Changes in contractile characteristics of rat skeletal muscles associated with P2-receptor activation after spinal cord transection

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Introduction

Traumatic spinal cord and peripheral nerve injuries are not unusual among people of working age and can be accompanied by severe and often irreversible motor disorders. Traumatic spinal cord injury (SCI) is characterized by immediate and irreversible tissue loss at the injury site followed by the secondary injury in adjacent tissues over time. Traumatic peripheral nerve injury is known to cause various changes in the expression of intracellular signaling molecules in the spinal cord [1], primarily in response to increased release of various mediators in activated spinal cord microglia [2], which may play an important role in the neuropathic pain development and maintenance [3].

Microglia activated by a trauma produces and releases pro-inflammatory cytokines and chemokines [4], which can stimulate neuronal activity. Adenosine triphosphoric acid (ATP) is an important pain mediator involved in the development of acute and chronic neuropathic pain after an injury [5]. Its excessive release by injured tissue activates high-affinity purinergic receptors in microglial cells, which may further affect the mechanisms of additional tissue damage, known as secondary injury [3].

Although the effects of ATP on the peripheral nervous system are relatively well understood, the pathophysiolo- gical role of purinergic signaling associated with spinalization remains unclear. So, the objective of the study is to evaluate the changes in contractile characteristics of rat skeletal muscles associated with P2-receptor activity after spinalization.

Materials and methods

Male Wistar rats aged 9–12 months, weighing 160–240 g, were used for the experiments. The objects of the study were the pelvic girdle and lower limb muscles, which are fundamentally important for motor activity (slow-twitch muscles [soleus muscle, m. soleus], fast-twitch muscles [extensor digitorum longus muscle, m. extensor digitorum longus, EDL], and functionally distinct respiratory muscle [diaphragm, m. diaphragma] with their corresponding neuromuscular synapses) isolated from intact rats and spina- lized animals.

One week prior to and during the experiments, rats were housed in individual cages at room temperature of 22ºC with a 12 h/12 h light/dark cycle, access to water and food ad libitum. All manipulations were performed at the same time of a day. Rats were divided into 2 groups of 12 animals each: the control group included intact animals and the spinalization group included animals after spinal cord transection.

The surgery was performed under aseptic conditions and combined intramuscular analgesia using zoletil (Zoletil 50, Virbac) at a dose of 0.5 mg/kg and xylavet (XylaVET, Pharmamagist Ltd.) at a dose of 0.5 ml/kg. After dissection of Th6–Th7 vertebrae, a laminectomy was performed to expose the spinal canal with subsequent transection of the spinal cord at this level (Fig. 1).

 

Fig. 1. Schematic diagram of spinalization at Th6–Th7.

 

Seven days after the surgery, the animals were anesthetized with sodium ethaminal (40 mg/kg intraperitoneally) and exsanguinated. M. soleus, m. EDL, m. diaphragma were isolated with nerve stumps fixed by both tendon ends, immersed in 10 ml beakers filled with Krebs–Henseleit solution [6].

The nerve stump of the isolated muscle was placed in a special nerve stump suction electrode for electrical stimulation [7]. Rectangular pulses of 10 V amplitude and 0.5 ms duration at a frequency of 0.1 Hz were applied for 2 min using D330 MultiStim System. Contractile force was recorded with a force displacement transducer (Linton FSG-01), the analog signal was digitized and processed using a Biopack MP100WSW data acquisition system.

The initial load on the myoneural preparations was 1 g on m. soleus and m. diaphragma and 0.5 g on m. EDL. The muscle preparations were kept in the solution for half an hour for adaptation, then the stability of the contractile responses was assessed twice at 5-minute intervals [8].

To study the effects of purinergic agonists and antagonists, 100 μM ATP was added to the bath and the muscle mechanical responses were assessed 10 minutes after. Further, after 20-minute washout of muscle preparations with Krebs–Henseleit solution, the electrical stimulation was repeated. To confirm the ATP effects on synaptic transmission, the muscles preparations were maintained in 100 μM suramin (non-selective P2-receptor antagonist) for 20 min, followed by the addition of 100 μM ATP (P2-receptor agonist), and mechanical muscle responses were again recorded. In control experiments, contractile responses to indirect electrical stimulation were recorded after 20-minute neuromuscular tissue incubation with 100 μM suramin [9].

The responses recorded within 2 min (12 contractions) were averaged and processed as a single value in % of the baseline results obtained at the beginning of the experiment. Statistical data analysis was performed with SPSS Statistics software. Conformity to normal distribution was checked using the Kolmogorov criterion. Statistical significance was assessed using multivariate analysis of variance (MANOVA) for independent and paired samples. The differences were considered significant at p < 0.05.

Results and discussion

After the spinalization, contractile responses in m. soleus and m. EDL changed divergently in contractile force and in time parameters (Fig. 2; Table 1). In contrast, amplitude-time parameter values in m. diaphragma remain stable, perhaps due to a higher position of phrenic motor neurons, which were less affected by spinalization.

 

Fig. 2. Traces of single contractile responses of the isolated rat m. soleus, m. EDL and m. diaphragma evoked by electrical stimulation in controls and in spinalized rats (selected representative traces are presented).

 

Table 1. Parameters of rat muscle contractility evoked by electrical stimulation in different experimental conditions, n = 10–12

Experimental conditions	n	Parameter	Control	ATP, 100 μМ	Suramin, 100 μМ	Suramin, 100 μМ + ATP, 100 μМ
M. soleus
Normal value	10	CF	100,0 ± 4,2	67,6 ± 5,2*	104,3 ± 3,9	98,5 ± 7,1
		CT	0,081 ± 0,004	0,083 ± 0,006	0,080 ± 0,004	0,079 ± 0,005
		RT/2	0,092 ± 0,007	0,105 ± 0,011	0,090 ± 0,006	0,093 ± 0,010
Spinalization	10	CF	119,8 ± 5,1#	114,0 ± 6,1#	120,2 ± 4,3#	121,8 ± 6,4#
		CT	0,073 ± 0,005	0,076 ± 0,007	0,071 ± 0,006	0,074 ± 0,004
		RT/2	0,101 ± 0,009	0,116 ± 0,010	0,098 ± 0,008	0,105 ± 0,010
M. EDL
Normal value	10	CF	100,0 ± 4,5	86,2 ± 3,9*	102,0 ± 6,1	98,7 ± 5,3
		CT	0,057 ± 0,003	0,056 ± 0,005	0,059 ± 0,004	0,058 ± 0,006
		RT/2	0,067 ± 0,005	0,069 ± 0,004	0,065 ± 0,007	0,068 ± 0,005
Spinalization	10	CF	88,7 ± 3,8#	83,1 ± 5,4	85,9 ± 4,8#	83,1 ± 6,7#
		CT	0,068 ± 0,005	0,069 ± 0,006	0,068 ± 0,006	0,067 ± 0,005
		RT/2	0,071 ± 0,006	0,073 ± 0,007	0,070 ± 0,005	0,073 ± 0,004
M. diaphragma
Normal value	12	CF	100,0 ± 3,7	114,6 ± 5,2*	98,3 ± 4,7	102,9 ± 6,2
		CT	0,065 ± 0,004	0,066 ± 0,003	0,064 ± 0,006	0,064 ± 0,004
		RT/2	0,075 ± 0,006	0,075 ± 0,005	0,074 ± 0,006	0,076 ± 0,004
Spinalization	12	CF	103,2 ± 4,1	112,7 ± 3,9*	102,0 ± 4,9	103,8 ± 7,5
		CT	0,071 ± 0,005	0,070 ± 0,003	0,069 ± 0,004	0,072 ± 0,004
		RT/2	0,074 ± 0,003	0,076 ± 0,006	0,074 ± 0,005	0,075 ± 0,006

Note. *р < 0.05 compared with the control group; #р < 0.05 compared with normal value. CF — contractile force (% from the level in the control group); CT — contractile time, s; RT/2 — half-relaxation time, sес.

 

Application of 100 µM ATP to muscle preparations of intact rodents modulates the contractility parameters: a 10-min exposure to ATP decreased the contractile force of locomotor m. soleus and m. EDL and increased the contractility of respiratory m. diaphragma. ATP had virtually no effect on the neuromuscular preparations from the spinalized animals. Only m. diaphragma remained sensitive to the study nucleotide (see Table).

Suramin (100 μM) as a non-competitive inhibitor of P2-receptors showed no significant effects. In the presence of suramin (100 μM), exogenous ATP (100 μM) activity was completely inhibited in all study objects (see Table).

Our findings demonstrate a significant suppression of the peripheral nervous system activity in the SCI animal model. Changes in synaptic signaling indicate axon degeneration after the injury of the spinal cord at its upper levels.

Understanding mechanisms underlying suppression of the peripheral nervous system is important to prevent functional decline and maintain a high potential for motor function recovery, especially with cellular therapies aimed at SCI repair.

Disorders of muscle function caused by SCI can result from a mechanical injury and from secondary injury caused by pathophysiological response to the initial trauma. For example, there are studies demonstrating abnormally high and persistent ATP release levels in peritraumatic tissues in SCI rat models, indicating P2-signaling involvement in the cascade of degenerative events, known as secondary injury, and neurodegeneration after the initial injury [10].

This cascade of injury-associated events include extensive hemorrhage, necrosis of cellular components of the central and peripheral nervous systems. The subsequent activation of astrocytes and other cells located in close proximity to the injury site results in extremely unfavorable conditions for axon repair. The concurrent activation of the immune system leads to additional tissue damage at the injury site by attracting immune inflammatory cells, such as neutrophils and macrophages. On the other hand, macrophages and T-helpers provide trophic support to damaged components of the CNS. All of the above processes lead to axon degeneration and the loss of communication between neurons, which primarily results in various functional muscle disorders [11].

The obtained data demonstrate significant differences in the mechanisms of contractility control in fast-twitch and slow-twitch skeletal muscles of warm-blooded animals, which is consistent with our earlier observations in spinal shock models [12]. The suppression of P2-receptors affecting muscle contraction associated with such a striking response to spinalization demonstrates the involvement of the purinergic signaling in the neurotrophic control and functioning of various motor units.

Activation of spinal microglia caused by trauma leads to an increased expression of P2-receptors. For example, P2X4R levels have been shown to increase in association with SCI, while P2X4R inhibition has been shown to reduce neuropa- thic pain [13]. Another ATP-sensitive purinergic receptor, P2X7, can form a macromolecular pore under repeated or prolonged exposure to high concentrations of ATP [14], which is of paramount importance taking into consideration that ATP release in peritraumatic regions rises massively. The role of this receptor is particularly important in understanding SCI pathophysiology due to its extensive expression in CNS neurons [10]. There are data indicating potential involvement of other receptor subtypes, namely, P2Y6, P2Y13 and P2Y14, in the physiological responses of microglia [15, 16].

Despite the severity of the damage, even with extensive SCI at the level of the thoracic segments, electrical stimulation applied slightly below the level of the injury allows to register stable rhythmic motor activity in the lower limbs, which was demonstrated in a number of animal models [17, 18].

Inhibiting purinergic receptors can improve outcomes in SCI patients. For example, intraspinal injection of a P2X7-receptor antagonist into the peritraumatic region reduced the damage caused by SCI [10]. P2X7R inhibition also reduced motor neuron loss and promoted subsequent functional recovery in injured animals.

On the other hand, axon membrane damage caused by an injury is associated with rapid changes in intracellular ion concentrations. The effects of ATP on spinal cord neurons cause their excitation leading to a persistent irreversible increase in Ca²⁺ levels resulting in a cell death [10].

Moreover, a number of fundamental animal model studies demonstrated pathological changes in skeletal muscles associated with SCI: the massive ATP release from damaged tissues provokes local and generalized inflammatory process with the release of proinflammatory cytokines (in particular, interleukins-1 and -6), which mediates muscle disorders, similar to muscle denervation atrophy [14]. ATP activates ionotropic P2XRs, particularly P2X7, which mainly leads to an increase in intracellular Ca²⁺ levels and induces cytoskeletal reorganization, inflammation, apoptosis/necrosis, and proliferation, usually in a long-term perspective [19].

Conclusion

Thus, all the data available by now only outline the ways to study the mechanisms of the effects we have described. Further studies of P2-signaling role in post-spinalization processes are required.

Abnormal ATP-mediated modulation of neuromuscular transmission demonstrated in this study indicates axon degeneration and suggests that transsynaptic degeneration of motor neurons may occur below the level of spinal cord injury after the traumas similar to the ones described in the study.

About the authors

Adel E. Khairullin

Kazan State Medical University; Kazan (Volga Region) Federal University

Author for correspondence.
Email: khajrulli@ya.ru
ORCID iD: 0000-0001-6155-622X

Cand. Sci. (Biol.), Assistant Professor, Department of biochemistry and clinical laboratory diagnostics, Kazan State Medical University; senior researcher, Research laboratory «Mechanobiology», Institute of Fundamental Medicine and Biology, Kazan Federal University

Russian Federation, Kazan; Kazan

Dina V. Efimova

Kazan State Medical University

Email: khajrulli@ya.ru
ORCID iD: 0009-0000-3966-0813

postgraduate student, Department of biochemistry and clinical laboratory diagnostics, Kazan State Medical University

Russian Federation, Kazan

Marat A. Mukhamedyarov

Kazan State Medical University

Email: marat.muhamedyarov@kazangmu.ru
ORCID iD: 0000-0002-0397-9002

D. Sci. (Med.), Professor, Head, Department of normal physiology, Kazan State Medical University

Russian Federation, Kazan

Maxim E. Baltin

Kazan (Volga Region) Federal University

Email: khajrulli@ya.ru
ORCID iD: 0000-0001-5005-1699

researcher, Research laboratory «Mechanobiology», Institute of Fundamental Medicine and Biology, Kazan Federal University

Russian Federation, Kazan

Tatiana V. Baltina

Kazan (Volga Region) Federal University

Email: khajrulli@ya.ru
ORCID iD: 0000-0003-3798-7665

Cand. Sci. (Biol.), Associate Professor, Head, Research laboratory «Mechanobiology», Institute of Fundamental Medicine and Biology, Kazan Federal University

Russian Federation, Kazan

Sergey N. Grishin

Kazan State Medical University

Email: sgrishin@inbox.ru
ORCID iD: 0000-0002-2851-579X

D. Sci. (Biol.), Professor, Department of medical and bio- logical physics with computer science and medical equipment, Kazan State Medical University

Russian Federation, Kazan

Airat U. Ziganshin

Kazan State Medical University

Email: airat.ziganshin@kazangmu.ru
ORCID iD: 0000-0002-9087-7927

D. Sci. (Med.), Professor, Head, Department of pharmacology, Kazan State Medical University

Russian Federation, Kazan

References

Supplementary files