there was a residual misalignment immediately after the links were removed, which was significantly greater for longer link periods, the link configuration did not have a statistically significant effect (Table 6). It is possible that a link configuration effect was hidden in this small sample because the residual PBAs were small compared with the linked PBAs. Lastly, we thought that the misalignment created while linked would decrease after the links were removed, possibly to stabilize at a minimal misaligned state in the extension jig or even approximate control values (Hypothesis 7). We further expected that the magnitude of this long-term residual misalignment would be different for the 3 link configurations and would be greater for longer link periods but smaller for longer unlinked periods. As expected, misalignment was greater for longer link periods, but as with the stiffness data, misalignment was also greater for longer unlinked periods. These data further support the notion that a progressively developing process affected the biomechanics of the experimentally fixed spine segments (L4–L6).
This minimal residual radiographic misalignment in the presence of increased intervertebral stiffness suggests that malposition (misalignment) is a dynamic phenomenon that cannot be simply characterized as a bone out of place on static films. Gatterman  commented on the assertion by some that the term subluxation should be reserved for radiographically measurable disrelationships of joint surfaces. She argued that some misalignments may be insufficient to be readily discernible by current radiographic technologies, and therefore, radiographically shown misalignment should not be the sole criterion for detecting subluxation. Rather, she emphasized the role of fixation in identifying the subluxation, “It seems probable that subluxation refers to impaired mobility with or without positional alteration …. Logic leads us to conclude that we are dealing with a functional entity involving restricted vertebral movement, because it is the movement-restriction component of manipulable subluxation that responds to thrust procedures.”
Quadruped vs Biped
It is widely accepted that quadruped spines are subjected to different loads than upright human spines. Consequently, the observations made in this quadruped model must be interpreted cautiously when making inferences about bipedal spine function. This consideration was discussed in our first article on this topic.  We concluded that fundamental biomechanical similarities between the rat and human spines permit critical studies of basic biomechanical mechanisms that are common to both species. Moreover, such studies can benefit from both the similarities and differences between species. Much of the work presented here is currently being replicated in “behaviorally induced” biped rats. Biped rats spend more time on their hind legs than typical quadruped rats and have been found by investigators to more closely mimic the biomechanical features of bipeds, such as humans. [42–45] Thoughtful application of the ELM to the study of spine function, with due consideration to important experimental advantages and limitations, offers great promise. Further study is also needed to determine if the experimental hypomobility and misalignment produced in the ELM has a homologue in either the rat or the human population. Consequently, we stress that although this study supports the theoretical construct that has been historically identified as the chiropractic subluxation, it does not establish it as a lesion that actually exists in either rats or people.
The findings from this study suggest that the ELM can be a valuable tool for studying biologically significant effects of spine fixation and misalignment. We showed that the external link system provided a means for producing both experimental spine fixation and misalignment, the 2 cardinal biomechanical features of subluxation. Both spine fixation and misalignment were marked while the external links were in place, and significant residual stiffness and misalignment remained after the links were removed. Most interesting was the apparently progressive effect of this experimental biomechanical lesion. After the links were removed, the spine segment continued to become stiffer over the subsequent 12–week period of the experiments. These observations are consistent with both subluxation theory and clinical chiropractic experience. In addition to studying the biologically significant effects of subluxation, the controlled application of specific treatment interventions to this model may also allow us to study the mechanisms by which spine treatments produce their effects.
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