2. Dynamic properties of skeletal muscle contraction in rats with diabetes

#### 2.1. Methods of experiment conduction

diabetic polyneuropathy increases with age and duration of diabetes mellitus [1]. According to recent data, in the last 10 years, in young patients with type 1 diabetes, an increase in the incidence of diabetic polyneuropathy from 24.2% to 62.9% has been observed [2]. Diabetic neuropathy correlates with a high risk of cardiovascular complications [3, 4]. Patients with diabetic polyneuropathy are also at risk for the formation of trophic ulcers that do not heal for a long time and often lead to amputating a limb [5–7]. In the USA, 15% of all patients with

Mortality due to diabetes mellitus, complicated by diabetic polyneuropathy, remains high in all countries of the world, regardless of their socioeconomic status [3]. Patients with diabetic polyneuropathy often require outside help, which, of course, is reflected on the quality of their

Metabolic, vascular and immune theories were proposed to explain the pathogenesis of diabetic polyneuropathy [11]. Independent causes of the risk of this serious complication of diabetes mellitus are age, male gender, unsatisfactory control of the level of glycaemia, elevated lipid levels in the blood, height, overweight and obesity, and insulin treatment [12–16]. Thus, the pathogenesis of diabetic polyneuropathy is multifactorial. It includes the increase of mitochondrial production of free radicals due to hyperglycemia-induced oxidative stress [1]. A number of other factors affect the activity of neurons, mitochondrial function, permeability of membranes and endothelial function. These include the activation of polyol aldose reductase pathway [17], activation of poly(ADP ribose) polymerase [18], and modified Na+/K+-ATPase

In diabetic polyneuropathy, autonomic, motor, large fiber and small fiber nerve functions are attacked [20]. The most frequent variant of defeat of peripheral nervous system at a diabetes is distal symmetric sensorimotor neuropathy [21]. As a rule, this complication occurs in a few years from the onset of the underlying disease [22]. This form of diabetic polyneuropathy develops slowly (chronically), the first symptoms (numbness and paresthesia) occur in the lower extremities, sometimes unilateral [23]. Distal symmetric sensorimotor neuropathy is the cause of the development of chronic neuropathic pain syndrome. Pain is the reason for 40% of patient visits in a primary care setting, and about 20% of these have had pain for more than 6 months [24]. In this form of neuropathy, poorly myelinated and thin nonmyelinated fibers are affected in various combinations. In most cases, at the onset of the disease, the neurological deficit is caused by the damages to fine fibers. Symptoms of their damages are manifested by burning or shooting pain, hyperalgesia, paresthesia, disturbances of pain and temperature sensitivity, ulceration of the feet and a decrease in pain sensitivity from the internal organs. With the defeat of myelinated (thick) fibers, there is a violation of deep and vibrational

Diabetic polyneuropathy affects both type 1 and type 2 diabetes patients, although specific differences exist in the underlying pathobiology, pathology and clinical expression of the disease [25]. In type 1 diabetes patients, diabetic polyneuropathy is more rapid and severe.

The nerve conduction study is a reliable and objective diagnostic method to evaluate the diabetic polyneuropathy treatment response [26]. Although a nerve conduction study is regarded as the

diabetes will develop foot ulcers [8].

120 Pathophysiology - Altered Physiological States

life [9, 10].

pump function [19].

sensitivity, and a decrease or loss of tendon reflexes.

The study was conducted on 20 white nonlinear laboratory male rats, which were divided into two groups of 10 animals each. The rats in the first group were used as control. Rats in the second group were induced type I diabetes by administration of streptozotocin (STZ) (65 mg/kg, i/р). Diabetes in rats was confirmed by the presence of hyperglycemia. On the 28th day of experiment, glucose loading test was conducted for the confirmation of diabetes presence. For the establishment of pain sensation, mechanical nociceptive test was conducted in rats (Randall-Selitto analgesiometer test) [28]. Also heat-induced rat tail-flick latency was determined as a measure for nociceptive pain [29].

the study, and to correct the algorithm of further investigation whether pathological

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2.2.2. The changes in time of muscle force response beginning caused by 10 consecutive stimulation

When the intensity of stimulation changes, the temporal parameters of the stimulation pools conduction in axon do not remain constant. The investigation of changes of time delays of impulses conduction with an increase number of stimuli makes it possible to assess the level of pathological changes in the neuromuscular preparation with prolonged, static reactions of the muscular system. High-frequency stimulation of peripheral afferents that form monosynaptic contacts with motor neuron causes an effective summation of successive action potentials and stable depolarization of the cell membrane. In this case, the pulse frequency is determined by the average level of membrane depolarization and increases with rise of frequency stimulation. During the development of pathological processes associated with diabetic polyneuropathy, the use of stimulation without relaxation of the corresponding long transsynaptic activation of motor neurons causes adaptive time decrease of stimuli conduction. Change in this parameter is the marker of pathological processes presence in the neuromuscular preparation while

We analyzed several basic biomechanical parameters during studying the myotonic response of the muscle. These parameters are universal markers that show the presence of biomechan-

Changes in each of described biomechanical parameters is an indicative marker of the dysfunction presence in the excitation-response chain of both the neuromuscular preparation and

We have designated the investigated segments of biomechanical responses for more favorable

The maximum force generation on which the active muscle is capable is an important indicator for fast, ballistic, nontargeted movements (Figure 1—Δt1) The changes of this indicator show the level of physiological dysfunction of neuromuscular preparation when it implements the

The stationary state of the active muscle is a temporal area of the contractile activity of the muscle tissue without the presence of a significant trend in one or the other direction, during the activation of the muscle (Figure 1—Δt2). Physiologically, the stationary state of the active muscle is the level of muscle force production that corresponds to the physiological state of the

2.2.4. The changes in time of achievement of stationary state of contraction, with the use of

changes are present.

pools with 10 s relaxation time between them

applying stimulation signals close to physiological parameters.

ical disturbances caused by factors of different nature.

description of the changes in the obtained curves (Figure 1).

2.2.3. The changes in time to reach maximum force response

the state of the organism as a whole.

maximum power tasks.

modulated stimulation signal

Animals were anesthetized (Ketamine (100 mg/kg "Pfizer", USA), and tracheotomy and connection to lung ventilator were performed. In the area of the popliteal fossa, musculus gastrocnemius was isolated and cut down to be attached to the force sensors. Further, the animal was fixed in a stereotaxic machine with the head, pelvis and extremities rigid fixation system. Nerve that innervate musculus gastrocnemius was fixed on a bipolar platinum wire electrode for further electrical stimulation. The parameters of stimulation signals were programmed. The skin edges (hind legs) around the incision were sutured to the machine tool and formed trays with the muscle and nerve and were filled with liquid paraffin. Heart rate and ECG amplitudes were monitored during surgery and experiments [30].

Before stimulation of the spinal cord, the ventral root muscle was connected to a load that did not stretch it because of unilateral mechanical limiter, and only shortening of the muscle was possible. After activation, the isometric growth of the force began until the muscular effort reached the external load, after that the isotonic shortening of the muscle started.

In the initial stage of the shortening, it was possible to distinguish a near-linear part of motion due to velocity measurement at which it was possible to establish the empirical dependence of the contraction rate on the level of isotonic loads. The pathological processes that occurred during the development of diabetic polyneuropathy modulated the muscular response registered by us. The level of this modulation was a qualitative characteristic of residual physiological disorders both at the neuropathic and at the myopathy level of pathology development. The statistical analysis of the data was conducted in the program Statistica 8.0. To approximate this empirical dependence, several analytic approaches were selected.

As a modulating component, a stimulating signal of different amplitudes and times characteristics was used and regarded as an input effect, and the output signal was the first harmonic of the muscle-developed effort and the subsequent realization of the modulated stimulation pool.

#### 2.2. The analysis of electro-physiological parameters used in work as indicators of pathological processes development during diabetic polyneuropathy

#### 2.2.1. The changes in time of muscle force response beginning caused by a single stimulation pool

Time between first and second mitotic response that was caused by successive stimulation with fixed meaning between them (2000 ms). This indicator makes it possible to assess the presence or absence of pathologies (neuropathy or myopathy) during the initial stages of the study, and to correct the algorithm of further investigation whether pathological changes are present.

#### 2.2.2. The changes in time of muscle force response beginning caused by 10 consecutive stimulation pools with 10 s relaxation time between them

When the intensity of stimulation changes, the temporal parameters of the stimulation pools conduction in axon do not remain constant. The investigation of changes of time delays of impulses conduction with an increase number of stimuli makes it possible to assess the level of pathological changes in the neuromuscular preparation with prolonged, static reactions of the muscular system. High-frequency stimulation of peripheral afferents that form monosynaptic contacts with motor neuron causes an effective summation of successive action potentials and stable depolarization of the cell membrane. In this case, the pulse frequency is determined by the average level of membrane depolarization and increases with rise of frequency stimulation. During the development of pathological processes associated with diabetic polyneuropathy, the use of stimulation without relaxation of the corresponding long transsynaptic activation of motor neurons causes adaptive time decrease of stimuli conduction. Change in this parameter is the marker of pathological processes presence in the neuromuscular preparation while applying stimulation signals close to physiological parameters.

We analyzed several basic biomechanical parameters during studying the myotonic response of the muscle. These parameters are universal markers that show the presence of biomechanical disturbances caused by factors of different nature.

Changes in each of described biomechanical parameters is an indicative marker of the dysfunction presence in the excitation-response chain of both the neuromuscular preparation and the state of the organism as a whole.

We have designated the investigated segments of biomechanical responses for more favorable description of the changes in the obtained curves (Figure 1).

#### 2.2.3. The changes in time to reach maximum force response

the second group were induced type I diabetes by administration of streptozotocin (STZ) (65 mg/kg, i/р). Diabetes in rats was confirmed by the presence of hyperglycemia. On the 28th day of experiment, glucose loading test was conducted for the confirmation of diabetes presence. For the establishment of pain sensation, mechanical nociceptive test was conducted in rats (Randall-Selitto analgesiometer test) [28]. Also heat-induced rat tail-flick latency was

Animals were anesthetized (Ketamine (100 mg/kg "Pfizer", USA), and tracheotomy and connection to lung ventilator were performed. In the area of the popliteal fossa, musculus gastrocnemius was isolated and cut down to be attached to the force sensors. Further, the animal was fixed in a stereotaxic machine with the head, pelvis and extremities rigid fixation system. Nerve that innervate musculus gastrocnemius was fixed on a bipolar platinum wire electrode for further electrical stimulation. The parameters of stimulation signals were programmed. The skin edges (hind legs) around the incision were sutured to the machine tool and formed trays with the muscle and nerve and were filled with liquid paraffin. Heart rate and ECG ampli-

Before stimulation of the spinal cord, the ventral root muscle was connected to a load that did not stretch it because of unilateral mechanical limiter, and only shortening of the muscle was possible. After activation, the isometric growth of the force began until the muscular effort

In the initial stage of the shortening, it was possible to distinguish a near-linear part of motion due to velocity measurement at which it was possible to establish the empirical dependence of the contraction rate on the level of isotonic loads. The pathological processes that occurred during the development of diabetic polyneuropathy modulated the muscular response registered by us. The level of this modulation was a qualitative characteristic of residual physiological disorders both at the neuropathic and at the myopathy level of pathology development. The statistical analysis of the data was conducted in the program Statistica 8.0. To approximate

As a modulating component, a stimulating signal of different amplitudes and times characteristics was used and regarded as an input effect, and the output signal was the first harmonic of the muscle-developed effort and the subsequent realization of the modulated

2.2. The analysis of electro-physiological parameters used in work as indicators of

2.2.1. The changes in time of muscle force response beginning caused by a single stimulation pool

Time between first and second mitotic response that was caused by successive stimulation with fixed meaning between them (2000 ms). This indicator makes it possible to assess the presence or absence of pathologies (neuropathy or myopathy) during the initial stages of

reached the external load, after that the isotonic shortening of the muscle started.

this empirical dependence, several analytic approaches were selected.

pathological processes development during diabetic polyneuropathy

stimulation pool.

determined as a measure for nociceptive pain [29].

122 Pathophysiology - Altered Physiological States

tudes were monitored during surgery and experiments [30].

The maximum force generation on which the active muscle is capable is an important indicator for fast, ballistic, nontargeted movements (Figure 1—Δt1) The changes of this indicator show the level of physiological dysfunction of neuromuscular preparation when it implements the maximum power tasks.

#### 2.2.4. The changes in time of achievement of stationary state of contraction, with the use of modulated stimulation signal

The stationary state of the active muscle is a temporal area of the contractile activity of the muscle tissue without the presence of a significant trend in one or the other direction, during the activation of the muscle (Figure 1—Δt2). Physiologically, the stationary state of the active muscle is the level of muscle force production that corresponds to the physiological state of the

2.2.6. The changes in time of maximum contraction force generation

2.2.7. The changes in minimum contraction force generation

2.2.8. The changes in integrated power of muscle contraction

analogue of the working capacity of the muscular system as a whole.

the maximum force response.

stimuli and stimulation time increase.

polyneuropathy

ence was described.

This marker is an indicator of the general dysfunction of the muscular system, index of decrease (with pathologies development) of the maximum possible force response (Figure 1—Fmax). The change in this parameter can be related either to a violation in the neuronal component or to the miotic components of the studied pathology. The dysfunction of this parameter can also be associated with a violation of the integrity of the signals that generate motor neurons in the synaptic current, and as a result, the violation of the summation of the transmembrane currents occurred in accordance with the internal membrane properties. That influences the pathological transformation of the sequence of action potentials that trigger a muscle contraction that causes

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125

This data show the maximum pathological changes caused by the pathological process during analyzing changes in contraction of each successive contractile act (Figure 1—Fmin). This marker is the main indicator of muscle dysfunction while performing simple one-joint movements. The phenomenological analysis of it makes possible to establish the presence of causeeffect relationships between the level of decrease in biomechanical activity of muscles, the basic mechanical parameters of movements and the level of development of the pathological process. The accuracy of such conclusions is enhanced due to multiple repetitions of these

The integrated power is subtracted from the total area of force curves (Figure 1—S) and is an indicator of the overall capacity of the muscle with the use of applied stimulation pools. Analysis of this value makes it possible to evaluate the mechanisms of the formation of muscular activity in the equilibrium system, in the force-external load system, that is, a physiological

2.2.9. The changes in fatigue processes of the neuromuscular preparation accompanied by diabetic

In condition of 1 and 2 Hz, unrelaxed stimulation of the analysis of fatigue processes development was made, which makes it possible to evaluate the development of fatigue in different time ranges. Fatigue evaluation was calculated by time intervals with achievements of 50 and 30% force responses, with stimulation irritations. It should be noted that in control, the change of this data had a long time frame, which complicated the description of fatigue processes development during pathology. Therefore, for more precise description of results, the change in control values was considered 100%, and while analyzing the data, the percentage differ-

Figure 1. Schematic representation of analyzed biomechanical parameters of muscles gastrocnemius contraction using a modulated stimulation signal.

neuromuscular preparation at this moment. The time of its establishment is a time of adaptation processes passage in the muscle during its activation by stimulating pools, to select the optimal amplitude-strength characteristics of the contracting muscle in order to realize the incoming stimulations with the least deviations from the CNS tasks.

#### 2.2.5. The changes in time of stationary state retention, with the use of modulated stimulation signal

The retention of stationary state is an indicator of adaptability of the muscular system to a new state of the neuromuscular system, altered by a pathological action (Figure 1—Δt3). In some cases, we could record relatively stable periodic changes in the level of stationary state at the applied pulse activity frequency, but without significant dependencies of these fluctuations to the level of pathology development or methods of drug administration. We consider the presence of oscillations at the phases of stationary state retention is a consequence of individual differences in the muscular system of experimental laboratory animals.

#### 2.2.6. The changes in time of maximum contraction force generation

This marker is an indicator of the general dysfunction of the muscular system, index of decrease (with pathologies development) of the maximum possible force response (Figure 1—Fmax). The change in this parameter can be related either to a violation in the neuronal component or to the miotic components of the studied pathology. The dysfunction of this parameter can also be associated with a violation of the integrity of the signals that generate motor neurons in the synaptic current, and as a result, the violation of the summation of the transmembrane currents occurred in accordance with the internal membrane properties. That influences the pathological transformation of the sequence of action potentials that trigger a muscle contraction that causes the maximum force response.

#### 2.2.7. The changes in minimum contraction force generation

This data show the maximum pathological changes caused by the pathological process during analyzing changes in contraction of each successive contractile act (Figure 1—Fmin). This marker is the main indicator of muscle dysfunction while performing simple one-joint movements. The phenomenological analysis of it makes possible to establish the presence of causeeffect relationships between the level of decrease in biomechanical activity of muscles, the basic mechanical parameters of movements and the level of development of the pathological process. The accuracy of such conclusions is enhanced due to multiple repetitions of these stimuli and stimulation time increase.

#### 2.2.8. The changes in integrated power of muscle contraction

neuromuscular preparation at this moment. The time of its establishment is a time of adaptation processes passage in the muscle during its activation by stimulating pools, to select the optimal amplitude-strength characteristics of the contracting muscle in order to realize the

Figure 1. Schematic representation of analyzed biomechanical parameters of muscles gastrocnemius contraction using a

2.2.5. The changes in time of stationary state retention, with the use of modulated stimulation signal

The retention of stationary state is an indicator of adaptability of the muscular system to a new state of the neuromuscular system, altered by a pathological action (Figure 1—Δt3). In some cases, we could record relatively stable periodic changes in the level of stationary state at the applied pulse activity frequency, but without significant dependencies of these fluctuations to the level of pathology development or methods of drug administration. We consider the presence of oscillations at the phases of stationary state retention is a consequence of individ-

incoming stimulations with the least deviations from the CNS tasks.

modulated stimulation signal.

124 Pathophysiology - Altered Physiological States

ual differences in the muscular system of experimental laboratory animals.

The integrated power is subtracted from the total area of force curves (Figure 1—S) and is an indicator of the overall capacity of the muscle with the use of applied stimulation pools. Analysis of this value makes it possible to evaluate the mechanisms of the formation of muscular activity in the equilibrium system, in the force-external load system, that is, a physiological analogue of the working capacity of the muscular system as a whole.

#### 2.2.9. The changes in fatigue processes of the neuromuscular preparation accompanied by diabetic polyneuropathy

In condition of 1 and 2 Hz, unrelaxed stimulation of the analysis of fatigue processes development was made, which makes it possible to evaluate the development of fatigue in different time ranges. Fatigue evaluation was calculated by time intervals with achievements of 50 and 30% force responses, with stimulation irritations. It should be noted that in control, the change of this data had a long time frame, which complicated the description of fatigue processes development during pathology. Therefore, for more precise description of results, the change in control values was considered 100%, and while analyzing the data, the percentage difference was described.

#### 2.2.10. Analysis of fusion index

To analyze the dynamics of real movements, we considered the peculiarities of the transformation of segmental and descending activity during the development of polyneuropathy. An important role in the realization of the motor function belongs dedicated to asymmetric nature of the muscle reactions as a result of increase in the level of incoming efferent activity. In our work, almost all movements are relatively simple and are provided with straight pattern of motor neuron populations. Since motor neurons directly control muscle contraction, the nature of the transformation of activity coming to them from multiple sources is largely predetermined by the peculiarities of muscle dynamics. The significant inertia of muscle contraction during the development of the pathological process requires motor neurons to have such dynamic properties that could compensate for the insufficiently high-speed parameters of muscle contraction. Thus, the slowdown of smooth tetanus appearance can be used as another parameter to describe the dynamics of pathologies development. We investigated the transition of active muscle force response from the state of the unfused tetanus to the fused one. We had also analyzed the time variation between the peaks of the force response and their maximum force. Two above-described parameters are important for the transition of the active muscle from the state of unfused tetanus to the fused one. The analysis of their changes shows us the peculiarities of dysfunction generation by individual motor units, and the consistent nature of their activation provides the possibility of smooth regulation of the force developed by the whole muscle.
