A Sensitive and Reliable Locomotor Rating Scale for ...

JOURNAL OF NEUROTRAUMA Volume 12, Number 1, 1995 Mary Ann Liebert, Inc., Publishers

A Sensitive and Reliable Locomotor Rating Scale for Open Field Testing in Rats D. MICHELE

BASSO, MICHAEL S. BEATTIE, and JACQUELINE C. BRESNAHAN

ABSTRACT Behavioral assessment after spinal cord contusion has long focused on open field locomotion using modifications of a rating scale developed by Tarlov and Klinger (1954). However, on-going modifications by several groups have made interlaboratory comparison of locomotor outcome measures difficult. The purpose of the present study was to develop an efficient, expanded, and unambiguous locomotor rating scale to standardize locomotor outcome measures across laboratories. Adult rats (n = 85) were contused at T7-9 cord level with an electromagnetic or weight drop device. Locomotor behavior was evaluated before injury, on the first or second postoperative day, and then for up to 10 weeks. Scoring categories and attributes were identified, operationally defined, and ranked based on the observed sequence of locomotor recovery patterns. These categories formed the Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale. The data indicate that the BBB scale is a valid and predictive measure of locomotor recovery able to distinguish behavioral outcomes due to different injuries and to predict anatomical alterations at the lesion center. Interrater reliability tests indicate that examiners with widely varying behavioral testing experience can apply the scale consistently and obtain similar scores. The BBB Locomotor Rating Scale offers investigators a more discriminating measure of behavioral outcome to evaluate treatments after spinal cord injury.

INTRODUCTION

Assessment

of long-term behavioral outcome after experimental spinal cord injury is necessary as arbiter final of therapeutic efficacy (Goldberger et al., 1990; Beattie and Bresnahan, 1989; Kunkela et Bagden al., 1992; Beattie et al., 1988; Hsu, 1992; Wrathall, 1989). Well-designed behavioral outcome measures may also identify potential mechanisms of functional recovery (Murray and Goldberger, 1974; Goldberger, 1988; Kunkel-Bagden et al., 1992). Several investigators have developed tests to measure locomotor, sensory, and reflex function in spinal cord-injured animals (Basso et al., 1993, 1994c; Wrathall, 1989; Gale et al., 1985; Goldberger et al., 1990; Kunkel-Bagden et al., 1992; Robinson and Goldberger, 1986; Goldberger, 1988; Stelzner and Cullen, 1991; Barbeau & Rossignol, 1987; Vierck, 1989; Tarlov and Klinger, 1954). These have ranged from simple qualitative descriptions of walking behavior, to elaborate combinations of tests aimed at detecting diverse but overlapping manifestations of CNS function. It is not generally agreed which type of behavioral assessment tool is the most useful or meaningful descriptor of recovery. The sensitivity of elaborate tests may make them best-suited for detailed investiga-

Department of Cell Biology, Neurobiology and Anatomy, 1

Ohio State

University, Columbus, Ohio.

BASSO ET AL.

tion of recovery mechanisms but generally impractical for use in preclinical trials. Qualitative tests, while being simple to use, usually have very limited sensitivity due to the reliance on subjective observations. For use in preclinical trials, the ideal assessment tool would be easy to use, sensitive to small, but important behavioral changes and capable of rapidly assessing general locomotor function in large numbers of experimental animals. Currently, the closest approximations to this ideal are qualitative or semiquantitative scales, such as that originally designed by Tarlov and Klinger (1954) and modified by later investigators (Bresnahan et al., 1987; Faden and Jacobs, 1983, 1984; Gale et al., 1985). The present study extends and optimizes a modified Tarlov locomotor scale (Bresnahan et al., 1987), which correlates highly with anatomical outcome (Beattie and Bresnahan, 1989; Behrmann et al., 1992) and physical descriptors of the injury (e.g., displacement, force, impulse momentum; see Behrmann et al., 1992; Stokes et al., 1992). Additionally, these locomotor measures were highly reproducible between trained raters. However, some subcategories of the modified-Tarlov scale were infrequently used and others were ambiguously defined. Scoring records of 177 rats from previous studies (Behrmann et al., 1992,1993, 1994) showed that some subcategories were rarely scored. More importantly, relatively large and consistent improvements in locomotion due to therapeutic intervention were expressed as small differences on the scale (Behrmann et al., 1993, 1994). These perceived problems may result from an inability of the scale to reflect the underlying recovery process. More precise, forced-choice operational definitions of locomotor elements should also facilitate accurate interlaboratory data transfer (Wolf, 1994). Thus we sought to improve the sensitivity and reliability of locomotor assessment by developing and evaluating a monotonie scale based on unambiguously defined categories of locomotor behavior observed during recovery from spinal cord contusion in the rat. A follow-up study (Basso et al., 1994b) evaluated the usefulness and reliability of the scale in the context of a multicenter preclinical trial. A preliminary report of some of the data included in the present paper has appeared in abstract form (Basso et al., 1994a).

METHODS

Subjects A total of 85 female rats (250-300 g) sustained spinal cord contusions and were used to develop or validate the BBB scale. Some rats were used for both scale development and validation while others were used exclusively for validation purposes as shown in Table 1. For instance, of 11 rats with mild OSU contusions, six were used for both development and validation (severity parameters). The remaining five rats were used solely for validation (severity, anatomy, and variability parameters). Sprague-Dawley (n = 71) and Long-Evans (n 13) strains were used (Harlan Sprague Dawley Inc., Indianapolis, IN). One was sacrificed during the first postoperative week due to autotomy and, one Sprague-Dawley rat died of unknown causes during the 11th postoperative week. rat Long-Evans -

Surgical Procedures All rats were subjected to surgical procedures at Ohio State University (OSU) as previously described in detail by Behrmann et al. (1992). Briefly, the rats were anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg) and were given prophylactic antibiotics (gentomycin sulfate 1 mg/kg). After a mid-thoracic laminectomy exposing T7, T8, or T9 cord segmental level, the rats were contused with the OSU impactor or the New York University (NYU) impactor provided by Dr. Wise Young. The groups will be referred to as OSU mild, OSU moderate, NYU mild, and NYU moderate. (For detailed descriptions of each device see Stokes et al., 1992; Grüner, 1992; Constantini and Young, 1994.) Briefly, the OSU impactor is an electromagnetic device that creates a contusion by displacing the cord surface a specified distance. In the present study, the spinal cord was displaced 0.8 or 0.9 mm for mild contusions and 1.1 mm for moderate contusions. The NYU impactor is a weight-drop device that releases a 10-g rod from various heights onto the exposed cord. Movements of the impact rod and vertebral column are recorded using optical potentiometers. For the present study, the rod was dropped from heights of 6.25 mm for mild contusions and 12.5 and 25 mm for moderate contusions. Two rats fell out of the spinal clamps upon impact from 12.5 or 25 mm heights and 2

BBB LOCOMOTOR RATING SCALE Table 1.

Sprague-Dawley and Long-Evans Rats Mild or Moderate Contusions2

with

Scale validation

Injury type OSU device I Mild

(«=1D Moderate

(n

=

Scale devel.

Interrater

Severity

Anatomy

SDbef

9/1/93

9/14/93

5 SDef

1 SDe

6SDb

11

11 SDC

12 SDcf

6 SDcf

1 SDf

0

42 SDd

0

0

0

0

2 LE«

4 LE«

0

4

5

SDef

variability

12)

Mild and Moderate n = 42 NYU device Mild

11 LE«

10 LE«

8 (2 LE« 6 SD)

6 SD«

(«= 11) Moderate

(n

=

8)

(2 LE« 2SD)

aThe number of Sprague-Dawley (SD) and Long-Evans (LE) rats with mild or moderate contusions produced by the OSU or NYU impactor used for development and validation of the BBB scale. Scale validation encompassed sensitivity to contusion severity (severity), the relationship between contusion size and behavioral performance (regression), and interrater variability measured on two occasions (variability). 'Tested daily for 10 days then 1-3 times a week for 3 weeks (n = 6). Tested daily for 21 days then 1-3 times a week for 7 weeks («=11). dMonitored weekly at various points between 1 and 70 dpo (« = 42). Tested 3 times a week for 5 weeks (n = 3). •Videotaped occasionally from 1 to 59 dpo; tested periodically between 60 and 90 DPO

(n 3). =

Tested 1 and/or 2 DPO then at least once a week for 3-12 weeks (n = 19).

the impactor hit bone rather than dura in another rat with a contusion from 12.5 mm. Hence, these rats tained less contusive force at impact and were included with the mild rather than the moderate group.

sus-

Pretesting Procedures Three open field environments were used in this study: a circular metal enclosure (106.7 cm diameter, cm wall height), a molded plastic wading pool with a dimpled floor (100 cm diameter, 21 cm wall height), and a smaller plastic wading pool with a smooth floor (90 cm diameter, 7 cm wall height). The rats were exposed to the testing environment daily for at least 10 days or twice a day for at least 5 days, with most sessions occurring consecutively. Several rats were placed in the open field at one time and were encouraged to locomote continuously without crawling up the sides of the enclosure. Each rat was handled several times during each 30-60 min session so that they became accustomed to being picked up and moved while in the open field. Pretesting sessions were continued until the rats no longer showed signs of fear (crouching, cowering away from the examiner, little or no locomotion, frequent defecation and urination, piloerection, vocalizations, and failure to groom).

61

Testing Procedures Two examiners participated in all open field tests and were positioned across from each other to observe both sides of the rat. Rats were tested either alone for 4 min or in pairs for 5 min. Experience indicated that 4 min sessions provided sufficient time to observe and record behavioral recovery of individual rats 3

BASSO ET AL. Table 2.

The 21-Point

Basso, Beattie, Bresnahan Locomotor Rating Scale Definitions

0 1 2

of

Categories

No observable hindlimb (HL) movement Slight movement of one or two joints, usually the Extensive movement of one joint

hip

and

and

Operational

Attributes

and/or knee

or

3 4 5 6 7 8

extensive movement of one joint and slight movement of one other joint Extensive movement of two joints Slight movement of all three joints of the HL Slight movement of two joints and extensive movement of the third Extensive movement of two joints and slight movement of the third Extensive movement of all three joints of the HL Sweeping with no weight support or

9

plantar placement of the paw with no weight support Plantar placement of the paw with weight support in stance only (i.e.,

when

stationary)

or

occasional, frequent,

10 11 12 13 14

or consistent weight supported dorsal stepping and no plantar stepping weight supported plantar steps, no forelimb (FL)-HL coordination Frequent to consistent weight supported plantar steps and no FL-HL coordination Frequent to consistent weight supported plantar steps and occasional FL-HL coordination Frequent to consistent weight supported plantar steps and frequent FL-HL coordination Consistent weight supported plantar steps, consistent FL-HL coordination; and predominant paw position during locomotion is rotated (internally or externally) when it makes initial contact with the surface as well as just before it is lifted off at the end of stance

Occasional

or

15

16

frequent plantar stepping, consistent FL-HL coordination, and occasional dorsal stepping Consistent plantar stepping and consistent FL-HL coordination; and no toe clearance or occasional toe clearance during forward limb advancement; predominant paw position is parallel to the body at initial contact Consistent plantar stepping and consistent FL-HL coordination during gait; and toe clearance occurs frequently during forward limb advancement; predominant paw position is parallel at initial contact and rotated at lift off

(continued on facing page)

with minimal risk of missing

key findings.

Rats

were

assessed

individually for

scale validation and inter-

reliability testing. During open field testing, rats were encouraged to continuously locomote. Rats that remained stationary for longer than 15-20 sec were enticed to move by having them follow a pencil or a piece of paper, or by lightly tapping or scratching on the side of the open field. If the animal failed to respond to these stimuli, it was picked up by the forequarters and placed in the center of the open field, which usually caused it to move toward the side. Special care was taken to avoid touching the tail and/or hindquarters during testing because the stimulation seemed to affect motor performance. In tests evaluating pairs of rats, observation periods were sometimes extended beyond 5 min for more accurate assessments of toe clearance, paw position, or forelimb-hindlimb coordination. In cases of borderline locomotor performance or disagreements between examiners, scores indicating greater deficits were assigned. All hindlimb movements were recorded except those that were obviously part of a reflex or were elicited by the touch of an examiner or another animal. For example, one unique reflex seen early after contusion but not scored as a locomotor component was flattening of the lumbar spine, curling of the tail, and extensive bilateral flexion of the hip, knee, and ankle joints. This reflex may have resulted from perineal stimulation as the rat pulled itself forward with the forelimbs. rater

Scale

Development

To develop the scale, we documented movement patterns displayed by rats during recovery. Mild and moderate groups were tested in pairs for 5-min periods as noted in Table 1. Forty-two rats used for scale 4

BBB LOCOMOTOR RATING SCALE Table 2. 17

18

19

20

21

(continued)

Consistent plantar stepping and consistent FL-HL coordination during gait; and toe clearance occurs frequently during forward limb advancement; predominant paw position is parallel at initial contact and lift off Consistent plantar stepping and consistent FL-HL coordination during gait; and toe clearance occurs consistently during forward limb advancement; predominant paw position is parallel at initial contact and rotated at lift off Consistent plantar stepping and consistent FL-HL coordination during gait; and toe clearance occurs consistently during forward limb advancement; predominant paw position is parallel at initial contact and lift off; and tail is down part or all of the time Consistent plantar stepping and consistent coordinated gait; consistent toe clearance; predominant paw position is parallel at initial contact and lift off; tail consistently up; and trunk instability Consistent plantar stepping and coordinated gait, consistent toe clearance, predominant paw position is parallel throughout stance, consistent trunk stability, tail consistently up

Definitions less than half the range of joint motion Extensive: movement through more than half of the range of joint motion Sweeping: rhythmic movement of HL in which all three joints are extended, then fully flex and extend again; animal is usually sidelying, the plantar surface of paw may or may not contact the ground; no weight support across the HL is evident No Weight Support: no contraction of the extensor muscles of the HL during plantar placement of the paw; or no elevation of the hindquarter Weight Support: contraction of the extensor muscles of the HL during plantar placement of the paw, or elevation of the hindquarter Plantar Stepping: The paw is in plantar contact with weight support then the HL is advanced forward and plantar contact with weight support is reestablished Dorsal Stepping: weight is supported through the dorsal surface of the paw at some point in the step cycle FL-HL Coordination: for every FL step an HL step is taken and the HLs alternate Occasional: less than or equal to half;