- Email: [email protected]
Neuromuscular evaluation using rat gait analysis
JtlUR?WOF ME ELSEVIER
Journal of Neuroscience Methods 61(1995) 79-84
Neuromuscular evaluation using rat gait analysis P.M. Santos ay* , S.L. Williams b, S.S. Thomas ’ of
a Division Otolaryngology, Southern Illinois University, Springfield, IL 62794, USA; ’ School of Veterinary Medicine, University of Illinois, Champaign, IL, USA, ’ Shriners Hospital Crippled Children, Portland, OR, USA
for
Received 16 September 1994; revised12December 1994;accepted 29January1995
Abstract We have developeda rat gait analysismodel to evaluate if ankle angleand other associatedgait parameterscould consistently define normal peroneal nerve and anterior tibialis musclefunction. The secondpart of the study was designedto determineif sucha model would be usefulto measurerecovery of function after a peronealnerve crush injury (NCI). A clear Plexiglastunnel was designedfor high-speedframe videotaping and subsequentcomputergraphicgait measurementand analysis.Normal gait patterns for ankle angle,back height, step and stride lengthsand the stance and swingtimes were determined in 8 rats. Data analysisdemonstratedno significant left/right differencesfor any of the variables(ANOVA) with the exception of step length. Subsequently,12 rats with a peronealNC1 were evaluated.All gait parametersevaluatedfrom the injured sidewere significantly different from the uninjured side after injury except stride length. Ankle anglewasthe most sensitiveoutcomevariable. Weekly gait analysisprovided objective measurementsas the ankle angle gradually returned to normal within 3 weeks. The rat gait model is a sensitiveand reproducible method for non-invasive evaluation of neuromuscular function during nerve recovery after
a peroneal crush injury. Keywords:
Gait; Nerve injury; Nerve regeneration;Model; Nerve evaluation
1. Introduction
The study of motor nerve injury and regeneration in rodent models is dependent on accurately determining meaningful outcome variables. A multitude of outcome variables have been described in animal models or techniques including axonal histological changes and electrophysiologic changes (including compound action potentials, conduction rates, electromyography, and the tension transduction device (Spyropoulos et al., 1994)). The sciatic function index (SF11 model was designed to test function of rat sciatic nerve and reinnervated muscle groups (de Medinaceli et al., 1982; Hare et al., 1992). A new model based on rat gait has been developed utilizing the same nerve muscle group, i.e., per-
Corresponding author: P.M. Santos, M.D., M.S., Division of Otolaryngology, P.O. Box 19230, Southern Illinois University, Springfield, IL 62794, USA. Tel.: (217) 524-0236; Fax: (217) 524-0253;
E-mail:[email protected]. ElsevierScienceB.V. SSD10165-0270(95)00026-7
oneal nerve and anterior tibialis muscle, as the tension transduction device. This study tested the hypothesis that the loss of dorsiflexion at the ankle joint results in an increased ankle angle compared to an internal control, i.e., the contralateral uninjured nerve. Furthermore, the ankle angle should return toward a normal ankle angle if nerve regeneration or healing were to occur. In order to test this hypothesis we developed equipment to record and analyze normal rat gait patterns as well as animals having sustained a relatively mild injury, i.e., a peroneal nerve crush. Compensatory gait changes frequently occur as a result of lower extremity nerve injuries. In bipeds, a foot drop during the swing phase results in circumduction of the leg about the hip, allowing the leg to swing through and prepare for the next step. In addition, there is a decrease in stride length, step length, swing phase times and an increase in stance phase times. We are unaware of any published gait research in the study of motor nerve injury which objectively quantifies the return of function in rodents.
80
P.M.
Sun~os et al. /Jound
of Neuroscience
Methods
61 (1995)
79-84
2. Methods
2.2. Animal
2.1. Equipment
Rats are trained to walk through the tunnel prior to actual test runs. Non-lighted boxes at each end of the lighted tunnel tend to attract them into the darkness. Two training periods are generally required for the rats to adapt to the tunnel. Light tapping on the boxes encourages the rats to walk to the opposite end of the tunnel into the box. The lower extremities are shaved and treated with Nair hair remover (Carter Products, New York, NY) after a brief anesthetic (2 days prior to videotaping) to improve the visual image obtained for recording and analysis. The testing protocol includes videotaping 5 separate runs viewing both right and left sides for each rat. This procedure is performed to obtain control/normal data and at regular time intervals after nerve injury. The rats in this study were evaluated before injury to obtain normal gait parameters and test consistency as well as 1, 2, 3 and 4 weeks after nerve crush injury (NCI).
A clear Plexiglas tunnel (70 cm long, 8.5 cm wide, 15.5 cm tall) was constructed and set on a box holding one F20T/12D Phillips 20 W fluorescent light. An identical light box rests on top of the Plexiglas tunnel with two lights. The tunnel is illuminated from above and below. Additional lighting is obtained from two 500 W quartz Model TSL 1000 halogen flood lights (Regent Lighting, Burlington, NC) placed 5 ft from the tunnel. Placed 172 cm from the tunnel and perpendicular to it the Toshiba microchip CCD KM40 camera (l/1000 shutter speed) (Toshiba, Tokyo, Japan), is fitted with a 50-mm lens and views the central 21 cm of the tunnel. The JVC super VHS recorder (BR-53784, JVC Elmwood Park, NJ) and Sony 13 inch Monitor PMV-13MD (Sony, Tokyo, Japan) are used to record and view the image. A Horita TRG-50 SMPTE time code generator (Horita, Mission Viejo, CA) is recorded onto the videotape audio signal allowing timing of gait parameters. The recorded image is captured with the Scion LG-3 Video Capture Card (Scion, Fredrick, MD) and Scion-adapted NIH public-domain software IMAGE version 1.51 (NIH, Bethesda, MD). The digitized image is analyzed on a 15 in. Apple Portrait Display Ml030 Monitor and driven by a Macintosh Centris 650 computer (Apple, Cupertino, CA) (Fig. 1).
handling
2.3. Gait parameters
Female Wistar adult rats (initial age range: 50-85 days; initial weight: 180-200 g) were tested. Rats were selected for the study because they are inexpensive, common and provide a peripheral nerve regeneration model compared to other animals. The videotaped
Fig. 1. The photograph demonstrates a’captured SvkIS video image of rat gait on the computer monitor angle is determined from the intersecting lines passing through the rat knee to ankle and rat metatarsal
for analysis of ankle head to ankle.
angle.
The ankle
P.M. Suntos et al. /Journal
of Neuroscience
images were studied for uninjured rats (n = 8) to determine within animal right and left variation as well as between-animal variation. Nerve crush animals (n = 12) were studied until function returned to normal. Only one leg was injured allowing the other side to serve as a control. The various gait parameters studied included ankle angle (degrees), back height (cm), stride length (cm), step length (cm), stance and swing phase (seconds). Ankle angle is defined by the intersection of lines extending from the knee to the ankle and the metatarsal head to the ankle. Back height is measured to determine the extent of compensation required to clear the foot during the swing phase. Stride length is defined as the distance traveled in centimeters of one foot through the gait cycle. Step length is defined as the distance in centimeters of the hind foot on the ipsilateral side to the hind foot of the contralateral side. Stance phase is the duration of time the ipsilatera1 toe touches the ground. Swing phase is the duration of time the ipsilateral toe is not touching the ground.
2.4. Surgical procedures
Anesthesia was administered with intraperitoneal injections of 40-80 mg/kg Ketamine (Fort Dodge Laboratories, Fort Dodge, IA) combined with 5-10 mg/kg of Xylazine (Lloyd Laboratories, Shenandoah, IA). Supplemental anesthesia was maintained with lo-20 mg/kg of Ketamine and 1-3mg/kg of Xylazine. This is the recommended technique by the veterinarians at our institution and is approved by the American Veterinary Medical Association. Nerve crush injury was carried out after administering the anesthetic, shaving the fur, cleansing the skin with betadine solution followed by alcohol, incising the skin and then the biceps femoris muscle thereby exposing the peroneal nerve. One millimeter width of the peroneal nerve was crushed with a no. 5 Dumont straight forceps 7 mm proximal to the nerve insertion into the muscle. The forcep was held closed with the index finger and thumb for 30 s. The nerve injury site was clear after the crush. Care was taken to avoid tearing or lacerating the nerve. The surgical field was closed with absorbable Vicryl 4-O suture in a single muscle layer and the skin closed with a staple gun.
Methods
61 (1995)
81
79-84
3. Results 3.1. Normal rat gait evaluation
Ankle angle was evaluated in 2 phases - midswing and terminal. The 8 normal animals demonstrated no significant left/right differences in ankle angle. Variability did exist between animals: Mid-Swing Phase significance level (P < 0.0001) (Fig. 2); Terminal-Swing Phase significance level (P < 0.04) (Fig. 3). Step length, the distance from ipsilateral toe touch to the next toe touch, demonstrated some left/right variability (P < 0.003) and between-animal variability (P < 0.005) (Fig. 4). Stride length, the greatest distance between the 2 contralateral toe touches, demonstrated no left/right variability or between animal variability (Fig. 5). Back Height, the greatest distance from the ground to the back of the animal during terminal-swing ankle angle phase, demonstrated no left/right variability but significant between animal variability (P < 0.0001) (Fig. 6). Stance Phase, the duration of time of ipsilateral toe touch to ground, demonstrated no left/right variability but significant between-animal variability (P < 0.0001) (Fig. 7). Swing Phase, the duration of time while the ipsilateral toe is between toe to ground touch, demonstrated no left/right variability but significant between-animal variability (P < 0.0001) (Fig. 8). 3.2. Nerve crush gait evaluation
After nerve crush all but one of the gait parameters demonstrated a significant difference (P < 0.001) between the control and the crushed peroneal nerve sides. The most obvious visual difference was seen for ankle angles. Dorsiflexion ankle angles (both mid- and terminal-swing phase) and step length were greater than control at 1,7, and 14 days (P < 0.0001) after NC1 and returned to the same values as contralateral uninjured control side by 22 days (P > 0.05) (Figs. 9 and 10). The other parameters (back height, stance time
2.5. Data analysis Animal
Measurements from each parameter were evaluated within and between groups using ANOVA and post hoc Tukey testing as needed.
Number
Fig. 2. Ankle angle, mid-swing phase: the graph demonstrates the ankle angle during the midswing phase of recovery for the 8 different animals tested. The range of degrees are specified based on the 95% confidence interval.
P.M. Santos er al. /Journal
of Nnrroscience
Methods
61 (1995)
79-54
12
Stride (M%
Length,
tz%anca8 Interval)
10 s 4 2
0,
, 1
, 2
, 3
Animal
, 4
, 5
, 6
, 7
0
,
12
8
3-45
Animal
Number
Fig. 3. Ankle angle, terminal-swing phase: the graph demonstrates the ankte angle during the terminal-swing phase of recovery for the 8 different animals tested. The range of degrees are specified based on the 95% confidence interval.
and swing time) were significantly different for 1 and (P < 0.0001) and 7 days (P = 0.0001) after NC1 and returned to the same values as contralateral uninjured control side by 14 days (P > 0.05, figures not shown). Stride length after nerve crush never varied from control. Within 3 weeks of the nerve injury, there were no visible or measurable differences between injured and uninjured sides for any gait parameter. All of the aforementioned parameters were also significant for change through time as well as an interaction of side and time.
6
78
Number
Fig. 5. Stride length: the graph demonstrates stride length for the 8 animals with 95% confidence interval.
'1'2'3'4.5'6.7.8.
Animal
Number
Fig. 6. Back height: the graph demonstrates the animals with 95% confidence interval.
back height of the 8
4. Discussion A rat gait analysis model has been developed and tested. It utilizes the rat peroneal nerve and anterior tibialis muscle group for study of gait parameters. Evaluation of the rat gait model with normal uninjured rats demonstrated general consistency of parameters. It is possible that the variability we found between animals is due to gait speed. Hruska et al. (1979) demonstrated rat gait stance time decreases and stride length increases as a function qf speed. Speed was not controlled for in this study but will be evaluated in
‘1.
Animal
step
length
for the 8
Nuntbw
Fig. 7. Stance phase: the graph demonstrates the stance the 8 animals tested with 95% confidence interval.
12
2'3'4'5.6.7-8. ARimmNumbu
Fii. 4. Step length: the graph demonstrates animals with 95% confidence intelvat.
1 -2'3'4'5'6'7'8.
3 4 Anfmal
5
B
7
phase
for
8
Number Fig. 8. Swing phase: the graph demonstrates the swing 8 animals tested with 95% confidence interval.
phase for the
P.M. Santos et al. /Journal
of Neuroscience
Methods
61 (199.5)
79-84
83
80 80 70 50
Terminal ” Ankle Angle 48 Btd Dev 38 20
Before Injury
* Significant
1 day
7days
14days
Mdays
28days I
Fig. 9. Nerve crush vs. control for ankle angle. This graph demonstrates the average ankle angle measured from the peroneal NC1 leg compared to the contralateral uninjured leg from 12 animals evaluated before injury, 1 day after injury, 7, 14, 22, and 28 days after injury. Ankle angle was significantly different between injured and controlled legs on days 1, 7, and 14. There was no significant difference on days 22 and 28 between injured and uninjured legs.
future studies. The variability of left and right uninjured sides was negligible for most parameters (e.g., ankle angle). As a result of this finding we elected to evaluate a unilateral crush injury to determine if the uninjured leg could serve as an internal control to the injured side. Within the second part of the study, the peroneal NC1 was quantified based on weekly ankle angle measurements and other gait parameters. As the paresis resolved the ankle angle returned to normal uninjured values. Of the various gait parameters measured after nerve crush, the ankle angle and step length appear to be the most sensitive outcome variables to evaluate injury because they were the last to return to normal. However, between these two parameters ankle angle is more useful since step length demonstrated some left right variability in uninjured animals and ankle angle did not. The finding that ankle angle is the most sensitive outcome variable for NC1 is reasonable since all of the other parameters are a result of compensatory changes to the increased ankle angle. As a result of this study we were able to conclude that the mea-
surement of ankle angles alone is sufficient for future studies since it is the most sensitive indicator or NC1 with the latest return to normal control values. The most common nerve model for study in rodents is the sciatic nerve. The sciatic nerve is surgically accessible and sufficiently large for various surgical procedures. However, the sciatic nerve is a mixed nerve, carrying motor axons for antagonistic muscle groups (ankle dorsiflexion and plantarflexion) and sensory axons to the lower extremity. Regeneration of a sectioned sciatic nerve results in severe synkinesis of motor axons into both anterior and posterior muscle groups. In addition, proximal motor axons may regenerate into distal sensory axons and vice versa. Unlike the sciatic nerve, the peroneal nerve innervates muscles of dorsiflexion and no antagonistic muscle groups, thus obviating some of the synkinesis attributable to motor nerves with antagonistic muscle function. However, aberrant nerve regeneration secondary to sensory and afferent spindle fibers still occurs if the axonal basal lamina has been interrupted as in a transection nerve injury. We attempted to avoid complicating this
10 9 8 StepLength, kSt?&w
3 5 4 3
q Control
:
a Injured 8 * Significant
gz
1 day
7days
14days
22days
28days I
Fig. 10. Nerve crush vs. control for step length. This graph demonstrates the step length before injury and after injury for the injured leg compared to the controlled leg. A significant difference existed on days 1, 7, and 14 between the injured and controlled legs. No significant difference was measured on days 22 and 28.
84
P.M. Santos et al. /Journal
of Neuroscience
stage of the gait analysis model evaluation by performing a NC1 and not a transecting injury. The evaluation of more complicated injuries are underway. SF1 or ‘rat tracking’ method evaluates the footprints of the rat after nerve injury. The technique serves as a useful means of evaluating the sciatic nerve as a function of the footprint, i.e., toe spread, stride length, etc. (de Medinaceli et al., 1982). Although not a direct measure of nerve muscle function, it does serve as a means of evaluating nerve regeneration. SF1 values are subject to measurement error associated with the complex motions of lower extremity swing. Since the sciatic nerve innervates antagonistic muscle groups a variable and significant amount of synkinesis occurs which increases SF1 variability. Furthermore, modifications of the SF1 for isolated tibia1 nerve injury or peroneal nerve injury have had moderate and limited success respectively (Bain et al., 1989; Hare et al., 1992). Difficulties with SF1 and the inability to directly measure muscle force led our laboratory to the development of the tension transduction device (Spyropoulos et al., 1994) and gait analysis model utilizing the rat peroneal nerve/anterior tibialis muscle. Two advantages of the gait analysis model over the tension transduction device are the ability to monitor nerve function without surgical exposure of the nerve and the capacity for multiple non-invasive evaluations during regeneration allowing for measurement of rate of recovery.
Methods
61 (1995)
79-84
Acknowledgements The author would like to thank Ms. Shirley Gossard for her assistance in preparing the manuscript, Ms. Caria Shaffer for data entry and Larry Hughes, Ph.D., for his assistance in managing the statistical data. This work was supported by funding from the Southern Illinois University, School of Medicine, Central Research Committee 1993 (Central Research Grant 3093).
References Bain, J.R., Mackinnon, S.E., and Hunter, D.A. (1989) Functional evaluation of complete sciatic, peroneal, posterior tibia1 nerve lesions in the rat. Plast. Reconstr. Surg., 83: 129-136. De Medinaceli, L., Freed, W.J., and Wyatt, R.J. (1982) An index of the functional condition of rat sciatic nerve based on measurements made from walking tracks. Exp. Neural., 77: 634443. Hare, G.M.T., Evans, P.J., Mackinnon, S.E., and Best, T.J. (1992) Walking track analysis: a long term assessment of peripheral nerve recovery. Plast. Reconstr. Surg., 89: 251-258. Hruska, R.E., Kennedy. S., and Silbergeld, E.K. (1979) Quantitative aspects of normal locomotion in rats. Life Sci., 25: 171-180. Spyropoulos, B.P., Williams. S., and Santos, P.M. (1994) Tension transduction device for functional evaluation of the rat peroneal nerve. J. Neurosci. Methods, 53: 95-100.
Journal of Neuroscience Methods 61(1995) 79-84
Neuromuscular evaluation using rat gait analysis P.M. Santos ay* , S.L. Williams b, S.S. Thomas ’ of
a Division Otolaryngology, Southern Illinois University, Springfield, IL 62794, USA; ’ School of Veterinary Medicine, University of Illinois, Champaign, IL, USA, ’ Shriners Hospital Crippled Children, Portland, OR, USA
for
Received 16 September 1994; revised12December 1994;accepted 29January1995
Abstract We have developeda rat gait analysismodel to evaluate if ankle angleand other associatedgait parameterscould consistently define normal peroneal nerve and anterior tibialis musclefunction. The secondpart of the study was designedto determineif sucha model would be usefulto measurerecovery of function after a peronealnerve crush injury (NCI). A clear Plexiglastunnel was designedfor high-speedframe videotaping and subsequentcomputergraphicgait measurementand analysis.Normal gait patterns for ankle angle,back height, step and stride lengthsand the stance and swingtimes were determined in 8 rats. Data analysisdemonstratedno significant left/right differencesfor any of the variables(ANOVA) with the exception of step length. Subsequently,12 rats with a peronealNC1 were evaluated.All gait parametersevaluatedfrom the injured sidewere significantly different from the uninjured side after injury except stride length. Ankle anglewasthe most sensitiveoutcomevariable. Weekly gait analysisprovided objective measurementsas the ankle angle gradually returned to normal within 3 weeks. The rat gait model is a sensitiveand reproducible method for non-invasive evaluation of neuromuscular function during nerve recovery after
a peroneal crush injury. Keywords:
Gait; Nerve injury; Nerve regeneration;Model; Nerve evaluation
1. Introduction
The study of motor nerve injury and regeneration in rodent models is dependent on accurately determining meaningful outcome variables. A multitude of outcome variables have been described in animal models or techniques including axonal histological changes and electrophysiologic changes (including compound action potentials, conduction rates, electromyography, and the tension transduction device (Spyropoulos et al., 1994)). The sciatic function index (SF11 model was designed to test function of rat sciatic nerve and reinnervated muscle groups (de Medinaceli et al., 1982; Hare et al., 1992). A new model based on rat gait has been developed utilizing the same nerve muscle group, i.e., per-
Corresponding author: P.M. Santos, M.D., M.S., Division of Otolaryngology, P.O. Box 19230, Southern Illinois University, Springfield, IL 62794, USA. Tel.: (217) 524-0236; Fax: (217) 524-0253;
E-mail:[email protected]. ElsevierScienceB.V. SSD10165-0270(95)00026-7
oneal nerve and anterior tibialis muscle, as the tension transduction device. This study tested the hypothesis that the loss of dorsiflexion at the ankle joint results in an increased ankle angle compared to an internal control, i.e., the contralateral uninjured nerve. Furthermore, the ankle angle should return toward a normal ankle angle if nerve regeneration or healing were to occur. In order to test this hypothesis we developed equipment to record and analyze normal rat gait patterns as well as animals having sustained a relatively mild injury, i.e., a peroneal nerve crush. Compensatory gait changes frequently occur as a result of lower extremity nerve injuries. In bipeds, a foot drop during the swing phase results in circumduction of the leg about the hip, allowing the leg to swing through and prepare for the next step. In addition, there is a decrease in stride length, step length, swing phase times and an increase in stance phase times. We are unaware of any published gait research in the study of motor nerve injury which objectively quantifies the return of function in rodents.
80
P.M.
Sun~os et al. /Jound
of Neuroscience
Methods
61 (1995)
79-84
2. Methods
2.2. Animal
2.1. Equipment
Rats are trained to walk through the tunnel prior to actual test runs. Non-lighted boxes at each end of the lighted tunnel tend to attract them into the darkness. Two training periods are generally required for the rats to adapt to the tunnel. Light tapping on the boxes encourages the rats to walk to the opposite end of the tunnel into the box. The lower extremities are shaved and treated with Nair hair remover (Carter Products, New York, NY) after a brief anesthetic (2 days prior to videotaping) to improve the visual image obtained for recording and analysis. The testing protocol includes videotaping 5 separate runs viewing both right and left sides for each rat. This procedure is performed to obtain control/normal data and at regular time intervals after nerve injury. The rats in this study were evaluated before injury to obtain normal gait parameters and test consistency as well as 1, 2, 3 and 4 weeks after nerve crush injury (NCI).
A clear Plexiglas tunnel (70 cm long, 8.5 cm wide, 15.5 cm tall) was constructed and set on a box holding one F20T/12D Phillips 20 W fluorescent light. An identical light box rests on top of the Plexiglas tunnel with two lights. The tunnel is illuminated from above and below. Additional lighting is obtained from two 500 W quartz Model TSL 1000 halogen flood lights (Regent Lighting, Burlington, NC) placed 5 ft from the tunnel. Placed 172 cm from the tunnel and perpendicular to it the Toshiba microchip CCD KM40 camera (l/1000 shutter speed) (Toshiba, Tokyo, Japan), is fitted with a 50-mm lens and views the central 21 cm of the tunnel. The JVC super VHS recorder (BR-53784, JVC Elmwood Park, NJ) and Sony 13 inch Monitor PMV-13MD (Sony, Tokyo, Japan) are used to record and view the image. A Horita TRG-50 SMPTE time code generator (Horita, Mission Viejo, CA) is recorded onto the videotape audio signal allowing timing of gait parameters. The recorded image is captured with the Scion LG-3 Video Capture Card (Scion, Fredrick, MD) and Scion-adapted NIH public-domain software IMAGE version 1.51 (NIH, Bethesda, MD). The digitized image is analyzed on a 15 in. Apple Portrait Display Ml030 Monitor and driven by a Macintosh Centris 650 computer (Apple, Cupertino, CA) (Fig. 1).
handling
2.3. Gait parameters
Female Wistar adult rats (initial age range: 50-85 days; initial weight: 180-200 g) were tested. Rats were selected for the study because they are inexpensive, common and provide a peripheral nerve regeneration model compared to other animals. The videotaped
Fig. 1. The photograph demonstrates a’captured SvkIS video image of rat gait on the computer monitor angle is determined from the intersecting lines passing through the rat knee to ankle and rat metatarsal
for analysis of ankle head to ankle.
angle.
The ankle
P.M. Suntos et al. /Journal
of Neuroscience
images were studied for uninjured rats (n = 8) to determine within animal right and left variation as well as between-animal variation. Nerve crush animals (n = 12) were studied until function returned to normal. Only one leg was injured allowing the other side to serve as a control. The various gait parameters studied included ankle angle (degrees), back height (cm), stride length (cm), step length (cm), stance and swing phase (seconds). Ankle angle is defined by the intersection of lines extending from the knee to the ankle and the metatarsal head to the ankle. Back height is measured to determine the extent of compensation required to clear the foot during the swing phase. Stride length is defined as the distance traveled in centimeters of one foot through the gait cycle. Step length is defined as the distance in centimeters of the hind foot on the ipsilateral side to the hind foot of the contralateral side. Stance phase is the duration of time the ipsilatera1 toe touches the ground. Swing phase is the duration of time the ipsilateral toe is not touching the ground.
2.4. Surgical procedures
Anesthesia was administered with intraperitoneal injections of 40-80 mg/kg Ketamine (Fort Dodge Laboratories, Fort Dodge, IA) combined with 5-10 mg/kg of Xylazine (Lloyd Laboratories, Shenandoah, IA). Supplemental anesthesia was maintained with lo-20 mg/kg of Ketamine and 1-3mg/kg of Xylazine. This is the recommended technique by the veterinarians at our institution and is approved by the American Veterinary Medical Association. Nerve crush injury was carried out after administering the anesthetic, shaving the fur, cleansing the skin with betadine solution followed by alcohol, incising the skin and then the biceps femoris muscle thereby exposing the peroneal nerve. One millimeter width of the peroneal nerve was crushed with a no. 5 Dumont straight forceps 7 mm proximal to the nerve insertion into the muscle. The forcep was held closed with the index finger and thumb for 30 s. The nerve injury site was clear after the crush. Care was taken to avoid tearing or lacerating the nerve. The surgical field was closed with absorbable Vicryl 4-O suture in a single muscle layer and the skin closed with a staple gun.
Methods
61 (1995)
81
79-84
3. Results 3.1. Normal rat gait evaluation
Ankle angle was evaluated in 2 phases - midswing and terminal. The 8 normal animals demonstrated no significant left/right differences in ankle angle. Variability did exist between animals: Mid-Swing Phase significance level (P < 0.0001) (Fig. 2); Terminal-Swing Phase significance level (P < 0.04) (Fig. 3). Step length, the distance from ipsilateral toe touch to the next toe touch, demonstrated some left/right variability (P < 0.003) and between-animal variability (P < 0.005) (Fig. 4). Stride length, the greatest distance between the 2 contralateral toe touches, demonstrated no left/right variability or between animal variability (Fig. 5). Back Height, the greatest distance from the ground to the back of the animal during terminal-swing ankle angle phase, demonstrated no left/right variability but significant between animal variability (P < 0.0001) (Fig. 6). Stance Phase, the duration of time of ipsilateral toe touch to ground, demonstrated no left/right variability but significant between-animal variability (P < 0.0001) (Fig. 7). Swing Phase, the duration of time while the ipsilateral toe is between toe to ground touch, demonstrated no left/right variability but significant between-animal variability (P < 0.0001) (Fig. 8). 3.2. Nerve crush gait evaluation
After nerve crush all but one of the gait parameters demonstrated a significant difference (P < 0.001) between the control and the crushed peroneal nerve sides. The most obvious visual difference was seen for ankle angles. Dorsiflexion ankle angles (both mid- and terminal-swing phase) and step length were greater than control at 1,7, and 14 days (P < 0.0001) after NC1 and returned to the same values as contralateral uninjured control side by 22 days (P > 0.05) (Figs. 9 and 10). The other parameters (back height, stance time
2.5. Data analysis Animal
Measurements from each parameter were evaluated within and between groups using ANOVA and post hoc Tukey testing as needed.
Number
Fig. 2. Ankle angle, mid-swing phase: the graph demonstrates the ankle angle during the midswing phase of recovery for the 8 different animals tested. The range of degrees are specified based on the 95% confidence interval.
P.M. Santos er al. /Journal
of Nnrroscience
Methods
61 (1995)
79-54
12
Stride (M%
Length,
tz%anca8 Interval)
10 s 4 2
0,
, 1
, 2
, 3
Animal
, 4
, 5
, 6
, 7
0
,
12
8
3-45
Animal
Number
Fig. 3. Ankle angle, terminal-swing phase: the graph demonstrates the ankte angle during the terminal-swing phase of recovery for the 8 different animals tested. The range of degrees are specified based on the 95% confidence interval.
and swing time) were significantly different for 1 and (P < 0.0001) and 7 days (P = 0.0001) after NC1 and returned to the same values as contralateral uninjured control side by 14 days (P > 0.05, figures not shown). Stride length after nerve crush never varied from control. Within 3 weeks of the nerve injury, there were no visible or measurable differences between injured and uninjured sides for any gait parameter. All of the aforementioned parameters were also significant for change through time as well as an interaction of side and time.
6
78
Number
Fig. 5. Stride length: the graph demonstrates stride length for the 8 animals with 95% confidence interval.
'1'2'3'4.5'6.7.8.
Animal
Number
Fig. 6. Back height: the graph demonstrates the animals with 95% confidence interval.
back height of the 8
4. Discussion A rat gait analysis model has been developed and tested. It utilizes the rat peroneal nerve and anterior tibialis muscle group for study of gait parameters. Evaluation of the rat gait model with normal uninjured rats demonstrated general consistency of parameters. It is possible that the variability we found between animals is due to gait speed. Hruska et al. (1979) demonstrated rat gait stance time decreases and stride length increases as a function qf speed. Speed was not controlled for in this study but will be evaluated in
‘1.
Animal
step
length
for the 8
Nuntbw
Fig. 7. Stance phase: the graph demonstrates the stance the 8 animals tested with 95% confidence interval.
12
2'3'4'5.6.7-8. ARimmNumbu
Fii. 4. Step length: the graph demonstrates animals with 95% confidence intelvat.
1 -2'3'4'5'6'7'8.
3 4 Anfmal
5
B
7
phase
for
8
Number Fig. 8. Swing phase: the graph demonstrates the swing 8 animals tested with 95% confidence interval.
phase for the
P.M. Santos et al. /Journal
of Neuroscience
Methods
61 (199.5)
79-84
83
80 80 70 50
Terminal ” Ankle Angle 48 Btd Dev 38 20
Before Injury
* Significant
1 day
7days
14days
Mdays
28days I
Fig. 9. Nerve crush vs. control for ankle angle. This graph demonstrates the average ankle angle measured from the peroneal NC1 leg compared to the contralateral uninjured leg from 12 animals evaluated before injury, 1 day after injury, 7, 14, 22, and 28 days after injury. Ankle angle was significantly different between injured and controlled legs on days 1, 7, and 14. There was no significant difference on days 22 and 28 between injured and uninjured legs.
future studies. The variability of left and right uninjured sides was negligible for most parameters (e.g., ankle angle). As a result of this finding we elected to evaluate a unilateral crush injury to determine if the uninjured leg could serve as an internal control to the injured side. Within the second part of the study, the peroneal NC1 was quantified based on weekly ankle angle measurements and other gait parameters. As the paresis resolved the ankle angle returned to normal uninjured values. Of the various gait parameters measured after nerve crush, the ankle angle and step length appear to be the most sensitive outcome variables to evaluate injury because they were the last to return to normal. However, between these two parameters ankle angle is more useful since step length demonstrated some left right variability in uninjured animals and ankle angle did not. The finding that ankle angle is the most sensitive outcome variable for NC1 is reasonable since all of the other parameters are a result of compensatory changes to the increased ankle angle. As a result of this study we were able to conclude that the mea-
surement of ankle angles alone is sufficient for future studies since it is the most sensitive indicator or NC1 with the latest return to normal control values. The most common nerve model for study in rodents is the sciatic nerve. The sciatic nerve is surgically accessible and sufficiently large for various surgical procedures. However, the sciatic nerve is a mixed nerve, carrying motor axons for antagonistic muscle groups (ankle dorsiflexion and plantarflexion) and sensory axons to the lower extremity. Regeneration of a sectioned sciatic nerve results in severe synkinesis of motor axons into both anterior and posterior muscle groups. In addition, proximal motor axons may regenerate into distal sensory axons and vice versa. Unlike the sciatic nerve, the peroneal nerve innervates muscles of dorsiflexion and no antagonistic muscle groups, thus obviating some of the synkinesis attributable to motor nerves with antagonistic muscle function. However, aberrant nerve regeneration secondary to sensory and afferent spindle fibers still occurs if the axonal basal lamina has been interrupted as in a transection nerve injury. We attempted to avoid complicating this
10 9 8 StepLength, kSt?&w
3 5 4 3
q Control
:
a Injured 8 * Significant
gz
1 day
7days
14days
22days
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Fig. 10. Nerve crush vs. control for step length. This graph demonstrates the step length before injury and after injury for the injured leg compared to the controlled leg. A significant difference existed on days 1, 7, and 14 between the injured and controlled legs. No significant difference was measured on days 22 and 28.
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stage of the gait analysis model evaluation by performing a NC1 and not a transecting injury. The evaluation of more complicated injuries are underway. SF1 or ‘rat tracking’ method evaluates the footprints of the rat after nerve injury. The technique serves as a useful means of evaluating the sciatic nerve as a function of the footprint, i.e., toe spread, stride length, etc. (de Medinaceli et al., 1982). Although not a direct measure of nerve muscle function, it does serve as a means of evaluating nerve regeneration. SF1 values are subject to measurement error associated with the complex motions of lower extremity swing. Since the sciatic nerve innervates antagonistic muscle groups a variable and significant amount of synkinesis occurs which increases SF1 variability. Furthermore, modifications of the SF1 for isolated tibia1 nerve injury or peroneal nerve injury have had moderate and limited success respectively (Bain et al., 1989; Hare et al., 1992). Difficulties with SF1 and the inability to directly measure muscle force led our laboratory to the development of the tension transduction device (Spyropoulos et al., 1994) and gait analysis model utilizing the rat peroneal nerve/anterior tibialis muscle. Two advantages of the gait analysis model over the tension transduction device are the ability to monitor nerve function without surgical exposure of the nerve and the capacity for multiple non-invasive evaluations during regeneration allowing for measurement of rate of recovery.
Methods
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Acknowledgements The author would like to thank Ms. Shirley Gossard for her assistance in preparing the manuscript, Ms. Caria Shaffer for data entry and Larry Hughes, Ph.D., for his assistance in managing the statistical data. This work was supported by funding from the Southern Illinois University, School of Medicine, Central Research Committee 1993 (Central Research Grant 3093).
References Bain, J.R., Mackinnon, S.E., and Hunter, D.A. (1989) Functional evaluation of complete sciatic, peroneal, posterior tibia1 nerve lesions in the rat. Plast. Reconstr. Surg., 83: 129-136. De Medinaceli, L., Freed, W.J., and Wyatt, R.J. (1982) An index of the functional condition of rat sciatic nerve based on measurements made from walking tracks. Exp. Neural., 77: 634443. Hare, G.M.T., Evans, P.J., Mackinnon, S.E., and Best, T.J. (1992) Walking track analysis: a long term assessment of peripheral nerve recovery. Plast. Reconstr. Surg., 89: 251-258. Hruska, R.E., Kennedy. S., and Silbergeld, E.K. (1979) Quantitative aspects of normal locomotion in rats. Life Sci., 25: 171-180. Spyropoulos, B.P., Williams. S., and Santos, P.M. (1994) Tension transduction device for functional evaluation of the rat peroneal nerve. J. Neurosci. Methods, 53: 95-100.