Time-related changes of motor unit properties in the rat medial gastrocnemius muscle after the spinal cord injury. II. Effects of a spinal cord hemisection

Journal of Electromyography and Kinesiology 20 (2010) 532–541

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Time-related changes of motor unit properties in the rat medial gastrocnemius muscle after the spinal cord injury. II. Effects of a spinal cord hemisection Jan Celichowski a,*, Katarzyna Krys´ciak a, Piotr Krutki a, Henryk Majczyn´ski b, Teresa Górska b, Urszula Sławin´ska b a b

´ , Poland Department of Neurobiology, University School of Physical Education, Poznan Nencki Institute of Experimental Biology, Warsaw, Poland

a r t i c l e

i n f o

Article history: Received 12 March 2009 Received in revised form 5 June 2009 Accepted 8 July 2009

Keywords: Spinal cord hemisection Motor units Rat

a b s t r a c t The contractile properties of motor units (MUs) were investigated in the medial gastrocnemius (MG) muscle in rats after the spinal cord hemisection at a low thoracic level. Hemisected animals were divided into 4 groups: 14, 30, 90 and 180 days after injury. Intact rats formed a control group. The mass of the MG muscle did not change significantly after spinal cord hemisection, hind limb locomotor pattern was almost unchanged starting from two weeks after injury, but contractile properties of MUs were however altered. Contraction time (CT) and half-relaxation time (HRT) of MUs were prolonged in all investigated groups of hemisected rats. The twitch-to-tetanus ratio (Tw/Tet) of fast MUs after the spinal cord hemisection increased. For slow MUs Tw/Tet values did not change in the early stage after the injury, but significantly decreased in rats 90 and 180 days after hemisection. As a result of hemisection the fatigue resistance especially of slow and fast resistant MU types was reduced, as well as fatigue index (Fat I) calculated for the whole examined population of MUs decreased progressively with the time. After spinal cord hemisection a reduced number of fast MUs presented the sag at frequencies 30 and 40 Hz, however more of them revealed sag in 20 Hz tetanus in comparison to control group. Due to considerable changes in twitch contraction time and disappearance of sag effect in unfused tetani of some MUs in hemisected animals, the classification of MUs in all groups of rats was based on the 20 Hz tetanus index (20 Hz Tet I) but not on the standard criteria usually applied for MUs classification. MU type differentiations demonstrated some clear changes in MG muscle composition in hemisected animals consisting of an increase in the proportion of slow MUs (likely due to an increased participation of the studied muscle in tonic antigravity activity) together with an increase in the percentage of fast fatigable MUs. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction The influence of spinal cord injury has been studied in numerous experiments where different properties of skeletal muscles were investigated. Until now, the effects of spinal cord injury in muscle and motor unit (MU) contractile properties were investigated mainly after total transection of the spinal cord performed by a surgery whereas other kinds of mechanical injuries of the spinal cord were not studied. The experimental design of the evaluation of muscle morphology and function was different in those reports. They included investigations of contractile properties and morphometric measurements of whole muscles (Lieber et al., 1986a), histochemical studies of muscles and motor units (Mayer et al., 1984), electrophysiological studies on motoneurones and

* Corresponding author. Address: University School of Physical Education, 55, Grunwaldzka St., 60-352 Poznan´, Poland. Tel.: +48 61 8355435; fax: +48 61 8355445. E-mail address: [email protected] (J. Celichowski). 1050-6411/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jelekin.2009.07.003

motor units (Cope et al., 1986; Munson et al., 1986) or on functionally isolated motor units (Celichowski et al., 2006). All these investigations demonstrated that the spinal cord transection leads to decreased muscle mass, muscle fiber atrophy and modification of several contractile properties such as force, contraction time, fatigue resistance (Mayer et al., 1984; Lieber et al., 1986b; Celichowski et al., 2006; Mrówczyn´ski et al., 2009). Spinal cord injury leads to dramatically limited locomotor activity. The hind limbs of spinal rats are paralyzed and do not support the bodyweight. During spontaneous behaviour spinal animals use fore limbs while the hind limbs are dragged behind the body. In spinal animals the hind limb muscle activity is dramatically altered and only a limited EMG activity can be recorded. In our previous paper we described changes in contractile properties of MUs in the rat medial gastrocnemius (MG) muscle one month after the spinal cord total transection (Celichowski et al., 2006). The progressive development of those changes in the same parameters at various time points after the total spinal cord transection were the subject of an accompanying paper (Mrówczyn´ski

J. Celichowski et al. / Journal of Electromyography and Kinesiology 20 (2010) 532–541

et al., 2009). We found that the total spinal cord transection induced significant changes in MUs properties. In the fast fatigable motor units (FF) the contraction time and the half-relaxation time were prolonged, the twitch-to-tetanus ratio was increased whereas the ability to post-tetanic potentiation was diminished and maximal tetanus force was reduced, while the MUs of FR and S type became more fatigable. Moreover, the sag phenomenon in unfused tetanic contraction of fast MUs almost totally disappeared one month after the spinal cord injury (Celichowski et al., 2006). When MU contractile properties were analyzed 14, 30, 90 and 180 days after the spinal cord transection (Mrówczyn´ski et al., 2009) all these changes in MU properties, mentioned above, developed progressively in time. Thus, in the MG muscle of spinal rats 180 days after spinal cord total transection a complete lack of S type and a higher proportion of FF type MUs were observed. As described above, effects caused by spinal cord injury were studied on various species of mammals (mainly on cats and rats). Some features of muscular tissue were changed in similar way in those species, and some showed interspecies differences. One might expect that similar changes observed in various mammals can also be attributed (in qualitative aspects) to human beings after spinal cord injury. In clinical practice various forms of partial injuries are obtained (Wernig et al., 1999; Wirz et al., 2001; Dietz and Harkema, 2004). Therefore, numerous investigations examining the effects of a partial spinal cord injury in animal models were employed (Hultborn and Malmsten, 1983a,b; Górska et al., 1993; Kuhtz-Buschbeck et al., 1996; Galea and Darian-Smith, 1997; Górska et al., 2007). However, so far still little is known about changes of MU contractile properties induced by partial spinal cord lesions. In rats 14 days following spinal cord hemisection changes in myosin regulatory light chain phosphorylation, depressed posttetanic potentation, and increased twitch-to-tetanus ratio, as well as lower tetanic force of the MG muscle (Tubman et al., 1997), and larger excitatory reflexes on the lesioned side in comparison to the contralateral side (Malmsten, 1983) were described. In comparison to the results of the total spinal cord transection, which evoked a complete lose of postural and locomotor hind limb functions, partial spinal cord injury induced less dramatic limitation in movement. After a period of just a few weeks a restitution of voluntary locomotor activity is usually observed. In cats, after the spinal cord hemisection first paresis and then considerable improvement of affected hindlimb locomotor function were observed (Hultborn and Malmsten, 1983a; Kuhtz-Buschbeck et al., 1996). However, several months after unilateral lesion various locomotor deficits were still present, that can be divided into three groups: (1) disturbed interlimb coordination, (2) reduced flexor capacity of the affected hindlimb, and (3) impaired timing of the flexion–extension events (Kuhtz-Buschbeck et al., 1996). Hultborn and Malmsten (1983a,b) reported an increase in mono- and polysynaptic reflexes as well as larger facilitatory effects in motoneurones on the lesioned side. In addition, several changes in motoneurone properties after thoracic hemisection were reported: normal or higher firing rates, decreased minimum firing rates, decrease in recruitment force and shorter afterhyperpolarization time. The firing rate alterations were more evident in motoneurones with higher axonal conduction velocity (fast), while afterhyperpolarization changes concerned mainly motoneurones with lower axonal conduction velocity (slow). All motoneurones tended to show patterns of discharge typical for lower threshold motoneurones in intact animals (Powers and Rymer, 1988; Carp et al., 1991). Since, there is a lack of data concerning changes in MU contractile properties after partial lesions of the spinal cord, the aim of our study was to investigate time related changes in MU contractile properties after the spinal cord hemisection. Locomotor activity of rats, as well as cats, after spinal cord hemisection is also not

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so dramatically disturbed as that of rats with their spinal cord totally transected (Malmsten, 1983). Nevertheless, their hind limb muscles do not work properly and it can be expected that this altered muscle activity might influence MU contractile properties.

2. Methods Adult female rats of Wistar strain (n = 21) with the weight 251 ± 48 g (mean ± SD) were used in these investigations and were divided on five experimental groups. Six rats formed the control group. In the remaining 15 animals the effects of the left-side hemisection at the Th9 level of spinal cord was tested 14 (n = 3), 30 (n = 3), 90 (n = 4), 180 (n = 5) days after injury. All procedures used in these experiments were carried out with the approval of the local Ethics Committee and followed EU guidelines on animal care. 2.1. Surgical procedure and post-operative care Spinal cord hemisection at a low thoracic level (Th9/10) was performed in fifteen 3-month-old Wistar rats under deep Equithesin anaesthesia (9.7 mg pentobarbitone, 7.6 mg ethyl alcohol, 42.5 mg chloral hydrate, 428 mg propylene glycol, 21 mg MgSO4 per millilitre sterilized H2O), the amount of anaesthetic was 0,3 ml/100 g, i.p. The spinal cord was exposed by laminectomy over a length of 6–8 mm at the low thoracic level (Th9–Th10). The bone was removed as far laterally on the left as possible. Dura was cut, under the dissecting microscope, laterally to the dorsal columns. The spinal cord was dissected using fine watchmaker’s forceps. The operative field was cleaned of fluids by gentle suction. Finally, the dura was left unsutured and the wound was closed in anatomical layers using sterile sutures (Mersilk 0.22 mm) (Sławin´ska et al., 2000; Majczyn´ski et al., 2005). The skin was closed with stainlesssteel surgical clips. An adequate level of anaesthesia during surgery was ensured by regular testing for the lack of cutaneous withdrawal reflexes of the forelimbs and an additional dose of anaesthetic (0.1 ml/100 g, i.p.) was given when needed. After surgery, the animals received a non-steroidal anti-inflammatory and analgesic treatment Tolfedine (tolfedine acid, V´etoquinol, France) (0.4 mg/100 g, i.p.). All animals recovered from anaesthesia within 2–3 h after surgery. During the following 10 days, the animals were routinely (daily) given antibiotics gentamicin (LEK, Poland) (0.2 mg/100 g) and Baytril (Enrofloxacin, Bayer Healthcare) (0.5 mg/100 g, i.p.). Before the acute experiments, the rats were observed during natural exploratory behaviour in the home cage to check the degree of hindlimb impairment. 2.2. Final electrophysiological experiment For all animals the same methods of single MUs experiments as in the accompanying paper (Mrówczyn´ski et al., 2009) were applied. The acute electrophysiological experiments were performed on animals under pentobarbital anaesthesia (initial dose of 60 mg/ kg, i.p., supplemented when necessary with additional doses of 10 mg/kg). Throughout the experiments the depth of anaesthesia was precisely verified by controlling the pinna and withdrawal reflexes. Experiments were performed on the MG muscles. The sciatic nerve and the examined muscle were isolated from surrounding tissues. Blood vessels and nerve branches supplying the MG muscle were left intact, whereas remaining muscles of the hindlimb were denervated. The contractile properties of MUs in the studied muscle were measured under isometric conditions. To achieve this the Achilles tendon was cut and connected to the force transducer. During experiments the MG muscle was maintained in passive

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tension of 100 mN when most of its MUs generate maximal twitch force (Celichowski and Grottel, 1992). The spinal cord was exposed by over L2–S1 segments laminectomy and ventral roots were cut proximally to the spinal cord. Steel clamps were attached in three points: tibia, sacral bone and L1 vertebra immobilized animal and the operated hindlimb. The examined muscle was immersed in warm paraffin oil. Over the exposed part of the spinal cord special pool formed by skin was made and it was filled by warm paraffin oil. The oil temperature as well as rats core temperature were maintained at the level 37 ± 1 °C throughout the experiment by an automatic heating system. 2.3. Recordings and analysis For isolation of individual MU the L5 and L4 ventral roots were divided with fine forceps into as thin as possible filaments, which were stimulated electrically. The criterion used to approve force and muscle fiber action potentials, as an activity of single MU was ‘‘all or none” type response at stimulation with rectangular electrical pulses of 0.1 ms duration and variable amplitude up to 0.5 V. The muscle fiber action potentials were recorded using silver-wire bipolar electrodes inserted into the muscle. The force was recorded with a force transducer (FT 510). Recording forces and action potentials were stored on a computer disc using analog-to-digital converter (RTI-800 Utilities). The sampling rates of 1 kHz for force and 10 kHz for action potentials were used. For each MU the same stimulation protocol and the same pattern of stimuli was applied as in our previous studies (Celichowski et al., 2006; Mrówczyn´ski et al., 2009). The stimulation protocol was controlled by the computer program cooperating with the S88 Grass stimulator and the SIU5 isolation unit, and recorded MU activity had the following order: (1) the averaged twitch contraction (5 pulses at 1 Hz); (2) the unfused tetanus (500 ms train of stimuli at 40 Hz); (3) the fused tetanus (200 ms train of stimuli at 150 Hz); (4) series of tetani at progressively increasing stimulation frequencies (500 ms trains of stimuli at 10, 20, 30, 40, 50, 60, 75, 100 and 150 Hz in 10 s time intervals); (5) the averaged twitch contraction (5 pulses at 1 Hz); (6) the standard fatigue test (trains of 14 pulses at 40 Hz, repeated every 1 s for 3 min) (Burke et al., 1973). All six elements described above were generated with 10 s intervals. The following contractile parameters were measured: contraction time (CT, measured from the beginning of the contraction to its force peak), half-relaxation time (HRT, measured from the force peak of the contraction to a half of this value), and twitch force (TwF, from the baseline to the peak force of the contraction). In the fused tetanic contraction at 150 Hz the maximal tetanic force (TetF, measured from the baseline to the peak of tetanic contraction) was measured. The two force parameters (TwF and TetF) were used to calculate twitch-to-tetanus (Tw/Tet) ratio. The presence of a sag phenomenon was verified in unfused tetanic contractions evoked at 20, 30 and 40 Hz stimulation. The post-tetanic potentation (PTP) was measured as a ratio of the twitch force recorded after a series of tetanic contractions (the potentiated twitch) to the initial twitch force. The fatigue test was used to calculate the fatigue index (Fat I) as a ratio of the force measured 2 min after the maximal initial force to this maximal force value at the beginning of the test (Kernell et al., 1983). For MU classification into slow and fast categories we used the 20 Hz Tet I, which was calculated as a ratio of the force of the last contraction of the tetanus at 20 Hz to the force of the first contraction in this tetanus (Krutki et al., 2008). In order to distinguish two types of fast MUs: FF and FR, the Fat I was applied. Fast MUs with the Fat I below 0.5 were classified as a fast fatigable (FF), whereas those with Fat I above 0.5 as a fast resistant to fatigue (FR) (Kernell et al., 1983; Celichowski, 1992). Statistical evaluation of the results was

made using Mann–Whitney U-test. Additionally, the ANOVA Kruskal–Wallis and Post Hoc tests were used for statistical analysis of the changes in the muscle mass. 2.4. Histological verification of the spinal cord lesion extent At the end of acute electrophysiological experiment under a lethal dose of pentobarbitone (180 mg/kg, i.p.) the rats were transcardially perfused with 0.1 M PBS followed by 4% paraformaldehyde dissolved in 0.1 M PBS. Spinal cords were removed and postfixed in 4% paraformaldehyde/0.1 M PBS for 24 h. Thereafter, they were routinely processed and embedded in paraffin. The histological verification of the extent of the lesion was made on transverse sections of a piece of spinal cord containing the lesion, cut serially every 10 lm. Every 10th section at the level of the lesion and every 20th section 3–6 mm below and above the lesion, were stained alternatively with Kluver–Barrera, Nissl and Welcke methods. All sections were examined by light microscopy to determine the maximum size of the lesion for each rat. 3. Results 3.1. Histological verification Histological examination of the spinal cord lesion extent revealed that in the majority (10 of 15) of hemisected rats the lesions were confined almost exclusively to the left part of the spinal cord. Only in some (3 of 15) rats the extent of the lesion was smaller, sparing part of the left ventral funiculus (Fig. 1A), while in a few (2 of 15) others the extent of the lesion was greater and asymmetrical, involving the right ventral funiculus and sparing a small medial part of the left dorsal funiculus (Fig. 1B). 3.2. Behavioural observations All the animals following the left spinal cord hemisection were carefully investigated to check the degree of hindlimb impairment during natural exploratory behaviour in the home cage and during four-limb locomotion on a horizontal pathway. One day after hemisection all the rats locomoted using their forelimbs, while the body and hindquarters were dragged behind them. In all hemisected rats the left hindlimb was paralyzed and hyperflexed while the right hindlimb was extended and only small movements at the hips were observed. One week after surgery most rats regained active movement in hip, knee, and ankle joints of left hindlimb, but no plantar stepping or weight bearing support was seen. The feet were kept on their dorsal surface. All the rats had slight body weight support and plantar stepping in right hindlimb. The rats recovered four-limb locomotion about 10 days after surgery, however the hind limbs were still in a crouch position. Two weeks after surgery locomotion of the rats was still unstable, with poor forehind limb coordination as a result of occasionally shorter forelimb and longer hind limb steps. One month after spinal cord injury most of hemisected rats regained stable locomotor pattern with wider base of support in hind limbs and their left hindlimbs abducted. These alteration of locomotion persisted up to 6 months after surgery. 3.3. Muscle and MUs investigations The analysis of muscle atrophy performed after the final acute experiments revealed that the mass of the medial gastrocnemius muscle did not change significantly after spinal cord hemisection in comparison to control animals. The ratio of the medial gastrocnemius muscle to the body mass of rats in all experimental groups

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Fig. 1. Extent of lesion (hatched) performed at low thoracic level of the spinal cord in rats with the smallest (A) and the greatest (B) lesion area. L, left; R, right side of the spinal cord.

was evaluated as 0.25 ± 0.03 for the control group whereas 0.27 ± 0.03, 0.25 ± 0.01, 0.25 ± 0.02, 0.27 ± 0.04 for hemisected rats studied 14, 30, 90, 180 days after injury, respectively. The differences between groups were statistically insignificant (p > 0.05, ANOVA Kruskal–Wallis and Post Hoc tests). 3.4. Differentiation of MUs The contractile properties of 260 MUs in the MG muscle were investigated in rats after the spinal cord hemisection, whereas 84 MUs in intact rats were examined as a control group. In the hemisected animals from four groups: 14, 30, 90, 180 days after the spinal cord injury the following numbers of motor units were investigated: 49, 58, 78 and 75 MUs, respectively. In the present study MUs were classified as fast or slow types on the basis of 20 Hz Tet I (Krutki et al., 2008). As we described in an accompanying paper the parameters, which are usually applied for MU classification (i.e., the contraction time and sag effect) cannot be used in the case of data collected for fast-slow differentiation due to considerable changes either in twitch contraction times or lack of sag effect in some MUs after spinal cord injury. Fig. 2 presents a distribution of two types of MUs (slow and fast) based on the relation between the contraction time and the 20 Hz Tet I. The horizontal doted lines indicate values of the Tet I, where a clear-cut border between fast and slow types of MUs was determined i.e., 2.0 (for details see Krutki et al., 2008). All MUs with values of 20 Hz Tet I below 2.0 were accepted as fast, whereas those with the Tet I above 2.0 as slow ones. In general, the 20 Hz Tet I for the majority of fast MUs was around 1.0, whereas values for slow units considerably exceeded 2.0 (Fig. 2). For intact animals the border value between the longest twitch contraction times for fast MUs and the shortest values for S units was visible at 18 ms and was used as an additional criterion for fast/slow MU division (Celichowski et al., 2006; Krutki et al., 2008). The same results for fast/slow division were obtained using the classification based on the 20 Hz Tet I (Fig. 2A). However, for MUs in rats after the spinal cord hemisection the contraction time could not be used as a criterion of division because of a significant prolongation of the twitch time parameters (the contraction time as well as half-relaxation time). Moreover, in almost all groups of animals with spinal cord hemisection, contraction times for slow and fast units overlapped, and the border values were not possible to determine (Table 1 and Fig. 2B–E). In the control group of intact animals all fast MUs presented a sag in unfused tetanic contractions at 30 and 40 Hz stimulation

frequency and 75% of FF and 60% of FR units additionally presented sag at 20 Hz as well. After the spinal cord hemisection the number of MUs with a sag phenomenon visible at frequencies 30 and 40 Hz diminished, but on the other hand, more units revealed sag in 20 Hz tetanus in comparison to control group (Fig. 3). In spite of the decrease in the relative occurrence of MUs with sag, almost all fast MUs showed the sag in at least one of the examined frequencies (there were only six MUs out of 260 studied with no sag in any of the three frequencies). Using the above criteria for MU type differentiations in animals with their spinal cord hemisected we analyzed changes in proportion to the three types of MUs in MG muscle resulting from the spinal cord injury (Fig. 4). There was an increase in the percentage of fast fatigable MUs (starting from 14 to 90 days after the spinal cord injury group) together with an increase in the proportion of slow MUs in groups 14, 30, 90 and 180 days after the hemisection. 3.5. Changes in MU contractile properties Table 1 presents mean values (±SD) of all the contractile parameters investigated in the three types of MUs for control animals and for rats of four groups studied at various time points after the spinal cord hemisection. The spinal cord injury induced a prolongation of the twitch contraction. The prolongation of contraction time and half-relaxation time was statistically significant in all four groups of injured animals (Mann–Whitney U-test p < 0.01, see Table 1). It should be also stressed that changes in contraction time and half-relaxation time were the most statistically significant out of all revealed alterations in contractile parameters. The twitch-to-tetanus ratio of fast MUs after the spinal cord hemisection increased in comparison to control animals; especially for FF type MUs (Fig. 5). Generally, the twitch force revealed the tendency to increase and the tetanus force to decrease in FF and FR MUs groups (Table 1). These changes resulted in the mentioned above elevation of twitch-to-tetanus ratio. For slow MUs values of twitch-to-tetanus did not change in the early stage after the spinal cord injury (14 and 30 days), but for 90 and 180 days groups statistically significant decrease of this parameter was observed (Mann–Whitney U-test, p < 0.01). Decreased twitch-to-tetanus ratio in two groups of slow MUs was an effect of decreased twitch force values while at the same time the tetanus force did not change significantly. The spinal cord hemisection only slightly influenced the ability of fast MUs to potentiate their forces. For FF and FR decrease of post-tetanic potentation particularly two weeks after the spinal

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A

A

10

[%] 100

sag phenomenon

2 1

control 0.1

B

10

FF

80 60 40 20

2 1

0

14 days after hemisection

B

20 Hz Tet I

10

D

2 1

30 days after hemisection

90

180

[days]

FR

80 20 Hz 60

30 Hz

40

40 Hz

20

10

0

C

14

30

90

180

[days]

time after the spinal cord hemisection

2 1

Fig. 3. The presence of sag phenomenon in unfused tetani at the three stimulation frequencies (20, 30, 40 Hz) in control (C) and four groups of rats with the spinal cord hemisected 14, 30, 90 and 180 days after hemisection for FF (A) and FR (B) motor units. Black columns – stimulation at 20 Hz, grey columns – stimulation at 30 Hz, white columns – stimulation at 40 Hz.

90 days after hemisection 0.1

E

30

100

0.1

10 2 1

slow units fast units 180 days after hemisection

0.1 0

10

20

30

40

50

CT (ms) Fig. 2. Relationship between the 20 Hz tetanus index (20 Hz Tet I) used for classification of motor units into fast and slow types and the contraction time: A – control rats; B, C, D and E – hemisected rats (14, 30, 90 and 180 days after the spinal cord hemisection, respectively). The horizontal dotted lines denote value of 2.0 of the 20 Hz Tet I used for fast/slow MUs classification. Black circles – fast motor units, white circles – slow motor units.

cord injury was visible, but this change in most of studied groups of MUs was not statistically significant (Mann–Whitney U-test, p > 0.05) (Fig. 5). In comparison to control group the ability of post-tetanic potentiation of slow type MUs in all groups after the spinal cord hemisection did not change as well. The hemisection reduced the fatigue resistance of all the types of MUs. Higher fatigability of each type of MUs was illustrated by lower values of Fat I (Table 1 and Fig. 6). Significant changes were noted particularly in FR and S type of MUs (except 30 days group of FR units) in comparison to control MUs (Mann–Whitney U-test, p < 0.01). Furthermore, Fat I calculated for the whole examined population of MUs decreased progressively with the time after the hemisection and amounted to 0.69 for control groups and 0.65, 0.63, 0.62, 0.62 for groups 14, 30, 90 and 180 days after the injury, respectively. In the accompanying paper (Mrówczyn´ski et al., 2009) changes in MU properties after the complete spinal cord transection are

proportion of motor units

C

14

[%]

sag phenomenon

0.1

C

[%] 70

60

FF

50 FR

40

S

30 20 10 0

[days]

C

14

30

90

180

time after the spinal cord hemisection Fig. 4. Proportions of the three types of motor unit in control group (C) and four subsequent groups of units studied 14, 30, 90, 180 days after the spinal cord hemisection. Black columns – fast fatigable motor units, grey columns – fast resistant motor units, white columns – slow motor units.

evaluated overtime. For comparison with those data statistical analysis of differences between effects of transection and hemisection was performed (Table 2). Statistically significant differences between groups of rats with transected and hemisected spinal cord concerned all the measured parameters, however they were not equally distributed in time. The differences in the development of changes of MU properties after the total transection and hemisection are analyzed in the Section 4 below. 4. Discussion The present study is a continuation of our previous studies of the effects on MU contractile properties after the total spinal cord

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Table 1 Basic contractile properties (mean values ± SD and ranges) of three types of motor units from control and four groups of rats hemisected 14, 30, 90 and 180 days before the final experiments. CT – the contraction time, HRT – the half-relaxation time, TwF – the twitch force, TetF – the maximal tetanic force, Fat I – the fatigue index, 20 Hz Tet I – tetanus index. Types of motor units: S – slow, FR – fast resistant, FF – fast fatigable. Types of MUs

Number of MUs

CT

HRT

TwF

TetF

Fat I

20 Hz Tet I

Control (6 rats) S

10

FR

43

FF

31

22.3 ± 1.7 (20–26) 13.1 ± 1.7 (10–17) 11.8 ± 1.6 (9–15)

34.6 ± 6.4 (27–43) 15.1 ± 4.0 (9–25) 11.3 ± 3.1 (8–22)

4.5 ± 1.8 (1.7–6.7) 11.9 ± 6.9 (1.2–27.1) 33.6 ± 17.5 (5.9–81.1)

35.3 ± 13.3 (14.5–57.3) 61.2 ± 27.5 (7.7–124) 120.2 ± 52 (34.6–233)

0.99 ± 0.01 (0.96–1.0) 0.85 ± 0.1 (0.55–1.0) 0.22 ± 0.1 (0.02–0.42)

5.7 ± 1.99 (2.32–8.54) 1.03 ± 0.15 (0.81–1.49) 0.94 ± 0.11 (0.79–1.26)

14 days after the spinal cord hemisection (3 rats) S 14 30.5 ± 6.6* (22–44) FR 12 15.3 ± 2.6* (11–20) FF 23 14.3 ± 2.1* (11–18)

53.2 ± 16.7* (28–83) 18.6 ± 5.6* (14–32) 16.7 ± 7.3* (11–43)

3.9 ± 2.3 (1.8–9.2) 14.1 ± 8.9 (3.8–33.9) 65.7 ± 50.7* (2.1–207.4)

26.2 ± 10.8 (15.7–55.5) 54.6 ± 23.6 (22.5–106.9) 161.3 ± 93.5 (7.8–417.7)

0.91 ± 0.07* (0.75–0.99) 0.76 ± 0.07* (0.68–0.87) 0.27 ± 0.11 (0.12–0.50)

6.49 ± 2.34 (2.05–11.49) 0.96 ± 0.06 (0.86–1.10) 0.95 ± 0.07 (0.84–1.09)

30 days after the spinal cord hemisection (3 rats) S 14 31.0 ± 6.3* (19–41) FR 18 13.9 ± 1.5 (12–17) FF 26 14.5 ± 1.6* (11–19)

51.9 ± 19.9* (29–98) 17.9 ± 3.9* (12–28) 17.5 ± 5.2* (11–32)

1.8 ± 0.7* (1.1–3.4) 10.0 ± 8.3(3.227.2) 31.3 ± 16.1 (10.0–68.2)

13.2 ± 4.2* (7.7–20.5) 38.7 ± 21.9* (16.8–95.3) 83.4 ± 33.0* (31.2–149.1)

0.99 ± 0.06 (0.92–1.09) 0.72 ± 0.09* (0.52–0.83) 0.17 ± 0.11 (0.02–0.48)

5.63 ± 1.66 (2.36–8.22) 0.99 ± 0.10 (0.81–1.22) 1.04 ± 0.09* (0.95–1.40)

90 days after the spinal cord hemisection (4 rats) S 20 35.9 ± 6.6* (25–50) FR 24 18.5 ± 4.1 (13–27) FF 34 16.6 ± 2.4* (13–23)

59.0 ± 21.3* (31–106) 22.2 ± 8.7* (11–46) 18.5 ± 7.2* (9–40)

3.4 ± 2.5 (1.2–11.8) 17.3 ± 10.3 (6.4–42.8) 53.1 ± 29.1* (12.0–119.4)

29.2 ± 8.5 (18.2–46.6) 76.8 ± 30.7* (35.1–165.0) 158.8 ± 76.1* (43.7–296.8)

0.94 ± 0.05* (0.85–1.01) 0.77 ± 0.08* (0.53–0.90) 0.16 ± 0.10* (0.01–0.42)

8.43 ± 2.52* (2.27–11.62) 1.11 ± 0.43 (0.80–2.46) 0.95 ± 0.16 (0.74–1.51)

180 days after the spinal cord hemisection (5 rats) S 32 30.9 ± 4.6* (22–43) FR 19 16.4 ± 2.1* (14–22) FF 24 15.3 ± 1.8* (10–18)

53.5 ± 13.3* (33–96) 19.4 ± 5.9* (13–32) 17.4 ± 3.6* (12–24)

3.1 ± 1.7* (1.1–9.2) 20.9 ± 19.4 (3.3–83.1) 52.2 ± 33.0* (2.2–122.3)

36.5 ± 15.3 (10.0–74.0) 84.2 ± 60.5 (12.0–306.0) 151.8 ± 79.6 (8.4–295.0)

0.91 ± 0.11* (0.57–1.00) 0.77 ± 0.05* (0.64–0.86) 0.18 ± 0.09 (0.07–0.36)

9.09 ± 2.86* (3.09–13.41) 1.08 ± 0.32 (0.68–1.86) 1.03 ± 0.16* (0.75–1.50)

*

p < 0.05 – difference significant in relation to the control group (Mann–Whitney U-test).

transection in rats (Celichowski et al., 2006; Mrówczyn´ski et al., 2009). At the same time this is the first analysis of the changes of MU properties evoked by spinal cord hemisection. Results obtained in these experiments are compared to control animals and to results concerning changes after a total spinal cord transection described in an accompanying paper (Mrówczyn´ski et al., 2009). These data provide a better understanding of effects of the spinal cord injuries. The majority of the spinal cord injuries in humans are partial and therefore studies of this kind of spinal cord injury are important for clinical practice. So far the following aspects of spinal cord injury have been studied: behavioural investigation (locomotor function and EMG analysis) (Górska et al., 1993; Górska et al., 2007; Kuhtz-Buschbeck et al., 1996), clinical examinations and observations on humans with spinal cord injuries (Wernig et al., 1999; Wirz et al., 2001; Dietz and Harkema, 2004). Results of these studies enabled only partial prediction of the expected changes of MU contractile properties. Our results indicate that spinal cord hemisection in the rat evokes the following changes of MU contractile properties in MG muscle: prolonged contraction time and half-relaxation time, increased twitch-to-tetanus ratio, slightly decreased post-tetanic potentiation, increased fatigability of MUs as well as a changed contribution of particular types of MUs in the muscle. Moreover, after the spinal cord hemisection the disturbances in characteristic profiles of unfused tetanic contractions of fast MUs appeared – the sag phenomenon disappeared in fast MUs subfused tetanic con-

tractions. In our previous studies (Celichowski et al., 2006; Mrówczyn´ski et al., 2009) we observed more dramatic changes of contractile properties of MUs occurring after total spinal cord transection in comparison to those obtained after the spinal cord hemisection (Table 2). The comparison of the effects of partial (hemisection) and total spinal cord transection revealed statistically significant differences in all types of MUs. The total spinal cord transection resulted in total disappearance of slow type of MUs while in the fast MU types more strongly expressed prolongation of twitch time parameters and deeper force modulation, increasing with time after injury (Table 2). Moreover, a more reduced ability to potentiate the force (particularly in FF MUs) was observed after the total spinal cord transection (Table 2). Both kinds of injury induced very similar changes in fatigability of all MUs types. However, the Fat I of FF MUs 3 and 6 months after injury was lower after the spinal cord hemisection in comparison to the effects of the total transection (p < 0.001 and p < 0.05, respectively, see Table 2). Two possible reasons of these differences should be considered. As discussed in the accompanying paper (Mrówczyn´ski et al., 2009), in spinal animals FF MUs are less subjected to plasticity caused by reduction of motor performance. On the contrary, after the hemisection the animals within a few weeks regain activity of the muscles innervated by motoneurones located below the hemisection. Another explanation concerns the opposite directions of MU transformation after the total and partial spinal cord injury (see the section below). The increased transformation of previously less fatigable MUs of the FR type into the group of

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A 0.8

*

*

B

*

*

FF

2

PTP

Tw/Tet

0.4

1 0.5

0.2

0

0 C

14

30

90

C

180

FR

0.8

*

0.6

14

90

180

*

1.5

0.4

30

2

* PTP

Tw/Tet

*

1.5

0.6

0.2

1 0.5

0

0 C

14

30

0.8

90

180

*

*

0.2

14

30

90

180

C

14

30

90

180

1.5

0.6 0.4

C 2

S

PTP

Tw/Tet

*

1 0.5

0

0

C

14

30

90

180

time after the spinal cord hemisection (days) Fig. 5. Mean values and their standard deviations of twitch-to-tetanus ratio (A) and post-tetanic potentiation (B) for FF, FR and S motor units in control group (C) and four groups of animals 14, 30, 90 and 180 days after the spinal cord hemisection.

FF units after the total transection might prevent overtime drop in the average values of the fatigue index of the whole group of FF units. After the hemisection the number of FF units was also increased overtime, however in parallel with the considerable increase in the number of S units (see Fig. 4). It should be noted that with reference to the whole population of MUs studied in present paper a decrease of Fat I developed progressively in time in rats with complete as well as hemisected spinal cords in similar way and all these changes can be considered as symptoms of the spinal cord injury, independently of how large is the injury. Significant prolongation of the contraction and relaxation times of motor units in the studied muscle as a result of the spinal cord injury was observed in all groups after the hemisection (14– 180 days, p < 0.05, see Table 1). Such a prolongation was also observed after total spinal cord transection (Celichowski et al., 2006; Mrówczyn´ski et al., 2009). Significant differences between hemisected and transected animals were observed for all types of MUs only 30 days after the injury, with considerably more prolonged contraction and relaxation times after the total spinal cord transection (p < 0.01, see Table 2). It seems that the slowing of contraction is one of the early symptoms of the spinal cord injury, independent of its size, and observed even two weeks after the in-

jury. However, similar results (i.e. the prolongation of twitch contraction) were observed in numerous experiments concerning effects of denervation or inactivity (Finol et al., 1981; Gundersen, 1985; Spector, 1985; St-Pierre and Gardiner, 1985; Buffelli et al., 1997) although the underlying mechanisms of the slowing the twitch response are not similar to those present in the hemisection study. It should be stressed that in our hemisected rats nearly normal locomotion was observed after few weeks. During movements and also in periods of inactivity, to keep body balance hind limb muscles on the side of the lesion must be even more active as in intact animals. This additional activity of motor units in the studied fast muscle can be considered as a reason of observed fastto-slow transformation. Moreover, as described in the Introduction spinal cord injury considerably disturbs the neuronal network (Hultborn and Malmsten, 1983a,b; Kuhtz-Buschbeck et al., 1996) which can considerably change normal recruitment order of motor units as well as the time of their activity. The presumable mechanism responsible for lengthening of contraction time after spinal cord injury is prolonged Ca2+ transient (Pette and Staron, 1990). Results obtained by Pette and Staron (1990) showed that increased activity of muscle, as e.g. an effect of electrical stimulation effected in prolonged Ca2+ transient. A similar mechanism may also be the reason of a slowdown of all

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A

Table 2 Statistically significant differences for most important contractile properties between groups of rats with transected spinal cord in comparison to hemisected groups. CT – the contraction time, HRT – the half-relaxation time, TwF – the twitch force, TetF – the maximal tetanic force, Fat I – the fatigue index, PTP – the post-tetanic potentiation. Types of motor units: S – slow, FR – fast resistant, FF – fast fatigable.

10

S

2 1

FF

FR

0.1

B

10

S

FF

C 20 Hz Tet I

10

S

TwF

TetF

Fat I

PTP

– – –

–

– – –

– – –

– –

– – –

**

–

**

** **

– – –

– – –

– – –

– –

**

– –

**

–

**

**

– –

–

**

–

*

**

**

*

**

90 days group FR FF

– –

180 days group FR FF

– –

**

–

– *

– –

*

**

*

FF

Difference significant at p < 0.05 (Mann–Whitney U-test). Difference significant at p < 0.01 (Mann–Whitney U-test). – Difference non-significant at p > 0.05 (Mann–Whitney U-test).

FR

10

S

2 1

FF

0.1

E

HRT

**

0.1

D

FR

2 1

CT

14 days group S FR FF 30 days group S FR FF

2 1

0.1

Types of MUs

FR

10

S

2 1

slow units fast units

FF

0.1 0

FR 0.5

1.0

Fat I Fig. 6. Relationship between the 20 Hz tetanus (20 Hz Tet I) index and the fatigue index (Fat I) for the three types of motor units in control group (A) and four groups of spinal rats with spinal cord hemisected 14, 30, 90 and 180 days before the study (B, C, D and E, respectively). Black circles – fast motor units, white circles – slow motor units. The horizontal dotted lines denote value of 2.0 of the 20 Hz Tet I used for fast/slow MUs classification whereas vertical interrupted lines denote value of 0.5 of the (Fat I) used for a division of fast motor units as FF and FR.

types of MUs and even an increase in the proportion of slow MUs after the hemisection, when MG muscle must be more active to maintain almost proper locomotor pattern. The increase of the contraction twitch-to-tetanus ratio of fast MUs after spinal cord hemisection is consistent with results presented by Tubman et al. (1997), showing that two weeks after the spinal cord hemisection in rats twitch-to-tetanus ratio for MG muscle was significantly higher than in the control and sham-treated animals. Similar results were obtained for rat MUs after the total spinal cord transection (Lieber et al., 1986a,b; Celichowski et al., 2006; Mrówczyn´ski et al., 2009). Higher twitchto-tetanus ratio in comparison to control animals is thought to be an indicator of muscle atrophy (St-Pierre and Gardiner, 1985). However, our results did not prove any statistically significant changes in the ratio of muscle to body mass and therefore the increase of a ratio of the twitch and tetanus forces seems to be rather an indicator of the functional muscle plasticity.

Results concerning partial spinal cord injuries are not in unanimity about atrophy. Atrophy of the muscle following hemisection is a feature mentioned in some studies on animals. The MG muscle atrophy exceeding 50% two weeks after spinal cord hemisection in the rat was suggested by Tubman et al. (1997). However, in some papers concerning effects of the spinal cord hemisection, atrophy of the muscles on the lesioned side was not observed. Kuhtz-Buschbeck et al. (1996) performed kinematic analyses of movement of cats on a treadmill, from ten days to eight months after spinal cord hemisection, and observed that locomotor deficits were present but muscular atrophy of the affected hindlimb was not visible in any hind limb muscle. The differences may depend on animal species, time after injury, and the extent of spinal cord lesion. Moreover, type and function of the muscle is very important. Gordon and Mao (1994) demonstrated in the hemisected cats the pronounced atrophy of muscles that normally bear body weight and cross single joint (e.g., soleus), but negligible changes in the mass of muscles that do not bear body weight or that cross more than one joint (e.g., tibialis anterior or medial gastrocnemius). The differences in post-operative treatment and behaviour of animals might be also considered. It should be remembered, that in the present study no atrophic changes were observed in the MG muscle, and the behavioural observation revealed that after a short (2–3 weeks) period of altered locomotion the animals with their spinal cord hemisected regained locomotor hind limb movement almost similar to that of intact rats. The ability to potentiate the force after conditioning activity was diminished in animals with hemisected spinal cord in a short time after spinal cord injury (14 days for FF and FR MUs). Tubman et al. (1997) 14 days after the spinal cord hemisection also obtained a decrease in post-tetanic potentation for MG muscle consistent with our findings. However, post-tetanic potentiation at later time points after a partial spinal cord injury has not been examined so far. The probable reason for restitution of the post-tetanic potentiation values to a range for intact rats is that after a few weeks of limited locomotion the rats started to move almost properly and properties of the MUs recovered. After the spinal cord hemisection MUs became more fatigable (the mean value of the fatigue index of all investigated MUs slightly decreased). However, this change after hemisection was definitely less clear in comparison to those seen in our previous

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studies on animals with the spinal cord total transection (Celichowski et al., 2006; Mrówczyn´ski et al., 2009). Reasons of increased fatigability after the spinal cord injury concern probably reduced neuro-muscular activity (Cope et al., 1986; Lieber et al., 1986a,b; Munson et al., 1986; Talmadge et al., 1995, 1999). The reduced activity including spinal cord total transection, limb immobilization with the muscle in a shortened position, reduced loading, or even inactivity (spinal cord isolation) resulted in diminished fatigue resistance (Talmadge, 2000). Moreover, alterations in the Ca2+ handling properties of the muscle fibers and the sarcoplasmic reticulum must be taken into account. Alterations in the ability of the sarcoplasmic reticulum to release and reuptake Ca2+ appeared to be related to the onset of fatigue (Fitts, 1994; Williams and Klug, 1995; Favero, 1999). The sag phenomenon was described as a characteristic feature for fast MUs in intact animals (Burke et al., 1973; Kernell et al., 1983; Grottel and Celichowski, 1990; Celichowski, 1992). After partial spinal cord injury the presence of the sag phenomenon was disturbed (see results) which indicates less effective summation of successive twitches into the tetanus contraction (Celichowski et al., 2005). These changes are in agreement with the observed increased twitch-to-tetanus ratio and prolongation of the contraction time (Carp et al., 1999).

4.1. Fast to slow MU transformation Analysis of contribution of individual types of MUs in the MG muscle of different groups of animals after spinal cord injuries from two weeks up to six month after the injury showed considerable differences between effects of total spinal cord transection and spinal cord hemisection. After the hemisection of the spinal cord an evident increase of the number of S MUs type was observed. Therefore, the fast-to-slow MU transformation was revealed. Most important, in contrast to the hemisection, the total transection of the spinal cord caused a gradual increase of FF MUs and parallel decrease of S units number (S MUs nearly totally disappeared in 90 days group) (Mrówczyn´ski et al., 2009). The slow-to-fast muscle transformation, well known as an effect of spinal cord transection, has been visible beginning one month after the spinal cord transection (Celichowski et al. 2006; Mrówczyn´ski et al., 2009). The likely reasons of the increased contribution of S MUs and another kind of MU transformation observed after the hemisection could arise due to behavioural observations. One week after the hemisection all the rats had slight body weight support and plantar stepping in right hind limb (see Section 3). One month after the hemisection they regained slightly modified, but stable locomotor pattern. Therefore, the possible explanation of the increase in the relative number of slow MUs is an increased participation of the studied muscle in tonic antigravity activity. The above-described results indicate that effects of partial spinal cord injury are very complex and very different in comparison to that of total spinal cord transection. It turned out, that MU properties after left-side hemisection of the spinal cord did not change as spectacularly as after the total transection. The following reasons might be responsible for this phenomenon: (1) rats after hemisection in a few days regained locomotor functions and normal daily activity, which was not observed in rats after total spinal cord transection; (2) after spinal cord hemisection neural tracts of half of the spinal cord were cut, although it should be stressed that some tracts have bilateral course, some are crossed; (3) the activity of the neuronal network was disturbed, the order of MUs recruitment was substantially changed and time of activity of individual MUs is dramatically reduced in total spinal cord injury whereas for some of them was increased in hemisected animals.

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Changes in motor units contractile properties of the rat medial gastrocnemius muscle after spinal cord transection. Exp Physiol 2006;91:887–95. Celichowski J, Pogrzebna M, Raikova RT. Analysis of the unfused tetanus course in fast motor units of the rat medial gastrocnemius muscle. Arch Ital Biol 2005;143:51–63. Celichowski J. Motor units of medial gastrocnemius in the rat during the fatigue test. I. Time course of unfused tetani. Acta Neurobiol Exp 1992;52:17–21. Cope TC, Bodine SC, Fournier M, Edgerton R. Soleus motor units in chronic spinal transected cats: physiological and morphological alterations. J Neurophysiol 1986;6:1202–20. Dietz V, Harkema SJ. Locomotor activity in spinal cord-injured persons. J Appl Physiol 2004;96:1954–60. Favero TG. Sarcoplasmic reticulum Ca2+ release and muscle fatigue. J Appl Physiol 1999;87:471–83. Finol HJ, Lewis DM, Owens R. The effects of denervation on contractile properties of rat skeletal muscle. J Physiol 1981;319:81–92. Fitts RH. Cellular mechanisms of muscle fatigue. Physiol Rev 1994;74(1):49–94. Galea MP, Darian-Smith I. Corticospinal projection patterns following unilateral section of the cervical spinal cord in the newborn and juvenile macaque monkey. J Comp Neurol 1997;381:282–306. Gordon T, Mao J. Muscle atrophy and procedures for training after spinal cord injury. Phys Ther 1994;74:50–60. Górska T, Bem T, Majczyn´ski H, Zmysłowski W. Unrestrained walking in cats with partial spinal lesions. Brain Res Bull 1993;32(3):241–9. Górska T, Chojnicka-Gittins B, Majczyn´ski H, Zmysłowski W. Overground locomotion after incomplete spinal lesions in the rat: quantitative gait analysis. J Neurotraum 2007;24(7):1198–218. Grottel K, Celichowski J. Division of motor units in medial gastrocnemius muscle of the rat in the light of variability of their principal properties. Acta Neurobiol Exp 1990;50:571–88. Gundersen K. Early effects of denervation on isometric and isotonic contractile properties of rat skeletal muscles. Acta Physiol Scand 1985;124: 549–55. Hultborn H, Malmsten J. Changes in segmental reflexes following chronic spinal cord hemisection in the cat. I. Increased monosynaptic and polysynaptic ventral root discharges. Acta Physiol Scand 1983a;119:405–22. Hultborn H, Malmsten J. Changes in segmental reflexes following chronic spinal cord hemisection in the cat. II. Conditioned monosynaptic test reflexes. Acta Physiol Scand 1983b;119:423–33. Kernell D, Eerbeek O, Verhey BA. Motor unit categorization on basis of contractile properties: an experimental analysis of the composition of the cat’s m. Peroneus longus. Exp Brain Res 1983;50:211–9. Krutki P, Celichowski J, Krys´ciak K, Sławin´ska U, Majczyn´ski H, Re˛dowicz MJ. Division of motor units into fast and slow on the basis of profile of 20 Hz unfused tetanus. J Physiol Pharmacol 2008;59:353–63. Kuhtz -Buschbeck JP, Boczek-Funcke A, Mautes A, Nacimento W, Weinhardt C. Recovery of locomotion after spinal cord hemisection: an X-ray study of the cat hindlimb. Exp Neurol 1996;137:212–24. Lieber RL, Fridén JO, Hargens AR, Feringa ER. Long-term effects of spinal cord transection on fast and slow rat skeletal muscle. II. Morphometric properties. Exp Neurol 1986a;91:435–48. Lieber RL, Johansson CB, Vahlsing HL, Hargens AR, Feringa ER. Long-term effects of spinal cord transection on fast and slow rat skeletal muscle. I. Contractile properties. Exp Neurol 1986b;91:423–34. Majczyn´ski H, Maleszak K, Cabaj A, Sławin´ska U. Serotonin-related enhancement of recovery of hindlimb motor functions in spinal rats after grafting of embryonic raphe nuclei. J Neurotraum 2005;22:590–604. Malmsten J. Time course of segmental reflex changes after chronic spinal cord hemisection in the rat. Acta Physiol Scand 1983;119:435–43. Mayer RF, Burke RE, Walmsley B, Hodgson JA. The effect of spinal cord transection on motor units in cat medial gastrocnemius muscle. 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J. Celichowski et al. / Journal of Electromyography and Kinesiology 20 (2010) 532–541 Pette D, Staron RS. Cellular and molecular diversities of mammalian skeletal muscle fibers. Rev Physiol Biochem Pharmacol 1990;116:1–76. Powers RK, Rymer VVZ. Effects of acute dorsal spinal hemisection on motoneuron discharge in the medial gastrocnemius of the decerebrate cat. J Neurophysiol 1988;59:1540–56. Sławin´ska U, Majczyn´ski H, Djavadian R. Recovery of hindlimb motor function in adult rats is enhanced by transplantation of embryonic raphe nucleus into the spinal cord below the transection. Exp Brain Res 2000;132:27–38. Spector SA. Trophic effects on the contractile and histochemical properties of rat soleus muscle. J Neurosci 1985;5:2189–96. St-Pierre D, Gardiner PF. Effect of ‘‘disuse” on mammalian fast-twitch muscle: joint fixation compared with neurally applied tetrodotoxin. Exp Neurol 1985;90:635–51. Talmadge RJ, Roy RR, Edgerton VR. Persistence of hybrid fibers in rat soleus after spinal cord transection. Anat Rec 1999;255(2):188–201. Talmadge RJ, Roy RR, Edgerton VR. Prominence of myosin heavy chain hybrid fibers in soleus muscle of spinal cord-transected rats. J Appl Physiol 1995;78(4):1256–65. Talmadge RJ. Myosin heavy chain isoform expression following reduced neuromuscular activity: potential regulatory mechanisms. Muscle Nerve 2000;23:661–79. Tubman LA, Rassier DE, MacIntosh BR. Attenuation of myosin light chain phosphorylation and posttetanic potentiation in atrophied skeletal muscle. Eur J Physiol 1997;434:848–51. Wernig A, Nanassy A, Müller S. Laufband (treadmill) therapy in incomplete paraplegia and tetraplegia. J Neurotraum 1999;16(8):719–26. Williams JH, Klug GA. Calcium exchange hypothesis of skeletal muscle fatigue: a brief review. Muscle Nerve 1995;18(4):421–34. Wirz M, Colombo G, Dietz V. Long term effects of locomotor training in spinal humans. J Neurol Neurosurg Psychiatr 2001;71(1):93–6.

Jan Celichowski was born in Poznan´, Poland, in 1960. He received an M.Sc. degree from A. Cieszkowski University School of Agriculture (1983) and habilitation in neurophysiology from Nencki Institute of Experimental Biology in Warsaw (1996). Since 1997 he has been the Professor of Neurophysiology, and since 2000 the Head of Department of Neurobiology at the University School of Physical Education in Poznan´. His main fields of research are motor units’ contractile properties and action potentials, plasticity of the neuro-muscular system, mechanomygraphy.

Katarzyna Krys´ciak was born in Poland in 1982. She graduated the Faculty of Physiotherapy at the University School of Physical Education in Poznan´ (2006). She is a Ph.D. student at the Department of Neurobiology. Her main fields of interest are motor units and plasticity of the neuro-muscular system.

Piotr Krutki was born in Poland in 1967. He graduated the Karol Marcinkowski University School of Medical Sciences in Poznan´ (1992), he received a Ph.D. degree (1997) and the habilitation in neurophysiology from the Nencki Institute of Experimental Biology in Warsaw (2001). Since 2003, he has been an Associate Professor at the Department of Neurobiology, University School of Physical Education in Poznan´. His main fields of research are: spinal neuronal networks, mechanisms of motor control, motor units and plasticity of the neuro-muscular system.

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Henryk Majczyn´ski is an Assistant Professor in the Department of Neurophysiology at the Nencki Institute of Experimental Biology (Poland). He received his master’s degree in Electronics from Warsaw Technical University and his Ph.D. degree and habilitation in neurophysiology from the Nencki Institute of Experimental Biology. His research is focused on control of locomotion and investigation of different methods improving the recovery after spinal cord injury.

Teresa Górska was born in Warsaw, Poland in 1932. She received the M.Sc. degree in Psychology from Warsaw University (1954) and the Ph.D.degree (1965) and habilitation in neurophysiology (1973) from Nencki Institute of Experimental Biology in Warsaw. Since 1983 she has been the Professor at the Department of Neurophysiology of the Nencki Institute and the Head of the Laboratory of Motor Control. Since 2002 she is an Emeritus Professor. Her main fields of interest were behavioural studies on the mechanisms controlling motor functions, such as e.g. comparative studies of the role of proprioception and pyramidal system in instrumental reactions in different species and ages, locomotor impairment after partial spinal lesions of different extent, role of serotonergic and noradrenergic system in locomotion.

Urszula Sławin´ska was born in Poland. She graduated in Physics in 1982 at the Warsaw University, later she received her Ph.D. in neurophysiology (1993) and D.Sc. in biology (2003) from Nencki Institute of Experimental Biology in Warsaw. Since 1998 she is Head of the Laboratory of Neuromuscular Plasticity and since 2007 Deputy Director for Scientific Research at the Nencki Institute. Her major research interests are related to investigation of the mechanisms responsible for the plasticity processes in the neuromuscular system during development or after central or peripheral nervous system injury. The main goal of her research is devoted to development of new methods enhancing the restoration of neuromuscular functions after nerve injury.