Excitability changes in the sciatic nerve and triceps surae muscle after spinal cord injury in mice
© Ahmed et al. 2010
Received: 20 December 2009
Accepted: 18 April 2010
Published: 18 April 2010
From the onset to the chronic phase of spinal cord injury (SCI), peripheral axons and muscles are subjected to abnormal states of activity. This starts with very intense spasms during the first instant of SCI, through a no activity flaccidity phase, to a chronic hyperactivity phase. It remains unclear how the nature of this sequence may affect the peripheral axons and muscles.
We set out to investigate the changes in excitability of the sciatic nerve and to characterize the properties of muscle contractility after contusive injury of the mouse thoracic spinal cord.
The following changes were observed in animals after SCI: 1) The sciatic nerve compound action potential was of higher amplitudes and lower threshold, with the longer strength-duration time constant and faster conduction velocity; 2) The latency of the onset of muscle contraction of the triceps surae muscle was significantly shorter in animals with SCI; 3) The muscle twitches expressed slower rising and falling slopes, which were accompanied by prolonged contraction duration in SCI animals compared to controls.
These findings suggest that in peripheral nerves SCI promotes hyperexcitability, which might contribute to mechanisms of spastic syndrome.
The studies of Sherrington and others showed that in chronic spinalized and decerebrated preparations reflexes were easily elicitable and responded violently to stimuli, which otherwise had no effect before injury [1, 2]. Hyper-reflexia and spasticity which is velocity dependent increase in muscle tone , are considered as signs for corticoreticulospinal system lesions [4, 5]. There is also evidence linking the development of spasticity and hyper-reflexia to changes in spinal α motor neurons excitability [6–8] spinal interneuronal hyperexcitability  and potentiated synaptic input with muscle stretch [10–14]. However, the exact pathophysiological mechanism which underlies muscle tone and abnormalities in reflexes is unknown. Although, there is the possibility that peripheral nerve physiology might be altered after spinal cord injury (SCI), there have been limited studies to investigate it directly. However, muscle contraction studies showed significant alteration in muscle properties after SCI [15, 16] suggesting that the physiology of the peripheral axons would be altered as a result of SCI and spasticity.
A recent study by Lin et al.,  demonstrated that the function of the peripheral nerves was altered after SCI in humans. They specifically found that peripheral nerves were of high threshold and sometimes were completely inexcitable. They attributed these results to changes in axonal structure and ion channels. However, there is always the possibility that these findings might reflect a lower motor neuron lesion in human subjects. Therefore, an investigation of axonal changes in a more controlled animal model may provide more unequivocal data.
In the present study, we asked, using an animal model, whether the nerve-muscle complex (sciatic-triceps surae) becomes hyperexcitable after spinal cord injury. We specifically hypothesized that excitability measures - amplitude, threshold, latency, conduction velocity, and stimulus-response curves of nerve and muscle - would demonstrate the characteristics of hyperexcitability in nerve-muscle complex after SCI. Moreover, we hypothesized that muscle twitches will demonstrate the properties of spastic muscle as reported by Harris et al., . The present investigation gives evidence that sciatic-triceps surae complex is indeed hyperexcitable after SCI. Thus, it may provide an additional mechanism for spastic syndrome that develops after SCI.
Experiments were carried out on CD-1 male and female adult mice in accordance with NIH guidelines, with all protocols approved by the College of Staten Island IACUC. Animals were housed under a 12 h light-dark cycle with free access to food and water.
Spinal cord contusion injury
Mice were deeply anaesthetized with ketamine/xylazine (90/10 mg/kg i.p.). A spinal contusion lesion was produced (n = 7) at spinal segment T13 using the MASCIS/NYU impactor . The impactor was fitted with a 1 mm-diameter impact head rod (5.6 g) released from a distance of 6.25 mm onto T13 spinal cord level exposed by a T10 laminectomy. After the injury, the overlying muscle and skin was sutured, and the animals were allowed to recover under a heating lamp at 30°C. To prevent infection after the wound was sutured, a layer of ointment containing gentamicin sulfate was applied. Following surgery, animals were maintained under pre-operative conditions for ~8 months before testing. The time of recovery was selected to ensure a stable chronic SCI during testing.
The following behavioral evaluation were performed just before the electrophysiological studies, approximately 8 months after SCI.
Basso mouse scale (BMS)
Motor ability of the hindlimbs was assessed by the categorical motor rating of BMS , using rating system of: 0, no ankle movement; 1-2, slight or extensive ankle movement; 3, plantar placing or dorsal stepping; 4, occasional plantar stepping; 5, frequent or consistent plantar stepping; no animal scored more than 5. Each mouse was observed for 4 min in an open space before a score was given.
Abnormal posture scale (APS)
After SCI, animals usually developed muscle tone abnormalities that were exaggerated during locomotion. We developed a posture scale to quantify the number of muscle tone abnormalities demonstrated by the animals. The rating scale ranges from 0 to 12 with a cumulative score based on the sum of the following abnormalities: limb crossing of midline, abduction, and extension or flexion of the hip joint, paws curling or fanning, knee flexion or extension, ankle dorsi or planter flexion. A score of one was given for each abnormality. The total score is the sum of abnormalities from both hindlimbs. Abnormal postures were usually accompanied by spasmodic movements of the hindlimbs.
Intact (n = 7) and SCI (n = 7) animals underwent a terminal electrophysiological experiment. Animals were anesthetized using ketamine/xylazine (90/10 mg/kg i.p). Electrophysiological procedures started approximately 45 min after the first injection to maintain anesthesia at moderate to light level . As needed, anesthesia was kept at this baseline level using supplemental dosages (~5% of the original dose).
The skin covering the two hindlimbs was removed. Both triceps surae muscles were partially separated from the surrounding tissue preserving blood supply and nerves. The tendon of each of the muscles was connected to the force transducers with a hook shaped 0-3 surgical silk thread. In addition, the sciatic nerve was cleared from the surrounding tissue from the knee to the hip joint. The tissue was kept moist by drops of saline.
Both hind and fore limbs and the proximal end of the tail were rigidly fixed to the base. Muscles were attached to force displacement transducers (FT10, Grass Technologies, RI, USA); the muscle length was adjusted to obtain the strongest twitch force (optimal length). The whole setup was placed on an anti-vibration table (WPI, Sarasota, FL, USA). Animals were kept warm during the experiment with radiant heat (27°C).
Stimulus-muscle and stimulus-nerve response curves were generated by delivering stimuli (1 ms duration), which were increased in steps (in Volts) starting from 0.05, and then increasing from 0.1, to 1.0, and from 2 to 10, in 0.1 V and 1 V increments, respectively. To determine the strength-duration time constant (SDTC), a test protocol was used with 17 stimuli of different durations (10, 20, 30, 40, 50, 70, 90, 150, 200, 300, 400, 600, 800, 1000, 2000 μs). The strength of the stimulation (mA) was adjusted accordingly for each of the durations tested to elicit minimal (all or none) triceps surae muscle response (contraction). In the same group of animals, a test stimulus of two durations (10 and 1000 μs) was used to measure the time constant of 40% of maximal muscle contraction. The threshold charge (threshold current × stimulus duration) was plotted against the stimulus duration. The time constant is given as the negative intercept of the linear regression line of the threshold charge against stimulus duration on the duration axis.
Where M is the latency of the M-response, F is the latency of the F-wave. The 1 ms term is a correction for the delay in re-excitation of the motoneuron .
We recorded the time from the start of the stimulus artifact to the onset of the first deflection of nerve compound action potential (nCAP) as well as muscle twitch. Latency of muscle twitch was also measured as the time from the earliest onset of nCAP to the earliest onset of muscle twitch. Measurements were recorded using a cursor and a time meter on LabChart software. The amplitude of sciatic nerve nCAP was measured as peak-to-peak. Analysis of muscle contractions were performed with peak analysis software (ADInstruments, Inc, CO, USA), as the height of twitch force measured relative to the baseline. Slopes for muscle contractions were extracted through Matlab-based calculations (MathWorks, Natick, MA).
All data are reported as group means ± SEM. One sample t-tests were used for single group. Two sample student's t-tests (or Mann-Whitney Rank Sum Test) was used for two groups; statistical significance at the 95% confidence level. To compare multiple measurements, we performed one way ANOVA with Solm-Sidak corrections for post hoc analysis. Statistical analyses were performed using SigmaPlot (SPSS, Chicago, IL), Excel (Microsoft, Redwood, CA), and LabChart software (ADInstruments, Inc, CO, USA).
We used BMS and APS to identify the animals with SCI those developed locomotor and muscle tone abnormalities. BMS showed that animals with SCI had significant locomotor abnormalities (22.22% ± 4.9% of control). In addition, APS showed that animals with SCI had significant increase in the number of muscle tone abnormalities (9.1 ± 0.59).
To further illuminate this finding, the response to stimulus ratio for all submaximal nCAP was calculated and was found (Figure 3D) to be significantly higher in SCI animals (1436.3 ± 531.2%) than in controls (206.3 ± 29.7%) (p < 0.05). nCAP latency in SCI was significantly shorter (2.4 ± 0.2 ms) than in controls (3.0 ± 0.2 ms) (p < 0.01, Figure 3D).
The results show that the spinal cord injury leads to increased excitability of nerve-muscle complex. Several measures of excitability were employed in the present study. An increase in nerve conduction velocity was accompanied by reduced threshold for nCAP generation and an increase in its amplitude. Thus, the muscle could be excited easier and faster. Moreover, reduction in the duration of PMCT indicates that post-injury axonal changes lead to an increase in the conduction velocity along the whole motor nerve from the spinal cord to the site of the recording electrode located very close to the muscle. These results confirm the finding in paralyzed rats by Cope et al , however these results contradict the finding in human with SCI .
Importantly, SDTC was significantly increased in the sciatic nerves of injured animals. This suggests demyelination and/or increased persistent sodium current . The analysis of properties of muscle twitch in SCI animals revealed slowness in the rate of muscle contraction and relaxation. Similar changes were reported by Harris and collaborators  investigating segmental tail muscle in the rats. Since our experiments were performed on triceps surae muscle in mice, one can conclude that spinal cord injury might cause similar changes in all spastic muscles and across species.
In the present study, all injured animals exhibited behavioral signs of spasticity and demonstrated spasms. This indicates that the SCI that leads to spasticity may also be responsible for the increase in excitability of axons and muscles. It is known that spinal cord injury or brain damage results in hyperexcitability of neuromuscular system (expressed as dystonia, spasticity, spasm and hyper-reflexia). Although possible mechanisms of hyperexcitability may include among others the increased excitability of spinal motoneurons [6–8], spinal interneuronal hyperexcitability and potentiated synaptic input to the muscle [10–14], the exact mechanism of this phenomenon remains largely unknown. Our results expand current views on the hyperexcitability-mediating mechanisms, demonstrating that the whole neuromuscular complex becomes hyperexcitable and may participate in the mechanisms of spastic syndrome and its expression. This notion contradicts recent findings described by Lin et al., , who reported higher axonal threshold in human subjects with SCI. These differences may be due to a complex pathophysiology of SCI in humans which may be additionally complicated by nerve root injury. While pathophysiology of SCI in humans has been subdivided into several different types which can involve both peripheral and central damage, experimental damage of the spinal cord in animals represents reproducible injury executed in a well controlled fashion. The lesions are usually localized and limited to the zone of approximately 700 μ without apparent root damage (Ahmed, unpublished observation). The effects of SCI can also depend on the type of the muscle innervated by a damaged spinal cord segment. While Lin et al.,  evaluated motor pathway of tibialis anterior, triceps surae muscle and its innervations was the subject of our research. In support of this notion Yoshimura and Groat  reported that in SCI rats there was an increase in the excitability of the afferent neurons innervating urinary bladder but there was no change in neurons innervating the colon.
Inferences from the present results point to lesion-induced intrinsic changes in the peripheral axons and muscles. SDTC reflects mostly passive properties of the membrane at the nodes of Ranvier . Its increase in SCI animals might indicate injury-induced demyelination and/or an increase of the expression of sodium channels (particularly persistent Na+ channel) at the nodes, similarly as reported by Yoshimura and Groat  in afferents to urinary bladder, and observed by us in the sciatic nerve (unpublished observation). While upregulation of the sodium channel expression, or an increase in their rate activation constant  could reflect additional processes responsible for the increased conduction velocity, it can also be influenced by changes in axon diameter, myelin capacitance, and axoplasmic conductance [29, 30]. An increase in any of these factors with the exception of myelin capacitance would increase the conduction velocity. An increase in the diameter of the spinal neurons (which enhances axoplasmic conductance) observed after SCI [31, 26] could also take place in our animals and be responsible for observed increase in conduction velocity. In addition to axonal diameter, the axoplasmic conductance (and subsequently conduction velocity) can be enhanced by limited hypomyelination of the axon especially at the internode regions . The hypomyelination could also induce up-regulated expression of the sodium channels and ensuing hyperexcitability, as reported for shiverer mouse brain .
There was difference in threshold between week and strong muscles in animals with SCI. In that, weaker muscles expressed lower threshold than stronger muscles, becoming hyperexcitable. However, it has been reported that the weakness of the muscle induced by disuse in intact animals does not lead to hyperexcitability [36, 37]. This implies that hyperexcitability is not induced by injury-related disuse of the weak and spastic muscles, but might result from interaction between the effects of disuse and lesion-induced processes.
In conclusion, we have demonstrated that the nerve-muscle complex becomes hyperexcitable in animals with SCI. The nCAP from the sciatic nerve was of higher amplitude, lower threshold, longer strength-duration time constant, and faster conduction velocity. In addition, the earliest onset of muscle contraction from the triceps surae muscle was shorter in SCI animals when compared to controls. Muscle twitches were of slower rising and falling slopes, with prolonged contraction duration in SCI animals compared to controls. These findings show that after SCI motor axons undergo excitability changes similar to their perikarya in the ventral horn of the spinal cord [6–8]. One might speculate that hyperexcitability of peripheral motor axons after SCI injury may partially underlie the expression of spastic syndrome seen after SCI.
This research was supported by NYS/DOH grant # CO23684 and PSC-CUNY grant 60027-37-39 to ZA.
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