Dariusz Górka

Regeneration of sciatic nerves of adult rats induced by extracts from distal stumps of pre-degenerated peripheral nerves

Despite numerous experimental and clinical attempts to reconstruct injuries of peripheral nerves, the methods developed until now have not been sufficiently effective. We examined the influence of extracts (postmicrosomal fractions) obtained from non‐pre‐degenerated or 7‐day‐pre‐degenerated distal segments of peripheral nerves on the regeneration of injured sciatic nerves of male adult rats. The extracts were introduced to the site of injury with autologous connective tissue chambers filled with fibrin. Reference groups were treated with brain‐derived neurotrophic factor (BDNF) or fibrin only. We examined DiI‐labeled motoneurons, toluidine blue‐labeled myelinated fibers in the mid‐part of the chambers, and AChE‐positive nerve endings to assess the regeneration intensity. In addition, the length of fibers regrowing within the chambers was measured. We found that extracts obtained from distal stumps of 7‐day‐pre‐degenerated peripheral nerves enhanced nerve regeneration as strongly as BDNF.

Predegenerated Peripheral Nerve Grafts Rescue Retinal Ganglion Cells from Axotomy-Induced Death

The inability of axons to grow across damaged central nervous system tissue is a well-known consequence of injury to the brain and spinal cord of adult mammals. Our previous studies showed that predegenerated peripheral nerve grafts facilitate neurite outgrowth from the injured hippocampus and that this effect was particularly distinct when 7-, 28-, and 35-day-predegenerated nerve grafts were used. The purpose of the present study was to use the above method to induce and support the regrowth of injured nerve fibers as well as the survival of retinal ganglion cells (RGCs). Adult Sprague-Dawley rats were assigned to three groups. In the experimental groups transected optic nerve was grafted with peripheral nerve (predegenerated for 7 days (PD) or nonpredegenerated). In the control group, the optic nerve was totally transected. RGCs and growing fibers labeled with fluorescent tracers were examined. They were counted and the results were subjected to statistical analysis. Retinal ganglion cells survived in the groups treated with predegenerated as well as nonpredegenerated grafts; however, the number of surviving retinal ganglion cells was significantly higher in the first one. In both groups the regrowth of the transected optic nerve was observed but the distance covered by regenerating fibers was longer in the PD group. No fibers inside grafts and no labeled cells in retinas were present in the control animals. On the basis of the obtained results we can state that the predegeneration of grafts enhance their neurotrophic influence upon the injured retinal ganglion cells.

Time-dependent regenerative influence of predegenerated nerve grafts on hippocampus

Our previous studies have revealed that the predegeneration facilitated the neurite outgrowth from hippocampus following the peripheral nerve grafts implantation. The aim of the present work is to find whether the stimulative power of peripheral nerve grafts depends on the time lapse after the transection. Autologous predegenerated distal stumps of the rat sciatic nerves were implanted into the hippocampus on the 7th, 14th, 28th, and 35th day following the transection. Six weeks later, horseradish peroxidase conjugated with fluorescein isothiocyanate was injected into the graft and frozen sections of brains were made. Fluorescence microscope examination has shown that FITC-HRP labeled cells were present among the hippocampal neurons in all the brains under examination, excluding these grafted with 14-day predegenerated peripheral nerves. The FITC-HRP labeled neurons were particularly numerous when the 7- and 35-day-old predegenerated stumps were used as grafts.

Neurotrophic effect of submicrosomal fractions obtained from pre-degenerated peripheral nerves

Submicrosomal fractions obtained from pre-degenerated distal stumps of sciatic nerves were implanted by means of connective tissue chambers into the injured hippocampus for 8 and 18 weeks. The nerve stumps were allowed to pre-degenerate for 7, 28 and 35 days. The neuronal outgrowth was examined by means of FITC-HRP injected into the chamber. Eight weeks postoperatively the greatest number of traced cells was present in brains treated with the fraction obtained from nerves pre-degenerated for 7 days. Eighteen weeks following implantation the greatest number of FITC-HRP positive cells was found in brains grafted with the fraction from nerves pre-degenerated for 35 days.

The changes in neurotrophic properties of the peripheral nerves extracts following blocking of BDNF activity

Abstract Objectives: Retinal ganglion cells (RGCs) of adult rats are unable to regenerate their axons after optic nerve injury and soon after they enter the pathway of apoptosis. They may, however, survive and regenerate new axons in response to application of specific peripheral nerve extracts that presumably contain a range of neurotrophic substances. One of the recognized substances of proven neurotrophic activity is brain-derived neurotrophic factor (BDNF). We have investigated whether blocking the BDNF activity in post-microsomal fractions obtained from 7 day pre-degenerated peripheral nerves would affect its neurotrophic properties towards RGCs after optic nerve transection in adult rats. Methods: Autologous connective tissue chambers sutured to the distal end of transected optic nerve served as active substances containers. Surviving RGCs were visualized using Dil. The number of myelinated outgrowing fibers within the chambers was evaluated in histologic sections. Results: BDNF and 7 day pre-degenerated nerve extracts, and also extracts with blocked BDNF activity, enhanced RGC fibers outgrowth. The regeneration was significantly weaker in the control group. Blocking the BDNF activity in the 7 day pre-degenerated peripheral nerve extract reduced its neurotrophic effects but the differences were insignificant in comparison with non-blocked extracts. Discussion: The regeneration intensities in groups receiving 7 day pre-degenerated peripheral nerve extracts (PD7) and BDNF were comparable. The number of surviving cells was higher in the PD7 group and there were more regenerating fibers in the BDNF group, which may be explained by the strong BDNF effect on axonal collateralization and sprouting.

Physiotherapeutic techniques used in the management of patients with peripheral nerve injuries.

Peripheral nerve injuries affect a wide range of functional, manual and social function, and frequently lead to constant disabilities. After complete transection of nerve, axonal degeneration process gives rise to a variety of symptoms including hyperesthesia, reduced or altered sensation, pain and atrophy (Lee and Wolfe, 2000). With an array of choices for surgical and treating peripheral nerve injuries, there is also, a lot of new, coherent strategies on rehabilitation and physiotherapy protocols - which should be indispensable after injury. Physiotherapy, with a view to compensate dysfunctions relieves in sensory symptoms and creates grater neuroplasticital potential, forms essential part of the treatment for people after peripheral nerve injuries (Inoue et al., 2003). In the present paper we present the physiotherapeutic methods, protocols, and strategy currently used for initiation and support of peripheral nerve regeneration after injuries. Kinetic therapy in peripheral nerve injuries: Impact of kinetic therapy on peripheral nerve repair after its damage is mostly determined by the time required for regeneration of nerve fibers as well as for muscle reinnervation. Stress put on a paralyzed muscle through stretching or strengthening delays, and may even prevent full nerve recovery, and such treatment should not be started until the late stage of nerve regeneration, when progressive strength return can be seen. After injury of the nerve, physiotherapeutic methods are dedicated to eliminate paresis and to restore normal function of muscles as well as to improve circulation and following energetic supply to the tissues. Table 1 presents an overview of the current methods most commonly used in physiotherapy after peripheral nerve injury. Table 1 The most commonly used methods of kinetic therapy in peripheral nerve damage Electrostimulation: Electric stimulation plays an important role in the treatment of various neuromuscular dysfunctions. With a wide range of applications and the possibility of combining this method with others, it is considered as one of the most effective. There are many types and ways of electrostimulation which differ one from another with the technical embodiment. The most common method is transcutaneous electrical nerve stimulation (TENS), which consists of transcutaneous stimulation pulses of electric current with a frequency of 90–130 Hz. Chen et al. (2001) showed that percutaneous electric stimulation of 2 Hz frequency enhanced the mean values of the axon density, blood vessel number, blood vessel area and percentage of blood vessel area in total nerve area in injured rat sciatic nerve. As studies show, stimulation current of low frequency (20 Hz) for 1 hour a day for 2 weeks after the injury shortens the period of axonal outgrowth of three nerve bundles through the implanted graft (Al-Majed et al., 2000; Gordon et al., 2003. It also showed that electrical stimulation has a positive influence on regeneration processes by stabilizing the cholinergic receptors at the neuromuscular junction. Electroacupuncture is a simple method of indirect application of an electrical stimulation to injured nerve. Pomeranz and Campbell (1993) revealed that the regeneration of injured nerve was enhanced by continuous electrical stimulation at the site of the injury via chronically implanted electrodes. However, Inoue et al. (2003) showed that it is unclear whether electroacupuncture enhanced the axonal regeneration processes. Most frequently used patterns of electrical stimulation of peripheral nerve trauma are presented in Table 2. Table 2 The most commonly used patterns of electrical stimulation of peripheral nerves after injury Magnetotherapy: For the treatment of damaged peripheral nerve, a pulsed low frequency magnetic field can also be applied. Magnetic field therapy has well-known effects on enhancing enzymatic activity, oxy-reductive processes and better blood circulation what results in better oxygenation and conduction characteristics of regenerating peripheral nerves. These mechanisms base on the influence of magnetic field on liquid-crystal structure of many membranes and cell organelles resulting in ion-channels transmission changes. Alteration in intra- or extracellular ion distribution leads to changes in electric potentials in organella membranes as well as in cellular membranes of living biological systems. Magnetic stimulation enhances the regeneration of nerve fibers, as the nerve conductivity increases as well as the amplitude of the action potential (Negredo et al., 2004; Mert et al., 2006). The pulsed electromagnetic field (PEMF) has a high clinical value, as applied immediately after peripheral nerve injury - shortens the duration of functional defects (Mert et al., 2006). Unlike electrical stimulation, magnetic stimulation carries no risk of infection due to electrodes pinned around the wound, and it is completely painless, even in patients with well-preserved sensation. Therapeutic effects of PEMF and CEMF in the case of peripheral nerve damage are comparable and may be used complementarily (Bannaga et al., 2006). Spatial magnetic field generator is one of most recent achievements among the magnetostimulators. Prototype generates magnetic field through 3 pairs perpendicularly arranged magnetic coils. This allows for the interference of fields and results in obtaining the rotational magnetic field focused in small area. This new method may be more effective than other widely used techniques of magnetostimulation and magnetotherapy, as shown in animal experiments where strong spatial alternating magnetic field exerted positive effect on peripheral nerve regeneration. This improvement was found in all experimental groups, with best outcome observed in group exposed to the strongest magnetic field. Also dorsal root ganglion survival rate and nerve regeneration intensity were significantly higher in the group treated with the strongest field (Suszynski et al., 2014). Magnetic fields used in treatment of peripheral nerve injury are shown in Table 3. Table 3 The use of magnetic fields in the treatment of peripheral nerve injuries Bio-laser stimulation: For the treatment of peripheral nerve injury, low energy biostimulation lasers are used, applied in the way of pulsatile (905 nm), continuous (808 nm), or pulsing-constant rays. Laser therapy increases the formation of ATP, and the energy of the ATP hydrolysis can be used by nerve cell to restore normal transmembrane potential, which facilitates the generation of electrical impulses and thereby restoring nerve conduction (bioelectric effect). Application of laser beams improves microcirculation and hence nutrition and regeneration of nerve cells – bio-stimulation effect – and increases the release of endorphins and the concentration of neurotransmitters in the synapses – analgetic effect. Laser radiation can also be used to rejoin the nerve stumps (Lanre method – laser-assisted nerve repair). Studies evaluating the use of this method show comparable or even more effective reconstruction then surgical treatment. Less scar formation is observed in the site of anastomosis, which creates favorable conditions for the regeneration of nerve fibers (Huang and Huang, 2006). There are also promising results of the coupled use of fiber membranes or Gore-Tex™ with laser beams. This aids in assembling the ends of the nerve, and affects the speed and efficiency of the regeneration process. Application of laser irradiation (Ga-As laser) in the site of the anastomosis inhibits the degeneration process, accelerate remyelination, and nerve function recovery (Bae et al., 2004; Miloro et al., 2002). One of the major complications of peripheral nerve damage is the formation of a neuroma at the end of the proximal stump. Biostimulation with CO2 or Neodymium-Yag lasers reduces the risk of its formation, or at least alleviates severe pain caused by the formation of a neuroma (Kuzbari et al., 1996). Therapeutic use of ultrasounds (US) gave some promising results in animal experiment (Raso et al., 2005). However, before this technique might be implemented in human therapy, it is indispensable to precisely elucidate the influence of the US on nervous tissue, as well as to determine the most effective and safest therapeutic protocol that could be used in clinical practice. In general, there is a lack of randomized, good quality data representing results of clinical application of particular therapeutic methods using standardized dozymetry. Current research encompassing treatment and intervention in nerve injuries is limited, consisting mostly of descriptive and exploratory studies. Especially nonsurgical or post-surgical physical therapy is poorly understood by many physical therapists and even physicians, many clinicians fail to recognize that such nerves often need considerable time to regenerate.

Experimental hyperthyroidism enhances the regeneration of central neurites promoted by peripheral nerve grafts in the hippocampus.

The aim of the present paper was to ascertain whether experimental hyperthyroidism promotes the regenerative action of predegenerated peripheral nerve grafts implanted into the transected hippocampus. Hyperthyroidism was induced by subcutaneous injections of T4. Autologous peripheral nerve grafts were implanted immediately, 7 and 35 days following transection of the sciatic nerve. Cells extending their neurites into the grafts were traced by means of horseradish peroxidase conjugated with fluoresceine isothiocyanate (FITC-HRP). Fluorescence microscope examination revealed that experimentally induced hyperthyroidism considerably enhanced the regenerative influence of peripheral nerve grafts. This effect was particularly pronounced in hyperthyrotic animals treated with either nonpredegenerated or 35 day predegenerated nerve grafts.

University Sports Association as the way to sport activity development among Silesian students

Introduction. There are two fundaments of healthy lifestyle philosophy: regular physical activity and proper diet. The development of physical activity among students is determined by many complex factors. We can distinguish certain aspects such as university profile, access to the sport facilities and the opportunity to participate in sport activities which are organized by sport clubs or University Sports Association. The aim of the study. The aim of the study was to analyze the factors that affect self-assessment of students physical activity. The following questions have been asked: 1.Is the choice of the university related to the access to sport activities which are organized by University Sport Association? 2.Does the participating in activities, which are organized by University Sport Association, help in physical development? 3.Is there any relation between the location, time, type, the way of sport activities which are offered by University Sport Association and participating in them? Material and methods. 395 students took part in research: 257 women (18-27 years old) (x=20,7 ; SD=1,4) and 108 men (18-26 years old) (x=21; SD=1,45). All the students are from five Silesian universities. Method used for the research was the anonymous questionnaire consisting of 37 questions which concerned: the way of the physical activity taken by students, the issue of University Sport Association and the influence of the university profile on physical activity development. The Likert scale was used in 21 questions. Statistical description included descriptive statistics. Degree of difference was defined by ANOVA analysis. Results. Descriptive analysis showed significant dependence between the type of the university and the location of sport facilities (p=0,04). The type of the university has no influence on the factors like: time (p=0,07) and type of sport activities offered by University Sport Association (p=0,39). There is also an important connection between the type of the university and resignation from doing sport (p=0,0002). Mostly – Medical University of Silesia students, least frequently – University of Silesia students. There is dependence between approach to doing sport among students and the type of university (p=0,01). The university which helps the most in sport career is University of Economics in Katowice. Conclusions: 1.Taking the physical activity by student depends on location of the university and sport facilities. 2.The factors such as time and type of sport activities are not related to the type of the university. 3.The type of the university influence the resignation from doing sport by students.