Trigeminal Sensor System of the Rat, Development and Morphofunctional Features

Introduction

A frequent object of experimental biomedical research, including the study of the nervous system, is the rat. For an adequate interpretation of the obtained results and subsequent extrapolation to humans, it is important to have a clear understanding of the timing of the development and organization of the rat nervous system. The trigeminal sensory system or trigemenal sensory system plays an important role in the life of animals, as it is associated with vibrissae – sensitive hairs on the snout and body, necessary for orientation in space and touch. In humans, the trigeminal nerve is the main sensory nerve of the face and oral cavity and, in addition, carries efferents to the masticatory muscles, controlling their movements [1-4]. The purpose of the review is to analyze and systematize literature data on the development and morphofunctional features of the trigeminal sensory system in rat.

Development of the Trigeminal Sensory System

The study of the distribution of zones of innervation of the trigeminal nerve in embryogenesis showed the important role of neurotrophic factors and their receptors in this process. Genetic studies have shown that peripheral target tissues of the trigeminal nerve secrete chemoattractants, promoting axon growth. Up to a certain stage, neurons do not depend on trophic factors, but from the moment the first branches of the neuropil reach the innervation zones, peripheral tissues begin the synthesis of attractants necessary for the further life of nerve cells [2,3]. Trigeminal nucleus neurons (ganglion neurons) form from ectodermal placodes and neural crest from days 9 to 14 of embryonic development. In the early stages of antenatal development (day 14), the nucleus of the trigeminal nerve adjoins the bend of the bridge, being in close proximity to the zone of innervation. Branching of the neuropil of neurons spread radially in the direction from the forebrain to the innervated organs. Over the next few days, the trigeminal ganglion rotates 90° so that the ventral mandibular neurons become lateral and the brainstem nuclei (also by 90º, so that the lateral neurons and the afferent pathways from the perikaryon to the mandible become dorsal) [2,3].

Central Vibrissae Tract

Vibrissa follicles develop from the mesenchyme by the 14th day of embryogenesis, and by the 16th day the area of their afferent innervation is approximately the same size as in adult rats. The dendrites of the neurons of the trigeminal nucleus reach the brain stem on the 11-15th day of prenatal development, the thalamus – on the 14-15th, the cerebral cortex (layer IV) – on the 16-21st. The vibrissa tract itself, that is, the vibrissa communication system – the nucleus of the trigeminal nerve – the overlying parts of the brain develops mainly postnatally and is formed sequentially first in the brain stem (20 days of embryogenesis – 1 day after birth), thalamus (2-3 days of postnatal ontogenesis) and the cerebral cortex (3-5 days after birth). The development of afferent terminals is preceded by a period of asymmetric growth of dendrites and the migration associated with it, followed by accumulation of neuronal perikaryons in the area of the trigeminal nucleus facing towards the innervated organs.

The first signals received from vibrissae can be registered in the brainstem starting from the first day after birth, and in the cerebral cortex – from the 6th day of postnatal ontogenesis. The dimensions of the receptive fields approximately correspond to those in adult rats, however, newborn rat pups are characterized by a longer refractoriness period and impulse delay. By the 1st week of postnatal ontogenesis, peculiar barrel-shaped clusters of neurons surrounded by fibrous structures form in the IV layer of the neocortex. The diameter of these barrel columns is 100-400 μm. Each such structural unit is associated with certain large rat hairs – vibrissae [4]. GABAergic and serotonergic activity of neurons of the trigeminal nerve nucleus is detected from an early age, although it matures quite slowly, at least for 2 months. Moreover, in the early postnatal period, γ-aminobutyric acid causes excitation in the brain stem, and inhibition in the thalamus and cerebral cortex. In case of damage to the vibrissae before the formation of connections between the nucleus of the trigeminal nerve and the overlying parts of the brain, the formation of the vibrissae tract is disturbed. The critical period in this regard is: 1st day after birth for the brain stem, 2-3rd day for the thalamus and 3-4th day for the neocortex [2,3,5,6].

Trigeminal Sensory System of Adult Rats

Peripheral Nerves and Receptors: The trigeminal nerve has three branches: ophthalmic, maxillary, and mandibular. In the rat, as in other mammals, the ophthalmic branch supplies the dorsum of the head, upper eyelid, supraorbital vibrissae, cornea, conjunctiva, nasal skin, and intranasal mucosa. The maxillary branch innervates the postorbital skin, upper lip, mystacial vibrissae, cheeks, palate, and upper teeth. The mandibular branch supplies the temporomandibular joint, the external auditory canal, the prioreceptors of the jaw muscles, the skin over the lower jaw, the lower lip, the mandibular mucosa, the teeth, and the anterior tongue. The dura mater and cranial blood vessels are innervated by afferents from all three branches. Sensory receptors are found in the skin and muscles of the rat’s snout, oral and nasal mucosa, joints, and tendons [1-4]. The mediators in the afferent nerve endings are substance P and calcitonin, and in the cornea – galanin and the pituitary peptide that activates adenylate cyclase [6]. Irritation of nociceptors of the nasal mucosa is caused by both protective reflexes (sneezing) and neurogenic inflammation or disturbance of cardiorespiratory rhythms. Neurogenic inflammation is associated with vascular vasodilation, plasma extravasation, and mast cell degranulation. It often causes vascular headache [7].

Vibrissa: Individual vibrissae are supplied by both deep and superficial nerves. In typical laboratory rats, each follicle receives approximately 250 nerve fibers. About one third of this sensory innervation consists of unmyelinated nerve fibers. In addition, the fur between the vibrissae is richly supplied with nerve endings [4]. The temporomandibular joint is innervated by afferents that form non-encapsulated receptors. Injections of irritants (eg, glutamate) into the joint cavity are used to simulate arthritis and deep craniofacial pain. Most often, female rats are used for this. In these animals, as in humans, there are gender differences in the sensitivity of the trigeminal nerve [8,9]. Teeth and periodontal ligaments receive innervation from myelinated and unmyelinated fibers. Nerve endings were found in the odontoblast layer, predentin, and pulp. The innervation of the incisors is the most complex due to the increased functional activity of these teeth in rats [10].

Tongue: Trigeminal afferents supply the surface epithelium, filiform and fungiform papillae of the anterior surface of the tongue, providing general somatic sensations. In addition, activation of the trigeminal nerve has been described in the perception of bitter taste (nicotine, caffeine).

Neurochemistry of Afferent Synapses

Most peripheral nerve endings contain substance P or a peptide associated with the calcitonin gene ((Calcitonin gene-related peptide (CGRP)) [1,11,7].

Trigeminal Ganglion: The bodies of the neurons of most trigeminal afferents are located in the trigeminal ganglion, which lies in the middle cranial fossa of the base of the skull. The exception is neurons that receive part of the afferent fibers from the chewing muscles and the periodontium. Their pericarions are located in the mesencephalic nucleus of the trigeminal nerve of the brainstem. Ganglion neurons are pseudounipolar and surrounded by satellite cells. They are divided according to the size of the perikaryons into large (type A) and small (type B). There is some correlation between cell type and their function. So, for example, large vibrissae afferents mainly go to type A neurons, while neurons innervating the cornea mainly belong to type B. Ganglion neurons are surrounded by a number of fibers including noradrenergic sympathetic axons, VIPpositive parasympathetic fibers, serotonergic fibers, and various peptidergic axons containing CGRP, substance P, cholecystokinin, galanin, and NO synthase [1,12].

Cytoarchitectonics of the Trigeminal Ganglion: The neurons that innervate the cornea lie in the anterior ganglion, and those that receive afferents from the lower jaw lie in the posterolateral. There is also a dorsoventral organization: in the dorsal region there are neurons supplying the cornea and supraorbital vibrissae, and in the ventral region - innervating the lower jaw [12].

Neurochemistry of the Trigeminal Ganglion: Large ganglion neurons (type A) are usually immunopositive for neuropeptide Y. Smaller and medium-sized neurons contain glutamate, substance P, CGRP, neurokinin A, somatostatin, VIP, GABA and galanin, chemokinin, melatonin, gastrin-releasing peptide. Substance P is usually localized in the cytoplasm of neurons with CGRP, in addition, a number of neurons simultaneously with CGRP can be immunopositive for both neuropeptide Y and enkephalins [12-16].

Sensory Nuclei of the Trigeminal Nerve

The sensory nuclei of the trigeminal nerve include several clusters of neurons located in the cervical spinal cord, medulla oblongata and midbrain. The spinal cord contains: the spinal nucleus of the trigeminal nerve and the paratrigeminal nucleus. The spinal nuclei receive most of their afferent inputs from vibrissae. In the brainstem are located: the main sensory nucleus, the motor nucleus of the trigeminal nerve, the supramedial nucleus and the intertubular nucleus, which controls the movements of the jaws. In the midbrain - the mesencephalic nucleus, which sends afferents to the periodontal teeth of both the upper and lower jaws [1,11,2].

Cytoarchitectonics: Most sensory nucleus neurons are pseudounipolar, but small multipolar cells are also present.

Neurochemistry of Sensory Nuclei: The neuropil of the sensory nuclei contains: glutamate, CGRP and substance P. GABA is mainly present in the spinal nuclei. The mesencephalic nucleus contains GABAergic, glycinergic neurons, as well as cells immunopositive for aspartate and glutamate, gastrin-releasing peptide [13,17,15,16].

Thalamic Switching Nuclei of the Trigeminal Nerve

The main tract passes through the medial part of the posterioventral and ventrobasal complex of somatosensory thalamic nuclei. Spinal sensory neurons also project to the intralaminar nuclei and the intermediate nuclei [1,11,18,19].

Cytoarchitectonics and Neurochemistry: The thalamic nuclei are composed of medium-sized multipolar GABAergic neurons [18].

Somatosensory Areas of the Zone of Innervation of the Trigeminal Nerve in the Neocortex: The vibrissa innervation zone has the most extensive representation in the neocortex. The fourth layer of the parietal cortex contains modules consisting of multipolar stellate GABAergic neurons and thalamic afferent fibers. Each module is associated with a specific vibrissa. Star-shaped neurons form synapses with cells of the 2nd, 3rd and 5th layers. Pyramidal neurons of the 5th layer of the neocortex, having received information from stellate cells, give efferents to the reticular thalamic nucleus, the superior striatum, the nuclei of the bridge and the sensory complex of the trigeminal nerve in the brain stem, providing the movement of vibrissae [18-22,1,5]. Thus, the data presented in this review on the development and morphofunctional features of the rat trigeminal sensory system will serve as the basis for further study of the nervous system of these animals with the possible implementation of the results obtained in practical medicine.

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Cryoanalgesia of the Tibial Nerve Neuroma after Failed Tarsal Release - Case Report

Introduction

Cryoanalgesia is a mini-invasive neurodestructive (ablative) procedure used to temporarily eliminate the pain signal conducted by the sensory branches through the action of low temperature. Due to the development of probes in the shape of long, rigid cannulas that allow the expansion of the gas (CO2, NO) circulating inside the probe, a temperature drop to -70 degrees C is achieved at the end of the active probe. This results in a mechanism called “heat theft” from the environment and the formation of the ice ball by freezing the water from the surrounding tissues. The mechanism of disruption of conduction is attributed to damage to Schwann cells while maintaining the continuity of the basement membrane and to micro embolisms in the nerve nutrient vessels. The method is used in the treatment of acute and chronic pain, and the denervation time is much longer than the duration of regional blocks, even with the use of continuous anesthesia catheters, and is even several months with minimal risk of infection and no risk of catheter removal [1,2]. The diameter of the ice ball formed at the top of the probe depends on the degree of tissue hydration, tissue blood supply, and freezing time, reaching even 1 cm in diameter [3]. Thanks to the coupling of the probe with the nerve stimulator and the use of ultrasound, X-ray, or CT imaging, very precise and safe percutaneous neuro destructive treatments have become possible. These treatments have been used for several decades in pain medicine and have found a very wide application, especially in the treatment of chronic pain in the spine of articular origin due to the possibility of precise identification and damage to the pain generator, i.e. medial branches departing from the dorsal spinal nerves without the risk of damaging the motor branches (nerve roots). Other applications include treatment of cervicogenic headaches, perineal pain, palliative denervation of peripheral joints in the course of severe osteoarthritis of the knee and hip (also preceding arthroplasty), and treatment of post-herpetic neuralgia or pain after rib fracture [4-12].

Cryoanalgesia, however, is also used wherever new pathological innervation arises, as in the case of a new network of periosteal sensory fibers in the sites of proliferative changes in osteoarthritis and musculotendinous attachments in tendinopathy and enthesopathy, or where, in the event of a change in the structure of the nerve, stump neuromas develop after amputations or in scars after unsuccessful surgeries to release the nerve entrapment syndromes [13-15]. The presented case relates precisely to such a situation where the chronic pain syndrome of the right foot fulfills the hallmarks of the failed tarsal release tunnel with the formation of a chronic pain syndrome of type II Complex Regional Pain Syndrome (CRPS) with typical allodynia, dysesthesia, and poor response for pharmacological treatment.

History of Complaints and Therapy

Female patient M.O. born in 2001 suffered a torsion right ankle injury in 2011. Treated conservatively, on an outpatient basis, locally and systemically anti-inflammatory with periodic plaster immobilization. The first available information sheet comes from 12/2014 when she was admitted to the children’s orthopedics department due to inflammation of the right ankle joint. In the epicrisis information on a three-year history of pain in the ankle joint previously treated with plaster casts, with CRP 16 mg / L, then 34 mg / L, was reported, and after antibiotic therapy and steroid therapy with temporary immobilization, the CRP was reduced to 1 mg / L. X-ray without evidence of bone destruction and without periosteal reactions, in scintigraphy, the hot area in the right ankle joint. Re-admitted to the same ward after 3 weeks with the diagnosis of Achilles tendonitis, and after local administration of steroid and plaster immobilization, she was discharged home with the recommendation of a follow-up in 2 weeks.

Another hospitalization on 02/2015 due to recurrent pain in the right ankle joint in the area of the medial ankle, laboratory tests showed low levels of Vit D (10 ng / ml), supplementation, and local physical treatment were included, and rheumatoid and neurological diseases were excluded. On 03/2015, a CT examination of the right ankle joint revealed an oval, hypodense lesion measuring 12 x 9 x 14 mm in the vicinity of the Flexor Halucis Longus tendon (FHL), but without specifying the diagnosis and possible relation to the symptoms. On 03/2015 stay in a rehabilitation center – for local physical treatment. On 04/2015, a recurrence of the pain - admission to the pediatric orthopedic department, treated with systemic and local anti-inflammatory drugs, and recommendation of orthopedic insoles - without making a specific diagnosis. On 10/2015, an MRI of the right ankle joint was performed with contrast, revealing an increased amount of fluid in the FHL tendon sheaths - otherwise, the image was normal.

Several visits to the emergency during an exacerbation of symptoms in 2016, but without elevated inflammatory markers, and treated with symptomatic anti-inflammatory treatment each time. On 05/2016, in the ultrasound examination, vascular disorders were excluded, but a hypoechoic lesion with a length of 8 mm was found along the tibial nerve, in the sinus of the steppe, with the conclusion of the description “possible scar after administration of the drug to the nerve”. The performed EMG (05/2016) revealed a slower conduction velocity in the medial plantar nerve. Another MRI scan found no damage to the tibial nerve. Nevertheless, in 06/2016 the patient was operated on and the indication for surgery was “tibial and medial plantar neuralgia and Baxter branch entrapment”. Relapse of the neuralgia occurred on 09/2016, Neurontin (Gabapentin) 3x300 mg was then recommended. The ultrasound examination on 10/2016 revealed extensive scarring involving the tibial nerve. Re-operated on 10/2016 - tibial nerve neurolysis and removal of scars complicated by postoperative wound dehiscence and prolonged skin healing. The prolonged postoperative pain was explained by the pressure of the tibial nerve entrapped into the scars, which was visualized in the next MRI examination in 2017. The attempt of botulinum injections and hyaluronidase in the area of the scar and pulsed radiofrequency of the tibial nerve in 2017 were unsuccessful. Due to the lack of improvement, for the third time the tibial neurolysis was performed on 11/2017, unfortunately without success.

In 2018, she was treated topically with the autologous conditioned serum (Orthokine) - 4 doses of ultrasound-guided infiltration of the tibial nerve surrounding without success. The only significant reduction in the intensity of neuropathic pain, sometimes to its complete abolition, although short-term, because only a few hours, was obtained after intravenous lignocaine infusions at a dose of 1.5 - 3 mg/kg / h calculated using the patient’s ideal body weight and given as an infusion over 20-30 min. Parallel to the lignocaine infusions, the mirror therapy used to desensitize the limb through behavioral patterns was also unsuccessful. The patient was qualified for spinal cord stimulator implantation with external stimulation, the procedure was performed on 06/2018 (with admission pain VAS 8-9, after its application pain reduction to VAS 4-5). Since then, the patient uses the pacemaker (Precision Montage™ MRI) at the time of exacerbation of symptoms, achieving a pain reduction by 2 points (with an average rating of 5-6), using a several-hour stimulation with the Burst program, but the pain has never been fully muted despite the constant intake of pregabalin (2x75 mg) and periodically Tramadol (50-100 mg).

Since 2019, the patient has undergone five cryoanalgesia treatments of tibial nerve neuroma in the area of postoperative scarring. According to the patient’s report, each of them reduces neuropathic pain with hyperalgesia by 3-4 points for several months (4-5 months on average), but it also does not completely eliminate it. For the patient, however, it is the only option so far to significantly reduce hyperalgesia and pain, without the risk of damage to the nerve trunk and loss of motor function of the foot.

Cryoanalgesia Technique

Depending on the type of pain generator, there are three main techniques for cryoanalgesia.

1) Classic technique - cryoablation of the sensory branch after effective sensory and motor stimulation - the most common model of the treatment in the area of the spine, where the goal is the medial branch innervating the intervertebral joints.

2) Periosteal technique - freezing of the pathological innervation around proliferative remodeling lesions on the tendon and ligament attachments or in the course of the osteoarthritis.

3) Indirect technique “in the lake” - in a situation that is impossible to identify with imaging techniques of a nerve branch due to its very small diameter (fascial or skin fine branches) or the edges of the neuroma in the postoperative scar, the technique of regional infiltration with lignocaine or saline and freezing in a block is used.

This third variant was used in the described case. In the first phase, the tibial nerve in the zone of its greatest tenderness was identified under ultrasound control, marking the boundaries of its course on the skin (Figures 1 & 2). Then, under ultrasound guidance, a thin needle was inserted towards the lateral wall of the neuroma, and lignocaine was infiltrated into the soft tissues forming scarring entrapment of the tibial nerve (Figures 3 & 4). A transcutaneous cannula was inserted through the path of the first needle and its position in the center of the fluid-soaked area was determined under ultrasound guidance (Figure 5). Then the cryoablation probe was inserted through the cannula and 2 cycles of 1.5 min freezing at minus 70 deg. C was performed with a 30-sec interval between the freezing cycles (Figure 6). During freezing, a growing ice ball was observed on the lateral wall of the tibial nerve in the area of the neuroma, and the skin was protected against frostbite by warming it with fingertips (Figures 7 & 8). After removing the probe, a pressure dressing was made.

Figure 1: US-guided identification of the target.

Figure 2: Tibial nerve neuroma - traced area.

Figure 3: US-guided infiltration of the neuroma.

Figure 4: US-guided infiltration of the neuroma with lidocaine.

Figure 5: Approaching the neuroma with the cannula.

Figure 6: US-guided cryoanalgesia.

Figure 7: Ice ball during cryoanalgesia.

Figure 8: Cryoanalgesia and preventing skin frostbite.

Discussion

Cryoablation of neuromas is one of the widely recognized methods of treating chronic neuropathic pain caused by them [16,17]. In most cases, they result from the cutting of nerves in the amputated limb or crushing of the nerves during high-energy trauma and as such can be cryoanalgesia over an extended period of time until they are completely destroyed. A different situation is created by partial injuries of active large nerve trunks containing motor fibers, which form intraneural neuromas with the ectopic activity of the damaged nerve, and which should be differentiated from tumor-like neurogenic lesions [18,19]. The reason for the production of such neuromas is partial nerve damage - cuts, injection injuries, scar entrapment with chronic ischemia due to pressure. Then the cryoablation must be weak enough not to damage the motor fibers and effective enough to break spontaneous discharges and conductivity in the emphases. For this reason, the cryoanalgesia probe cannot directly adhere to the nerve trunk itself, and the lowered temperature zone transmits like an echo from adjacent infiltrated tissues sufficiently effectively to damage the thin unmyelinated newly formed pain-conducting fibers and not damage the myelin sheath and Schwann cells of the motor fibers. Thanks to ultrasound control, it is possible to infiltrate the area around the damaged nerve and to dose the freezing time in such a way (from 30 sec to 1.5 min) so that, by following the dimensions of the ice ball, it does not cause irreversible damage to the motor fibers. A similar technique can be successfully used in post-herpetic pain syndromes for cutaneous and fascial branches, without the need for cryoablation of the intercostal nerves, thanks to which there is no denervation of the intercostal muscles, the subpatellar branch of the saphenous nerve without the need to freeze the main nerve trunk or finally cryoanalgesia the suprascapular nerve without risk of the supraspinatus and infraspinatus denervation.

Conclusion

The indirect cryoablation technique can be used to treat chronic neuroblastoma pain syndromes even in the immediate vicinity of a large nerve trunk without the risk of damaging the motor fibers.

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The Effects of Sustain Loads on the EMG Activity of the Leg Muscles During Soldier’s Quasi-Static Posture Control

Introduction

During military activity, professionals are sometimes required of their physical capacity to carry out cargo transportation. This activity is performed using basic equipment for the military to last in action, such as: the combat backpack, in which the individual will carry the items needed for personal survival, and his or her weaponry that will pro-vide you with security and protection. The high military physical effort, the specific tasks performed and the great weight transported, the military are subject to a higher risk of suffering some type of injury. The increase of this transported weight reflects in the increase of the tension of the muscles of the lower limbs, which are strictly related to the injuries [1-4]. In the load carriage using packs, the military is subjected to a natural imbalance, at this point he will seek postural control to keep himself balanced. this capacity is defined as the property for maintaining an upright posture, determined by the movement of the human body’s pressure center [5]. The most important thing in this activity is the location that the individual will carry such a load in his body. It is precisely in this decision that will reflect how the muscles of the lower limbs will behave to maintain the balance already mentioned above. The way of load carriage using backpacks will also bring reflexes to the military, with regard to biomechanics and human physiology. In this case, showing that distributing the weight in the anterior and posterior part of the body, such as a double backpack, will re-quire less energy from the military; promoting lower inclination of the body, but limiting the movement of the arms and increasing body temperature [3,6].

Another analysis, the high load distribution also alters the ground reaction force, promoting the balance of the body. In its conclusion, it showed the progressive increase of this force in relation to a load of 60% of body weight and with a focus on transportation; also reporting on military weapons [7]. With all this, in our study, to measure this orthostatic equilibrium in the face of a load variation, we used surface electromyography (EMG). This choice was made taking into account that this exam will provide the motor units activated during the individual’s overload and the discharge rate produced, showing the muscular behavior [8-10]. In this part of the literature, little has been said about the effect of the support of the military backpack and the armament on the orthostatic position [11,12], analyzed by the electromyographic behavior. This gap needs to be deepened, since it is the basic military activity, it will soon bring a benefit to the Brazilian Army. Generating adaptations, or suggestions, to improve the postural control of our military. All with the purpose of reducing injuries already men-tioned above; thus, improving the combative capacity of the terrestrial force. Therefore, the aim of this study was to evaluate the effects of backpack support and military weaponry on the electromyographic behavior of the leg muscles in maintaining standing position.

Materials and Methods

It is an experimental, applied study, cross-sectional and quantitative data. The participants were composed of experienced service members and were submitted to three conditions: The first, supporting only the combat backpack (CTRL), the second condition sup-porting the combat backpack with the machine gun simulator (MAG) and the third condition supporting the combat backpack with the rifle (FZL). These moments were compared based on the electromyographic signal variables of gastrocnemius lateralis (GNL) and tibialis anterior (TBA) muscles.

Subjects

The sample was of the non-probabilistic type of voluntary character and consisted of 16 participants (male sex; age: 27.5±4.9 years; total body mass: 77.2±9.3 kg; height: 176.8±5.1 cm), experienced service members (> 6 years of service and experience in cargo support). All participants were students in the instructor course of the Brazilian Army Physical Education School. The study protocol was approved by the Ethical Committee of the Salgado de Oliveria University (file: CAEE 48000321.3.0000.9433). All participants were fully informed about the content of the study and gave their written consent.

Equipment’s and Instruments

In the reference condition, the participants wore t-shirts, shorts, boots, socks, and a military backpack. Afterward, all subjects added a rifle and a machine gun according to the test. The individual combat equipment that was used by the 16 volunteers was composed of: 01 (one) large capacity Alice campaign backpack with two liter pet bottles with sand, totaling the weight of 15kg, 01 (one) Mauser carbine model 1935 with two shin guards, one of 3kg and another of 4 kg, weighing on average 10.8kg (simulating the weight of the MAG machine gun), together with a bandolier to assist in weight control), 01 (one) Model 1935 Mauser carbine with a 1kg shin and an extra weight of 0.5kg, weighing on average 4.8kg (simulating the weight of the FAL 7.62 rifle), along with a bandolier to assist in weight control) and 01 (one) personal boot, shirt and shorts.

Procedures

Data were collected from June to September of 2021 in the Biosciences laboratory of the Brazilian Army Physical Education School. The volunteers were scheduled to only collect 4 individuals per day. At first, the ICF and anamnesis were completed. After completing the mandatory documents, each military member had his or her stature (EST) and total body mass (MCT), measured using the military physical training uniform. It was then asked that the military put the boots, for a new conference of MCT and EST. They were then instructed on the procedures to be carried out. On the days of collection the MCT was again measured, but in the control conditions with backpack (MCT_CTRL), with backpack and rifle (MCT_FZL) and with backpack and machine gun (MCT_MAG). For the acquisition of biological signals (surface electromyography - sEMG), wireless surface electrodes (Trigno Wireless System, Delsys Inc., USA) were used, amplified by a signal acquisition module ((Delsysinc., USA, 2.4GHz transmission frequency, 1kHz sam-pling frequency, common rejection mode >80dB, 10Hz high pass filter and 450Hz low pass, total gain 1000 times). The electrodes were positioned on both sides of the lower limbs in the anterior tibialis (TBA) muscle, one-third to one-fourth of the distance from the knee to the ankle, in the largest palpable muscle mass, we palpated the area while the individual performed the dorsi-flexion of the foot. In the gastrocnemius lateralis muscle (GNL), the electrodes were positioned approximately two centimeters laterally in relation to the midline of the gastrocnemius muscle [9,12,13].

Myoelectric activity recorded from: gastrocnemius lateralis (GNL) and anterior tibialis (TBA) normalized for the maximum amplitude of myoelectric activity obtained during the maximum isometric voluntary contraction test (MIVC). The CIVM was performed with the individual seated on a stretcher with the trunk at 80º flexion in relation to the hip and with the knee positioned at 90º flexion and suspend-ed on the side of the table. After positioning, the participants performed the dorsi-flexion and plantar flexion against resistance imposed by the evaluator. This resistance was maintained for 5 seconds for each movement in both lower segments. After that, they climbed onto the power platform and looked for the upright and static posture, staring at a target on the wall in front of them, at a distance of 3 meters. The positioning of the feet, on the platform, was standardized, using a plastic wedge with an angle of 30º, making the heels stick together and the tips of the feet distant. Each participant was submitted to three conditions of estabilometric evaluation, being sustaining the combat backpack, sustaining the combat backpack and the Mauser carbine and sustaining the combat backpack and the MAG machine gun, in each of these positions were made 3 collections. Each measurement lasted 90 seconds, with 30 seconds (15 initial seconds and 15 final seconds) being discarded where sEMG analyses occurred in 60 seconds. The sEMG signals of the muscles and the platform of force were sincronized by means of an accelerometer positioned in the dorsal region of the boot (Table 1).

Table 1: Descriptive statistics with mean, standard deviation (SD), minimum (Min.) and maximum (Max.) of the total body mass (kg) in the conditions with backpack, backpack and rifle and with backpack and machine gun.

Note:

1Significant difference between MCT_CTRL and MCT_MAG condition.

2Significant differences between MCT_CTRL and MCT_FZL condition.

The signals were analyzed in specific software in which the test parameters consisted of continuous collection and thus initially stored in files on the computer hard drive for the processing of digital signals in MATLAB environment (R2015a) version 8.5.0 (The Math works Inc Natick, Massachusetts, USA) that provided root Mean square (RMS) and full-time analysis (iEMG) related time domain data. The RMS values are summed on each side according to the analyzed muscle. This sum generated a unique value for the TBA and GNL muscle in the respective conditions (CTRL, FZL and MAG). The same calculation was made for iEMG. After these values were normalized by the peak of the RMS and iEMG of the MIVC of each muscle.

Statistical Analysis

All data were stored and analyzed using the statistical program Statistical Package for the Social Sciences for Windows (SPSS) version 20.0 (SPSS Inc. Chicago, Illinois, USA). Shapiro Wilk’s normality test rejected the hypothesis of equality of EMG variables for different load conditions, and Friedman’s two-way test was performed to analyze the variance of related samples. All significance values (p value) were determined as <0.05. Descriptive statistics (mean, standard deviation, maximum and minimum) were calculated for each data set if presented in graphic form using Graphpad Prism software version 8.0.1 (Graphpad Software Inc. San Diego, California, USA).

Results

The total body mass (MCT) obtained initially was that of the control condition (93.56±9.16 kg). Then we obtained the MCT of the FZL condition, with the military carrying the simulacrum of the Rifle (98.36±9.16 kg), in which already presented a statistically significant difference between the CTRL condition (p=0.014). Finally, the MCT of the MAG condition, with the military carrying the MAG machine gun weight (104.36±9.16 kg), finally, this last condition was demonstrated with a statistically significant difference, both from the CTRL condition (p=0.0001), and from the FZL condition (p=0.014).When performing the maximum voluntary isometric contraction (CIVM), we obtained the measure adopted as 100% of the RMS (1.0 in u.a.), represented in the ordinal axis of Figures 1 & 2. When performing the sEMG analysis in the time domain, In Figure 1, we used the square root of the mean signal obtained (RMS) to quantify our muscle activation in relation to the maximum military activation, all of the two muscles under analysis, anterior tibialis (TBA) and gastrocnemiuslateralis (LNG). With this, we found that the CTRL condition (TBA: 0,204 [0,062 - 0,366]; LNG: 0,209 [0,067 - 0,371]) did not present a significant difference. The RMS values in the FZL condition (TBA: 0,195 [0,008 - 0,381]; LNG: 0,204 [0,013 - 0,385]) and in the GAM condition (TBA: 0,221 [0,072 - 0,293]; LNG: 0,225 [0,077 - 0,298]) were not statistically significant differences. However, there is a low-er maximum variation of this muscle activation in the latter condition. Another form of sEMG quantification is the integral of the entire area, in the frequency spectrum filled by the signal [7]. In this condition of Figure 2, we obtained for CTRL condition the result of 0.225 (0.064 - 0.368) for TBA and 0.231 (0.069 - 0.373) for GNL, both muscles without significant differences. For FZL condition, the results were 0.213 (0.012 - 0.382) for TBA and 0.218 (0.015 - 0.387) for LNG, also without significant differences for both muscles. Finally, in the GAM condition, the results were 0.219 (0.074 - 0.295) for TBA and 0.224 (0.079 - 0.300) for GNL, as well as the previous ones without significant difference. Results similar to Figure 1 with the treatment of MRH, corroborating the reliability of the data obtained in the time domain.

Figure 1: Root mean square (RMS) of the electromyographic signal in arbitrary unit (u.a.) comparing the conditions with backpack (CTRL), backpack and rifle (FZL) and backpack and machine gun (MAG) of the anterior tibialis (TBA) and gastrocnemius lateralis (GNL) muscles.

Figure 2: Electromyographic signal integral (iEMG) in arbitrary unit (u.a.) comparing the conditions with backpack (C-TRL), backpack and rifle (FZL) and backpack and machine gun (GAM) of the anterior tibialis (TBA) and gastrocnemius lateralis (GNL) muscles.

Discussion

Military activity requires the transportation of heavy cargo, whether backpack, equipment, or weaponry, for extended periods of time. We have shown in our study that the addition of the rifle and the MAG machine gun were statistically significant for the difference in total body mass (MCT), this is a potential risk factor for the occurrence of lesions in the locomotor system [14]. However much this difference in weight, when we add the MAG and compare it with the CTRL situation, it is significant, it is an increase of less than 20% of the MCT. The backpacks with less than 20% of body weight were not sufficient to activate the lower limbs muscles in the static position, noting a significant difference only in the rectus abdominis [15-17]. With the results of our sEMG, we observed that there was no statistically significant difference when comparing the three situations tested. All of them had about 20% of the muscular activation when compared to the total maximum voluntary isometric contraction. Several may be the reasons for this event, first we performed the measurement in the static position, while Simpson, et al. [18] collected these variables in displacement and found an increase in gastrocnemius lateralis activity, suggesting that a possible collection in displacement be more reliable the real employment situation of military troops [19].

In our research we analyzed the tibialis anterior and gastrocnemius lateralis muscles, in both results they were similar and did not present significant statistical differences. Corroborating with Lindner, et al. [1], in his study, found that these muscles, already mentioned above, also did not demonstrate significant muscle activation when transporting military equipment, however, in this same research, the greatest increase in electromyographic activity was in the rectus femoris muscle after adding the backpack. Birrel, et al. [7] presented in their research that there are statistically significant differences in the ground reaction force when walking with a rifle, but when dealing with muscle activation measured by sEMG. We are talking about the recruitment of muscle fibers to perform a certain activity, that is, the greater the difficulty of the task required, the greater the recruitment of muscle fibers. In our research, when we added the rifle or the MAG machine gun, the activation was similar to the CTRL moment, which would be the individual with only the backpack, it is worth noting that both the rifle and the MAG were inserted with the bandolier wrapping behind the neck with the armament resting in front of the body which suggests a greater demand of the upper back muscles due to the location closest to which weight was supported, thus requiring little muscle recruitment of the lower extremities, the previous statement is confirmed by the study by Lindner, et al. [1] which showed that the weight of the rifle showed no significant difference in muscle activation in the lower limbs, when the weight of the armament is carried by the upper limbs. Confirming our thesis Thuresson, et al. [18] showed that the weight of a helmet placed on the head did not reflect on the lower limbs, but on the muscles of the neck and upper back due to its proximity to the place of weight inserted.

Contributing to the findings of our research, Majumdar, et al. [20] in his study on the transport of loads in military personnel, found that the body adopts some postural changes to decrease muscle overload, but that if this load is carried is low, between 6.5% and 27.2% of body weight will not cause orthostatic changes, confirming our results, since the load we added in the military was well below 25% of the MCT. Our sample was composed of experienced service members, all with at least seven years of military service, with good physical fitness, being a very restricted and specific sample. Therefore, also attributed this factor to one of the causes of low muscle activation and concluded that experienced service members supported larger loads due to better training, physical condition and greater strength, supporting loads between 47% and 64% of your body weight while maintaining a normal gait pattern [21,22]. The present study has limitations such as: the small sample collected; the evaluation only in the static position; the weight of the additional transported load is relatively low; the collection time is small. The study did not evaluate other possible factors that contributed to the low electromyographic activity found in the research, such as: muscle strength of the lower limbs, and a strength test of this muscle group may have been performed previously; activity of other synergetic muscles, which may be collected in later studies, adding sEMG in the biceps femoris for example; finally, postural changes of individuals, and photogrammetry software may be used to quantify these changes. It is suggested to carry out more studies in the area, a crosssectional survey with military personnel during a march, with the continuous monitoring of the electromyographic activity of the lower limbs. Another suggestion would be a longitudinal study, analyzing the sample with the manipulation of the transported load variable and verifying the behavior of the military with the increase of this variable.

Conclusion

The body mass of the total FZL and MAG condition were significantly different. The sEMG of the TBA and GNL muscles in the CTRL condition showed about 20% of muscle activation in relation to VSD, the other two conditions resembled the control condition and did not present a statistically significant difference for both muscles measured. A similar result was observed in iEMG, in which the CTRL condition was not significantly different from the FZL and MAG condition, both of TBA and GNL, serving to corroborate with sEMG. These results indicate that the addition of the rifle load and the MAG machine gun, were not sufficient to significantly activate the muscles of the lower extremities, during the maintenance of the quasi-static posture with combat backpack.

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