JOURNAL OF NEUROTRAUMA Volume 9, Number 2, 1992 Mary Ann Liebert, Inc., Publishers
A Monitored Contusion Model of Injury in the Rat
Spinal Cord
JOHN A. GRUNER
successful clinical evaluation of methylprednisolone (MP) as a has been to focus attention on the ability of experimental models of SCI injury to evaluate treatment efficacy as well as mechanisms of injury and actions of various therapeutic regimens. That is, an ideal model not only should be capable of showing what causes cells to die or that a given therapy is capable of saving axons, but also it should be able to (1) demonstrate chronic neurophysiologic and behavioral efficacy, (2) determine the optimum dosage and window of application; and (3) evaluate efficacy in combination with other drugs or treatments. The large numbers of animals required for studies of treatment efficacy virtually mandate that injury models be standardized to avoid unnecessary duplication of effort and allow for direct comparison of results across a range of injury parameters, especially when related studies are performed in different laboratories. The contusion model of SCI, although it may not simulate all aspects of the wide range of clinical SCI, has been used successfully to bring effective therapies to clinical practice. In developing models for experimental SCI, it is important to consider the injury process in its broadest sense, from the time of anesthesia and laminectomy to months, even years after injury, since all the méthodologie and physiologic variables introduced during this period will affect the ultimate outcome. The injury itself initiates a complex chain of events. Beginning with the delivery of the prescribed energy to the spinal cord, the biomechanical characteristics of the cord (tissue geometry and such characteristics as elasticity and viscosity) determine how the energy impinging on the cord is distributed at the cellular level, which in turn determines the extent of cellular damage. Unfortunately, relatively little is known about this aspect of the injury model. Next, the initial trauma sets in motion many physiologic sequelae, including changes in blood flow, pH, and extracellular ion concentrations, production of free radicals, and other factors predicated to underlie secondary cell damage. Finally, intraneuronal and interneuronal processes of reorganization will determine the ultimate functional capacity of the nervous system, i.e., the animal's behavioral capabilities. Because of the multiplicative effect of variability at each of these stages, controlling variability, particularly at the early stages of the injury, may significantly improve statistical power for a given outcome measure, especially at longer periods after injury. Ideally, by measuring additional injury variables (e.g., cord compression) and incorporating them into the injury model as parameters, their contribution to outcome variability can be diminished. With these considerations in mind, we have developed a weight-drop contusion model of SCI, incorporating sensors to monitor the parameters of the impact and the biomechanical response of the tissue. Monitoring the injury process serves four purposes. First, the absolute accuracy of the device can be determined. Ideally, measured values (e.g, impact velocity) should correspond to theoretical values based on free fall. This feature permits anyone to reproduce the same calibrated injury. Second, the reliability of the device can be monitored. Monitoring allows impacts that do not achieve expected values to be legitimately discarded. Third, monitoring enables one to assess the immediate biomechanical response of the tissue to the impact.
Anspinal
recently
important effect of the cord (SCI) treatment
Department of Neurosurgery, New York University Medical Center, New York, New York. 123
GRÜNER
Differences in tissue response, such as might result from variations in spinal cord diameter, can influence the extent of tissue damage. If it can be shown that one or more of the postimpact biomechanical response parameters (e.g., tissue compression) improves prediction of tissue survival above that of the primary parameters (e.g., impact velocity), such data could be used to increase the statistical power of treatment comparisons. Likewise, it might be possible to use data from animals even when the primary impact parameters were outside the normal range by calculating the amount of tissue damage expected from a given set of impact and biomechanical response parameters. Fourth, by investigating the relationship among impact
parameters, tissue response, and primary tissue damage,
one might elucidate mechanisms of primary and tissue damage. secondary The device described is designed to be reliable, accurate, simple to operate, and inexpensive. It consists of a 10 g impactor that can be raised 6.25, 12.5, 25, 50, or 75 mm above the surface of the cord, producing a range of chronic injuries from very mild to complete paraplegia. When released, the weight is accelerated by the force of gravity until it hits the exposed spinal cord. The contact surface of the impactor is round and slightly chamfered at the edge to prevent tearing the dura. The diameter is chosen to be slightly less than that of the cord to clear the edges of the vertebral canal as the impactor compresses the cord. Using Long-Evans hooded rats, a diameter of 2.5 mm is used for animals of 200-350 g and 3.0 mm above 350 g. The weight is threaded onto a rod, which directs the weight as it falls and permits linkage to an optical potentiometer that indirectly measures the height of the weight (±0.025 mm resolution, sampled at 100 kHz). The weight, rod, and linkages produce a static load of 10 g, although this can be varied if desired by changing weights. Use of optical potentiometers allows the impact parameters and tissue response to be measured without the use of an impact interface or button. Such an interface can significantly affect the impact dynamics, particularly in rat models, since it can equal a significant percentage of the impact mass itself, thus reducing the energy actually delivered to the tissue. Also, it may produce significant initial loading. Finally, it makes precise reproduction of the injury in other laboratories virtually impossible unless an identical device is used. Although the original Allen weight drop model used an unsupported vertebral column, most models support the vertebrae in order to minimize the amount of impact energy absorbed by the ribs and the body mass underneath and to prevent movement of the animal, which would decrease the accuracy of the impact, particularly when an impact interface is not used. As a result of the impact, many of the axons are depolarized, producing extensive activation of limb and axial motoneurons. This causes the animal to extend its limbs and arch its back. If the vertebral column is not adequately fixed, additional injury may be produced when the cord is pushed up against the impactor. In the cat, when the vertebral column is rigidly supported, histologie and behavioral outcome is more severe for a given injury level because a greater percentage of the energy is absorbed by the cord. In the rat, extensive exposure of the transverse vertebral processes to place supporting rods against the vertebra at the impact site itself is difficult. Therefore, most rat contusion studies support the vertebral column using clamps attached to the dorsal spinous processes one or two segments rostal and caudal to the injury site. In the present model, a laminectomy is performed on the T10 vertebra, the dorsal process of T8 is clamped, and the Tl 1 vertebra is clamped around the transverse processes. When the column is fixed in this manner and the animal's belly is supported such that the legs cannot push against any surface, only a few millimeters of vertebral movement occurs. The impactor can rebound and hit the cord a second time, but the height is generally < 10% of the initial height, and the impact velocity is much lower, so that injury from the second bounce should be minimal. We do not believe that the benefits of preventing the second hit are warranted by the additional complexity of the device required. This method of fixing the vertebral column permits slight vertical movements of the cord under the impact site, which typically occur during breathing, as well as under the force of the impactor. Therefore, to determine the exact height and time of impact, it is necessary to monitor the exact instant the impactor contacts the cord. This is achieved by using an electronic circuit that senses a change in voltage at the impactor when the weight contacts the cord. To determine the biomechanical tissue response parameters after impact (particularly cord compression), it is necessary to measure the movement of the surface of the cord relative to the underlying vertebra. Vertebral movement is monitored using a rigid wire shaped to rest on a transverse process of the vertebra to be hit. The wire is attached via a rod to a second potentiometer. A computer and special program are used to sample the impactor and vertebral position sensors and the cord contact sensor for 200 ms after release of the weight, store the raw data, and display the impact curves on a
124
CONTUSION MODEL OF SCI monitor. The actual height, time, and velocity of the impact, cord compression, and other derived parameters computed and saved separately to disc for statistical analysis. Additional important factors that affect the biomechanical response of the tissue are the geometry of the cord and its viscous and elastic characteristics. Unfortunately, these are difficult to measure. Even the cord diameter cannot be determined easily without x-ray imaging. Preliminary studies using measurements of sodium and potassium at 24 h after spinal contusion suggest that the contusion model described can effectively distinguish treatment effects in groups of as few as 10 rats. In one such study, 60 female rats were divided randomly into six groups of 10 animals [(12.5, 25, or 50 mm contusion) x (MP or saline treatment)]. Animals were anesthetized with Nembutal (40 mg/kg), and a laminectomy was performed to expose the cord at T10. After impact, the rats were placed in a controlled temperature incubator (24°C). Fifty-eight rats survived until they were killed 24 h later. Treatment consisted of four doses of 30 mg/kg or an equal volume of saline delivered at 15 min and 6, 12, and 18 h after injury via an IV catheter inserted in the femoral vein. The cell volume fraction (CVF), i.e., the ratio of intracellular/ extracellular tissue volume, was determined at killing 24 h after injury. The value of CVF was calculated from the values of total tissue sodium and potassium in a 9 mm length of cord centered on the injury site, measured by atomic absorption spectroscopy. Analysis of the primary impact data from this study revealed that the impact height and velocity parameters in the three injury severity groups were not significantly different for the two treatment groups. As expected, the velocity vs impact height data fit a second order polynomial with r > 0.99 for both groups. The linear correlation coefficient for compression vs impact height was smaller (r 0.72) because of greater variation in the compression values. There was also a significant (p 0.016, unpaired r-test) difference in the mean values of cord compression in the 12.5 mm contused animals across the two treatment groups (unrelated to age or weight of the animals) but no differences for the other injury levels (p > 0.05). This would suggest that the 12.5 mm MP group received a slightly more severe injury. The impact data thus showed either no significant differences between treatment groups or, in the case of the 12.5 mm injury, possibly a slightly more severe injury in the MP group. Despite these results, a clear treatment difference was found in that the mean CVF values for each of the MP-treated groups were significantly higher than for the corresponding control groups. Comparison of the treatment x impact height groups using ANOVA showed them to be significantly different (F 33.4; p 0.0001). These data illustrate several advantages of monitoring weight drop contusions. First, the data demonstrate that the impact velocity is very close to that expected from a pure free fall, and, therefore, the results should be comparable with those of any other laboratory using a similar weight-height combination. Second, the reliability and consistency of the impacts were verified. In initial studies using prototype devices, some impacts did not achieve the expected impact velocity because of mechanical problems. Measurement of impact velocity itself was the only unambiguous measure that suggested a problem with those particular are
=
=
=
=
experiments. The third advantage of monitoring is to use data on the biomechanical response of the tissue to improve the statistical power of treatment comparisons. In the data described, differences in cord compression that might have resulted in significant outcome differences by themselves were noted. Without monitoring the tissue response to impact, one cannot be certain that different groups of animals, either within or between different laboratories, are being subjected to the same cord deformation. We have looked in detail only at ionic measurements of cell survival at 24 h postinjury, and at that time, the CVF was most highly correlated with impact velocity; i.e., cord compression or other measures did not add significantly to the correlation. The earlier an outcome measure is obtained after injury, the greater its statistical power is likely to be. We have found significant between-group treatment differences with as few as 10 animals using our contusion model and CVF as an outcome measure. In contrast, preliminary behavioral data (e.g., motor scores) using the same model indicate that at least 15 animals will be required to achieve significance. Incorporation of injury variables, such as cord compression, into the injury model may enable statistical significance to be achieved
with fewer animals. The fourth advantage of monitoring is to facilitate investigation of the mechanisms of injury. A critical issue in evaluating animal models is whether the parameters of the model that determine the outcome in terms of a given measure of tissue damage are, in fact, also those most important in determining the ultimate degree of deficit or recovery. For example, outcome has been shown to correlate with injury intensity over a broad range 125
GRÜNER
of injury levels, but in the severe injury range, where 90%-95% of axons are destroyed, there appears to be little correlation between numbers of surviving axons and locomotor recovery. It is hoped that a better understanding of the mechanisms of primary and secondary cell damage and improvements in measurement of chronic behavioral outcome will facilitate determination of effective therapies.
REFERENCES ALLEN, A.R. (1911). Surgery of experimental lesions of spinal cord equivalent to crush injury of fracture dislocation of spinal column. JAMA 57, 878-880. ANDERSON, D.K. (1990). Pharmacological treatment of acute, experimental spinal cord injury, in: Neural Monitoring. Salzman S.K.fed.). Humana Press, Inc.: Baltimore. BLIGHT, A.R. (1988). Mechanical factors in experimental spinal cord injury. J. Am. Paraplegia Soc. 11, 26-34.
BLIGHT, A.R., DECRESC1TO, V. (1986). Morphometric analysis of experimental spinal cord injury in the cat: The relation of injury and intensity to survival of myelinated axons. Neuroscience 19, 321-341. DOHRMANN, G.I., PANJABI, M.M., and BANKS, D. (1978). Biomechanics of experimental
Neurosurg. 48,993-1001.
spinal cord trauma. J.
FEHLINGS, M.G., TATOR, C.H., and LINDEN, R.D. (1989). The relationships among the severity of spinal cord injury, motor and somatosensory evoked potentials, and spinal cord blood flow. Electroenceph. Clin. Neurophysiol.
74, 241-259.
KEARNEY, P.A., KIDELLA, S.H., VIANO, D.C., and ANDERSON, T.E. (1988). Interaction of contact velocity and cord compression in determining the severity of spinal cord injury. I. Neurotrauma 5, 187-208. RAINES, A., DRETCHEN, K.L., MARX, K., and WRATHALL, I.R. (1988). Spinal cord contusion in the
Somatosensory evoked potentials as a function of graded injury.
YOUNG, W., DECRESCITO, V., FLAMM, E.S., Studies of high dose
et
J. Neurotrauma
rat:
5, 151-160.
al. (1988). Pharmacological therapy of acute Clin. Neurosurg. 34, 675-697.
spinal cord injury:
methylprednisolone and naloxone.
Address reprint requests to: John A. Grüner, Ph.D.
Department of Pharmacology Cephalon, Inc. 145 Brandy Wine Parkway W.
Chester, PA 19380-4245
DISCUSSION
Young: I might comment that the initial point of our looking for a model and an end point measure was a model where you can do as many as 5 to 10 experiments per day. The sodium and potassium measures are extremely easy to do. You can run 100 or 200 specimens and obtain the results on the same day. We are not saying that this should be the ultimate measure. There are a number of highly quantifiable measures that can be used to obtain results within the same day. We wanted to head in that direction and be able to relate the various parameters and mechanical injuries to an outcome that we have confidence in and that we can get large numbers of data points for quickly. In the past year, we have tested more than 1000 animals, which is a record for us in testing paradigms. Dr. Lucas: In our models, the fate of the cells at a lesion 100 p,m from the perikarya is determined in 2 h. We have looked at 24 h, and we find there is no delayed cell death with this injury. The results can be assessed very quickly. Dr. Blight: I think this is a wonderful technique and a great step forward. It gives confidence that something can be done to look at a lot of drugs very rapidly. I am still concerned, however, that we really do not know Dr.
to establish
126
CONTUSION MODEL OF SCI what we are talking about when we talk about secondary injury. We do not know when it occurs or exactly what it is. I would be concerned with this method—whether you can resolve to some extent when the secondary damage is occurring. I wonder whether you have looked at various times after injury to see if you get gradual increase in the cell volume or decrease in the intracellular volume fraction. I realize you will have problems with that because of the diffusion issues and blood flow issues, but really I am not sure if we know when we should be looking to detect the end point of secondary damage. Is it 1 day, is it 1 week? Dr. Grüner: We have done ion studies between 6 and 24 h and see that there are index changes, indicating that more cells have died at 24 h than at 6 h. There are technical problems in interpreting this measure at the different times because of the way cells die—or the way we hypothesize they die. Axons may die back over a period of time, for example. We have shown data for 24 h because we have a fairly high confidence that the measure is an accurate predictor of actual cell volume loss at that time. Dr. Blight: I think it is very hard to distinguish secondary from primary damage at 24 h with this kind of measure, but I imagine you could distinguish pretty well whether there was any secondary loss between 24 h and 1 week. Dr. Grüner: We can show treatment effects. That alone speaks to the issue of secondary damage. The problem that I wanted to raise is that we do not know what is killing the cells initially or what the time course of primary cell death is. Therefore, we do not know how to subtract that out from the secondary effects. Dr. Blight: There are many things killing the cells, which is one of the difficulties in dissecting these models of injury. There are so many possible mechanisms. Dr. Anderson: Secondary injury is multifactorial, and I think it occurs over a long period of time. There is evidence that some of the indicators of biochemical injury occur within a minute after injury. In addition, there are other aspects of what we call secondary injury (for instance, inflammatory responses) that may take place weeks or months later. Dr. Stokes: We have shied away from some of these acute issues at Ohio State for the very reasons mentioned—the issue of what actually is doing the killing. But very quickly after the injury (this may be a day or so later) one enters a phase of chronic behavioral recovery. One of the primary things we advocate is use of multiple independent measures of behavioral outcome in our mildly injured animals to give us multiple ways of evaluating the injury process. We may need different outcome measures in characterizing early changes and others for later times, which may be more reflective of different phases of this secondary damage. Dr. Grüner: One of the advantages of most behavioral measures is that you have repeated measurements on the same animal, and that gives you added statistical power. As the animal recovers, you can look at the rate of recovery and differences in the rates of recovery in animals in various treatment groups. In our methylprednisolone data, for example, walking scores show that there is a trend toward improved outcome, particularly at 7 and 14 days, in the methylprednisolone-treated animals. The differences are not significant because the standard deviations are large. The important point is that on the first day we test (day 2), there is no difference because animals are basically paralyzed. After 3 weeks, they have all recovered equally well, but between 2 days and 3 weeks, we see differences in the rate of recovery. Dr. Tator: You have shown a new version of the impact or weight drop model, and now you show us a new outcome measure to try to validate the new injury model. I am not sure if the new model is an improvement over previous models, and I do not think you have made the case effectively. In terms of the outcome measure, how can you use the new model to prove that the outcome measure is working? Dr. Grüner: This model is basically the same that Allen started with. The difference is our ability to determine whether or not we are able to reproduce the injury the same in every animal that we injure and have the same results between laboratories. This is very important if we are going to do treatment studies and will save a lot of time and effort to reproduce treatment studies in different laboratories. Monitoring gives us the added power to look at our data and determine if the tissue responded differently in any animal. That should enable us to better estimate the primary injury. With the current version of our impactor, there are significant correlations between the ionic measures and primary impact variables (height, velocity). Thus far, however, correlations of ionic measurements with derived impact parameters (e.g., compression, impulse) have not been better than correlations with height or velocity. We hope that improvements in the model will change that. When the mean motor scores of 36 animals studied behaviorally were compared against mean 24 h Na minus K measurements for a separate group of 19 rats as a function of injury level (12, 25, or 50 mm), linear 127
GRÜNER correlations as high as r2 0.97 (day 28 motor score against Na minus K) were obtained, so behavioral outcome does appear to be closely related to the ionic measurements. More careful studies comparing ionic measurements against behavioral outcome will be essentially to validate this model. Dr. Faden: I think all of us feel that it is important to have as much information as we can about a given injury—whether it is the actual impact velocity or the tissue deformation, and so forth. But the big question for those who have used the simple Allen technique with little change from 1911 is how much better are these newer models in terms of reliability, in terms of tightness, response, and so forth? Both the NYU and Ohio State University groups have had extensive experience before developing these newer models. I would ask each of you to comment on whether you have looked at the actual data in terms of tightness of the response or variability of the given hit that you might use for pharmacologie evaluations. What of other issues that might be important, such as training time for a technician to use the technique and consistency of responses across technicians? These are key questions on the practical use of these techniques. Could you elaborate on the differences between your new technique and the former approaches that were used so widely elsewhere? Dr. Grüner: I think that the reliability of our impact device is evident from the data I presented, and probably anyone can achieve similar results with a weight drop impactor. Variability is probably insignificant in impact velocity, for example, compared to the biologic variability in both the response of the tissue and the secondary factors that produce changes in tissue outcome. However, it is still incumbent on experimenters to demonstrate, one way or the other, that their device is producing the intended injury with negligible variability. This essentially is the old technique. We just know what our mechanical variables are now. It's not that we are hitting the cord with any different kind of energy. Dr. Blight: I think I can make the point that Dr. Grüner has already made—that derived parameters are better correlated than the height parameters, which is fundamentally a better handle on what is happening than the weight-height combination. Dr. Stokes: One of the difficulties that Dr. Grüner did not deal with is the accuracy of measuring devices and how one determines these factors. With the weight drop technique, there is no real measure of what is going on in the cord compartment, and one must rely on some of the modeling that has been done by Lighthall and others to get some of the parameters (e.g., velocity) that you would like to use for comparison. What one must rely on is the variability of outcome measures as an index of reliability of impact parameters. Our ability to make very accurate changes in the biomechanical predictors has markedly reduced the variability of behavioral outcome measures. We think this has great significance for preclinical trials. =
128
A Monitored Contusion Model of Injury in the Rat
Spinal Cord
JOHN A. GRUNER
successful clinical evaluation of methylprednisolone (MP) as a has been to focus attention on the ability of experimental models of SCI injury to evaluate treatment efficacy as well as mechanisms of injury and actions of various therapeutic regimens. That is, an ideal model not only should be capable of showing what causes cells to die or that a given therapy is capable of saving axons, but also it should be able to (1) demonstrate chronic neurophysiologic and behavioral efficacy, (2) determine the optimum dosage and window of application; and (3) evaluate efficacy in combination with other drugs or treatments. The large numbers of animals required for studies of treatment efficacy virtually mandate that injury models be standardized to avoid unnecessary duplication of effort and allow for direct comparison of results across a range of injury parameters, especially when related studies are performed in different laboratories. The contusion model of SCI, although it may not simulate all aspects of the wide range of clinical SCI, has been used successfully to bring effective therapies to clinical practice. In developing models for experimental SCI, it is important to consider the injury process in its broadest sense, from the time of anesthesia and laminectomy to months, even years after injury, since all the méthodologie and physiologic variables introduced during this period will affect the ultimate outcome. The injury itself initiates a complex chain of events. Beginning with the delivery of the prescribed energy to the spinal cord, the biomechanical characteristics of the cord (tissue geometry and such characteristics as elasticity and viscosity) determine how the energy impinging on the cord is distributed at the cellular level, which in turn determines the extent of cellular damage. Unfortunately, relatively little is known about this aspect of the injury model. Next, the initial trauma sets in motion many physiologic sequelae, including changes in blood flow, pH, and extracellular ion concentrations, production of free radicals, and other factors predicated to underlie secondary cell damage. Finally, intraneuronal and interneuronal processes of reorganization will determine the ultimate functional capacity of the nervous system, i.e., the animal's behavioral capabilities. Because of the multiplicative effect of variability at each of these stages, controlling variability, particularly at the early stages of the injury, may significantly improve statistical power for a given outcome measure, especially at longer periods after injury. Ideally, by measuring additional injury variables (e.g., cord compression) and incorporating them into the injury model as parameters, their contribution to outcome variability can be diminished. With these considerations in mind, we have developed a weight-drop contusion model of SCI, incorporating sensors to monitor the parameters of the impact and the biomechanical response of the tissue. Monitoring the injury process serves four purposes. First, the absolute accuracy of the device can be determined. Ideally, measured values (e.g, impact velocity) should correspond to theoretical values based on free fall. This feature permits anyone to reproduce the same calibrated injury. Second, the reliability of the device can be monitored. Monitoring allows impacts that do not achieve expected values to be legitimately discarded. Third, monitoring enables one to assess the immediate biomechanical response of the tissue to the impact.
Anspinal
recently
important effect of the cord (SCI) treatment
Department of Neurosurgery, New York University Medical Center, New York, New York. 123
GRÜNER
Differences in tissue response, such as might result from variations in spinal cord diameter, can influence the extent of tissue damage. If it can be shown that one or more of the postimpact biomechanical response parameters (e.g., tissue compression) improves prediction of tissue survival above that of the primary parameters (e.g., impact velocity), such data could be used to increase the statistical power of treatment comparisons. Likewise, it might be possible to use data from animals even when the primary impact parameters were outside the normal range by calculating the amount of tissue damage expected from a given set of impact and biomechanical response parameters. Fourth, by investigating the relationship among impact
parameters, tissue response, and primary tissue damage,
one might elucidate mechanisms of primary and tissue damage. secondary The device described is designed to be reliable, accurate, simple to operate, and inexpensive. It consists of a 10 g impactor that can be raised 6.25, 12.5, 25, 50, or 75 mm above the surface of the cord, producing a range of chronic injuries from very mild to complete paraplegia. When released, the weight is accelerated by the force of gravity until it hits the exposed spinal cord. The contact surface of the impactor is round and slightly chamfered at the edge to prevent tearing the dura. The diameter is chosen to be slightly less than that of the cord to clear the edges of the vertebral canal as the impactor compresses the cord. Using Long-Evans hooded rats, a diameter of 2.5 mm is used for animals of 200-350 g and 3.0 mm above 350 g. The weight is threaded onto a rod, which directs the weight as it falls and permits linkage to an optical potentiometer that indirectly measures the height of the weight (±0.025 mm resolution, sampled at 100 kHz). The weight, rod, and linkages produce a static load of 10 g, although this can be varied if desired by changing weights. Use of optical potentiometers allows the impact parameters and tissue response to be measured without the use of an impact interface or button. Such an interface can significantly affect the impact dynamics, particularly in rat models, since it can equal a significant percentage of the impact mass itself, thus reducing the energy actually delivered to the tissue. Also, it may produce significant initial loading. Finally, it makes precise reproduction of the injury in other laboratories virtually impossible unless an identical device is used. Although the original Allen weight drop model used an unsupported vertebral column, most models support the vertebrae in order to minimize the amount of impact energy absorbed by the ribs and the body mass underneath and to prevent movement of the animal, which would decrease the accuracy of the impact, particularly when an impact interface is not used. As a result of the impact, many of the axons are depolarized, producing extensive activation of limb and axial motoneurons. This causes the animal to extend its limbs and arch its back. If the vertebral column is not adequately fixed, additional injury may be produced when the cord is pushed up against the impactor. In the cat, when the vertebral column is rigidly supported, histologie and behavioral outcome is more severe for a given injury level because a greater percentage of the energy is absorbed by the cord. In the rat, extensive exposure of the transverse vertebral processes to place supporting rods against the vertebra at the impact site itself is difficult. Therefore, most rat contusion studies support the vertebral column using clamps attached to the dorsal spinous processes one or two segments rostal and caudal to the injury site. In the present model, a laminectomy is performed on the T10 vertebra, the dorsal process of T8 is clamped, and the Tl 1 vertebra is clamped around the transverse processes. When the column is fixed in this manner and the animal's belly is supported such that the legs cannot push against any surface, only a few millimeters of vertebral movement occurs. The impactor can rebound and hit the cord a second time, but the height is generally < 10% of the initial height, and the impact velocity is much lower, so that injury from the second bounce should be minimal. We do not believe that the benefits of preventing the second hit are warranted by the additional complexity of the device required. This method of fixing the vertebral column permits slight vertical movements of the cord under the impact site, which typically occur during breathing, as well as under the force of the impactor. Therefore, to determine the exact height and time of impact, it is necessary to monitor the exact instant the impactor contacts the cord. This is achieved by using an electronic circuit that senses a change in voltage at the impactor when the weight contacts the cord. To determine the biomechanical tissue response parameters after impact (particularly cord compression), it is necessary to measure the movement of the surface of the cord relative to the underlying vertebra. Vertebral movement is monitored using a rigid wire shaped to rest on a transverse process of the vertebra to be hit. The wire is attached via a rod to a second potentiometer. A computer and special program are used to sample the impactor and vertebral position sensors and the cord contact sensor for 200 ms after release of the weight, store the raw data, and display the impact curves on a
124
CONTUSION MODEL OF SCI monitor. The actual height, time, and velocity of the impact, cord compression, and other derived parameters computed and saved separately to disc for statistical analysis. Additional important factors that affect the biomechanical response of the tissue are the geometry of the cord and its viscous and elastic characteristics. Unfortunately, these are difficult to measure. Even the cord diameter cannot be determined easily without x-ray imaging. Preliminary studies using measurements of sodium and potassium at 24 h after spinal contusion suggest that the contusion model described can effectively distinguish treatment effects in groups of as few as 10 rats. In one such study, 60 female rats were divided randomly into six groups of 10 animals [(12.5, 25, or 50 mm contusion) x (MP or saline treatment)]. Animals were anesthetized with Nembutal (40 mg/kg), and a laminectomy was performed to expose the cord at T10. After impact, the rats were placed in a controlled temperature incubator (24°C). Fifty-eight rats survived until they were killed 24 h later. Treatment consisted of four doses of 30 mg/kg or an equal volume of saline delivered at 15 min and 6, 12, and 18 h after injury via an IV catheter inserted in the femoral vein. The cell volume fraction (CVF), i.e., the ratio of intracellular/ extracellular tissue volume, was determined at killing 24 h after injury. The value of CVF was calculated from the values of total tissue sodium and potassium in a 9 mm length of cord centered on the injury site, measured by atomic absorption spectroscopy. Analysis of the primary impact data from this study revealed that the impact height and velocity parameters in the three injury severity groups were not significantly different for the two treatment groups. As expected, the velocity vs impact height data fit a second order polynomial with r > 0.99 for both groups. The linear correlation coefficient for compression vs impact height was smaller (r 0.72) because of greater variation in the compression values. There was also a significant (p 0.016, unpaired r-test) difference in the mean values of cord compression in the 12.5 mm contused animals across the two treatment groups (unrelated to age or weight of the animals) but no differences for the other injury levels (p > 0.05). This would suggest that the 12.5 mm MP group received a slightly more severe injury. The impact data thus showed either no significant differences between treatment groups or, in the case of the 12.5 mm injury, possibly a slightly more severe injury in the MP group. Despite these results, a clear treatment difference was found in that the mean CVF values for each of the MP-treated groups were significantly higher than for the corresponding control groups. Comparison of the treatment x impact height groups using ANOVA showed them to be significantly different (F 33.4; p 0.0001). These data illustrate several advantages of monitoring weight drop contusions. First, the data demonstrate that the impact velocity is very close to that expected from a pure free fall, and, therefore, the results should be comparable with those of any other laboratory using a similar weight-height combination. Second, the reliability and consistency of the impacts were verified. In initial studies using prototype devices, some impacts did not achieve the expected impact velocity because of mechanical problems. Measurement of impact velocity itself was the only unambiguous measure that suggested a problem with those particular are
=
=
=
=
experiments. The third advantage of monitoring is to use data on the biomechanical response of the tissue to improve the statistical power of treatment comparisons. In the data described, differences in cord compression that might have resulted in significant outcome differences by themselves were noted. Without monitoring the tissue response to impact, one cannot be certain that different groups of animals, either within or between different laboratories, are being subjected to the same cord deformation. We have looked in detail only at ionic measurements of cell survival at 24 h postinjury, and at that time, the CVF was most highly correlated with impact velocity; i.e., cord compression or other measures did not add significantly to the correlation. The earlier an outcome measure is obtained after injury, the greater its statistical power is likely to be. We have found significant between-group treatment differences with as few as 10 animals using our contusion model and CVF as an outcome measure. In contrast, preliminary behavioral data (e.g., motor scores) using the same model indicate that at least 15 animals will be required to achieve significance. Incorporation of injury variables, such as cord compression, into the injury model may enable statistical significance to be achieved
with fewer animals. The fourth advantage of monitoring is to facilitate investigation of the mechanisms of injury. A critical issue in evaluating animal models is whether the parameters of the model that determine the outcome in terms of a given measure of tissue damage are, in fact, also those most important in determining the ultimate degree of deficit or recovery. For example, outcome has been shown to correlate with injury intensity over a broad range 125
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of injury levels, but in the severe injury range, where 90%-95% of axons are destroyed, there appears to be little correlation between numbers of surviving axons and locomotor recovery. It is hoped that a better understanding of the mechanisms of primary and secondary cell damage and improvements in measurement of chronic behavioral outcome will facilitate determination of effective therapies.
REFERENCES ALLEN, A.R. (1911). Surgery of experimental lesions of spinal cord equivalent to crush injury of fracture dislocation of spinal column. JAMA 57, 878-880. ANDERSON, D.K. (1990). Pharmacological treatment of acute, experimental spinal cord injury, in: Neural Monitoring. Salzman S.K.fed.). Humana Press, Inc.: Baltimore. BLIGHT, A.R. (1988). Mechanical factors in experimental spinal cord injury. J. Am. Paraplegia Soc. 11, 26-34.
BLIGHT, A.R., DECRESC1TO, V. (1986). Morphometric analysis of experimental spinal cord injury in the cat: The relation of injury and intensity to survival of myelinated axons. Neuroscience 19, 321-341. DOHRMANN, G.I., PANJABI, M.M., and BANKS, D. (1978). Biomechanics of experimental
Neurosurg. 48,993-1001.
spinal cord trauma. J.
FEHLINGS, M.G., TATOR, C.H., and LINDEN, R.D. (1989). The relationships among the severity of spinal cord injury, motor and somatosensory evoked potentials, and spinal cord blood flow. Electroenceph. Clin. Neurophysiol.
74, 241-259.
KEARNEY, P.A., KIDELLA, S.H., VIANO, D.C., and ANDERSON, T.E. (1988). Interaction of contact velocity and cord compression in determining the severity of spinal cord injury. I. Neurotrauma 5, 187-208. RAINES, A., DRETCHEN, K.L., MARX, K., and WRATHALL, I.R. (1988). Spinal cord contusion in the
Somatosensory evoked potentials as a function of graded injury.
YOUNG, W., DECRESCITO, V., FLAMM, E.S., Studies of high dose
et
J. Neurotrauma
rat:
5, 151-160.
al. (1988). Pharmacological therapy of acute Clin. Neurosurg. 34, 675-697.
spinal cord injury:
methylprednisolone and naloxone.
Address reprint requests to: John A. Grüner, Ph.D.
Department of Pharmacology Cephalon, Inc. 145 Brandy Wine Parkway W.
Chester, PA 19380-4245
DISCUSSION
Young: I might comment that the initial point of our looking for a model and an end point measure was a model where you can do as many as 5 to 10 experiments per day. The sodium and potassium measures are extremely easy to do. You can run 100 or 200 specimens and obtain the results on the same day. We are not saying that this should be the ultimate measure. There are a number of highly quantifiable measures that can be used to obtain results within the same day. We wanted to head in that direction and be able to relate the various parameters and mechanical injuries to an outcome that we have confidence in and that we can get large numbers of data points for quickly. In the past year, we have tested more than 1000 animals, which is a record for us in testing paradigms. Dr. Lucas: In our models, the fate of the cells at a lesion 100 p,m from the perikarya is determined in 2 h. We have looked at 24 h, and we find there is no delayed cell death with this injury. The results can be assessed very quickly. Dr. Blight: I think this is a wonderful technique and a great step forward. It gives confidence that something can be done to look at a lot of drugs very rapidly. I am still concerned, however, that we really do not know Dr.
to establish
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CONTUSION MODEL OF SCI what we are talking about when we talk about secondary injury. We do not know when it occurs or exactly what it is. I would be concerned with this method—whether you can resolve to some extent when the secondary damage is occurring. I wonder whether you have looked at various times after injury to see if you get gradual increase in the cell volume or decrease in the intracellular volume fraction. I realize you will have problems with that because of the diffusion issues and blood flow issues, but really I am not sure if we know when we should be looking to detect the end point of secondary damage. Is it 1 day, is it 1 week? Dr. Grüner: We have done ion studies between 6 and 24 h and see that there are index changes, indicating that more cells have died at 24 h than at 6 h. There are technical problems in interpreting this measure at the different times because of the way cells die—or the way we hypothesize they die. Axons may die back over a period of time, for example. We have shown data for 24 h because we have a fairly high confidence that the measure is an accurate predictor of actual cell volume loss at that time. Dr. Blight: I think it is very hard to distinguish secondary from primary damage at 24 h with this kind of measure, but I imagine you could distinguish pretty well whether there was any secondary loss between 24 h and 1 week. Dr. Grüner: We can show treatment effects. That alone speaks to the issue of secondary damage. The problem that I wanted to raise is that we do not know what is killing the cells initially or what the time course of primary cell death is. Therefore, we do not know how to subtract that out from the secondary effects. Dr. Blight: There are many things killing the cells, which is one of the difficulties in dissecting these models of injury. There are so many possible mechanisms. Dr. Anderson: Secondary injury is multifactorial, and I think it occurs over a long period of time. There is evidence that some of the indicators of biochemical injury occur within a minute after injury. In addition, there are other aspects of what we call secondary injury (for instance, inflammatory responses) that may take place weeks or months later. Dr. Stokes: We have shied away from some of these acute issues at Ohio State for the very reasons mentioned—the issue of what actually is doing the killing. But very quickly after the injury (this may be a day or so later) one enters a phase of chronic behavioral recovery. One of the primary things we advocate is use of multiple independent measures of behavioral outcome in our mildly injured animals to give us multiple ways of evaluating the injury process. We may need different outcome measures in characterizing early changes and others for later times, which may be more reflective of different phases of this secondary damage. Dr. Grüner: One of the advantages of most behavioral measures is that you have repeated measurements on the same animal, and that gives you added statistical power. As the animal recovers, you can look at the rate of recovery and differences in the rates of recovery in animals in various treatment groups. In our methylprednisolone data, for example, walking scores show that there is a trend toward improved outcome, particularly at 7 and 14 days, in the methylprednisolone-treated animals. The differences are not significant because the standard deviations are large. The important point is that on the first day we test (day 2), there is no difference because animals are basically paralyzed. After 3 weeks, they have all recovered equally well, but between 2 days and 3 weeks, we see differences in the rate of recovery. Dr. Tator: You have shown a new version of the impact or weight drop model, and now you show us a new outcome measure to try to validate the new injury model. I am not sure if the new model is an improvement over previous models, and I do not think you have made the case effectively. In terms of the outcome measure, how can you use the new model to prove that the outcome measure is working? Dr. Grüner: This model is basically the same that Allen started with. The difference is our ability to determine whether or not we are able to reproduce the injury the same in every animal that we injure and have the same results between laboratories. This is very important if we are going to do treatment studies and will save a lot of time and effort to reproduce treatment studies in different laboratories. Monitoring gives us the added power to look at our data and determine if the tissue responded differently in any animal. That should enable us to better estimate the primary injury. With the current version of our impactor, there are significant correlations between the ionic measures and primary impact variables (height, velocity). Thus far, however, correlations of ionic measurements with derived impact parameters (e.g., compression, impulse) have not been better than correlations with height or velocity. We hope that improvements in the model will change that. When the mean motor scores of 36 animals studied behaviorally were compared against mean 24 h Na minus K measurements for a separate group of 19 rats as a function of injury level (12, 25, or 50 mm), linear 127
GRÜNER correlations as high as r2 0.97 (day 28 motor score against Na minus K) were obtained, so behavioral outcome does appear to be closely related to the ionic measurements. More careful studies comparing ionic measurements against behavioral outcome will be essentially to validate this model. Dr. Faden: I think all of us feel that it is important to have as much information as we can about a given injury—whether it is the actual impact velocity or the tissue deformation, and so forth. But the big question for those who have used the simple Allen technique with little change from 1911 is how much better are these newer models in terms of reliability, in terms of tightness, response, and so forth? Both the NYU and Ohio State University groups have had extensive experience before developing these newer models. I would ask each of you to comment on whether you have looked at the actual data in terms of tightness of the response or variability of the given hit that you might use for pharmacologie evaluations. What of other issues that might be important, such as training time for a technician to use the technique and consistency of responses across technicians? These are key questions on the practical use of these techniques. Could you elaborate on the differences between your new technique and the former approaches that were used so widely elsewhere? Dr. Grüner: I think that the reliability of our impact device is evident from the data I presented, and probably anyone can achieve similar results with a weight drop impactor. Variability is probably insignificant in impact velocity, for example, compared to the biologic variability in both the response of the tissue and the secondary factors that produce changes in tissue outcome. However, it is still incumbent on experimenters to demonstrate, one way or the other, that their device is producing the intended injury with negligible variability. This essentially is the old technique. We just know what our mechanical variables are now. It's not that we are hitting the cord with any different kind of energy. Dr. Blight: I think I can make the point that Dr. Grüner has already made—that derived parameters are better correlated than the height parameters, which is fundamentally a better handle on what is happening than the weight-height combination. Dr. Stokes: One of the difficulties that Dr. Grüner did not deal with is the accuracy of measuring devices and how one determines these factors. With the weight drop technique, there is no real measure of what is going on in the cord compartment, and one must rely on some of the modeling that has been done by Lighthall and others to get some of the parameters (e.g., velocity) that you would like to use for comparison. What one must rely on is the variability of outcome measures as an index of reliability of impact parameters. Our ability to make very accurate changes in the biomechanical predictors has markedly reduced the variability of behavioral outcome measures. We think this has great significance for preclinical trials. =
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