Early Stages Of Reinnervation Research Articles

Experimental Study of the Bone Marrow Aspirate Injection Effect into Denervated Muscle (Clinical Manifestations of Limb Denervation)

Relevance. Peripheral nerve injuries are potentially disabling lesions, which account for about 2-3% of all injuries. In order to study the clinical manifestations of limb denervation in the early and late stages, we conducted an experimental study.
 Objective: in the experiment, to study the effect of bone marrow aspirate injection into the target muscles on denervation-reinnervation processes by studying the clinical manifestations of denervation (presence of trophic ulcers, edema, or muscle wasting).
 Materials and Methods. The experiment was performed on 36 rabbits, which were divided into four groups: a group of pseudo-operated animals, group 1 (neurotomy and sciatic nerve suture), group 2 (on-time injection of bone marrow aspirate), and group 3 (delayed injection of bone marrow aspirate). Detection of hypotrophy or edema of the tibia was performed by determining the percentage of leg circumference of the operated on and intact limbs. Clinical manifestations of limb denervation were recorded during the examination of experimental animals before their euthanasia.
 Results. When comparing the number of complications in group 1 and group 2, more complications of the denervation process was observed in group 1. At the same time, no difference was found between group 1 and group 3, as well as between group 2 and group 3. When comparing the indicators of hypotrophy, the difference between group 1 and group 2, as well as a significant difference (p<0.05) between group 1 and group 3, with a predominance of hypotrophy in group 2 and group 3 were revealed.
 Conclusions. The injection of bone marrow aspirate into the target muscles during surgery and in the early stages of reinnervation helped to reduce the clinical manifestations of the denervation process. Delayed administration of bone marrow aspirate to target muscles significantly (p<0.05) helped to reduce edema of denervated target muscles.

STUDY OF BONE MARROW ASPIRATE INJECTION EFFECT ON DENERVATED MUSCLE ACCORDING TO ELECTROMYOGRAPHY STUDIES

The aim. To study in experiment the effect of bone marrow aspirate injection on the course of denervation-reinnervation processes in skeletal muscle using neurophysiological research method. Materials and methods. Experimental study was performed on 36 mature rabbits weighing 3-4 kg. Experimental model of the peripheral nerve acute injury and neurorrhaphy is taken as a basis. Animals were divided into 4 groups of 9 animals per group (3 animals for each experimental period). At 8, 12, and 16 weeks after surgery, all rabbits underwent needle EMG of the target muscles (m. gastrocnemius, and m. tibialis anterior) to study denervation-reinnervation changes. Results. Results of target muscles electromyographic examination are shown. As norm, we took motor unit potentials parameters, which were registered during the study of contralateral (not operated) pelvic limbs. Largest number of registered MUPs at different stages of the denervation-reinnervation process was observed, and was significantly higher (α = 0.07) in the groups in which bone marrow aspirate was injected in relation to group without bone marrow aspirate injection. Changes in the parameters of the registered MUPs in all groups corresponded to the general characteristics of the denervation-reinnervation process. Conclusions. Injection of bone marrow aspirate into the target muscles during surgery and in the early stages of reinnervation (in experimental study it is 7 weeks after surgery) – reliably (α = 0.07) promotes improvement of reinnervation processes in muscles, which is manifested by registration of more motor unit potentials.

Reinnervation of the soleus muscle by its own or by an alien nerve

The rates of reinnervation of the rat soleus muscle by its own or an alien nerve were compared using physiological and morphological criteria. The muscle's own nerve was more effective than the alien nerve in establishing functional connections with the original endplates. The relative ineffectiveness of the alien nerve during early stages of reinnervation was more apparent using physiological rather than morphological criteria. Five to six weeks after nerve injury there was no longer any difference between the muscles supplied by either nerve.

Transitional properties of afferents reinnervating mechanoreceptors in the human glabrous skin

The technique of percutaneous microneurography was used to record in man from single mechanoreceptive afferents during the early stages of reinnervation. Although response properties were in several way comparable to normal and to those of afferents studied long after nerve repair, two abnormalities were found: multiple receptive field innervation by single afferents (RA, SAI), and response fatigue with repeated stimulation (SAI). These abnormalities are considered transitional, disappearing when reinnervation is complete.

Changes in MEPP and EPP amplitude distributions in the mouse diaphragm during synapse formation and degeneration.

Miniature end-plate potential (MEPP) and end-plate potential (EPP) amplitude histograms were examined in the mouse diaphragm during degeneration, deterioration, re-innervation and neonatal development. MEPPs and EPPs were recorded with conventional electrophysiological techniques. Control MEPP amplitude distributions from mice 21-30 days old showed two classes of MEPPs. The larger class composed 80-90% of the MEPPs and formed a bell-shaped distribution (bell-MEPPs). The smaller class (skew-MEPPs) formed a skewed distribution with a peak 1/7 to 1/15 that of bell-MEPPs. Usually, MEPP amplitude distributions did not change during the course of nerve degeneration or during deterioration in the bath. MEPP amplitude distributions from newly re-innervated fibers were composed mainly of skew-MEPPs. At later stages of re-innervation the relative numbers of skew-MEPPs decreased. Many fibers from neonatal mice (2-3 days old) also showed mainly skew-MEPPs. Rise time vs amplitude plots were constructed from neonatal and re-innervating preparations. The skew-MEPP time-to-peak measurements fell on or below the regression line calculated from the time-to-peak data of the bell-MEPPs. This indicates that the skew-MEPPs originated from the same site as the bell-MEPPs. Unitary EPPs were recorded from neonatal and re-innervating preparations by reducing the evoked response with cobalt ions (4 mM). Distributions of unitary EPPs were similar to those of bell-MEPPs. It is concluded that there are two classes of spontaneous quanta. The skew-MEPP class dominates MEPP amplitude distributions during the early stages of re-innervation and early neonatal preparations. In all stages of development the unitary evoked EPPs have the same mean amplitude and time-to-peak as the bell-MEPPs. The data suggest that the skew class is not available for evoked release.

Transmitter release properties along regenerated nerve processes at the frog neuromuscular junction

Reinnervation following nerve crush was examined in the frog cutaneous pectoris muscle using histological and electrophysiological techniques. Functional recovery, as monitored by miniature endplate potential (mepp) frequency and quantal content, was slower than morphological recovery. To understand this discrepancy, extracellular recording electrodes were used to determine the amount of transmitter released from localized areas of nerve terminals in both unoperated and experimental muscles. There was no increased tendency over controls for the proximal portion of a regenerated nerve process to release more quanta (spontaneous or nerve evoked) than the distal portion. Moreover, extracellular recording at the growing nerve tip showed that this portion of nerve terminal was capable of transmitter release upon nerve stimulation. These results suggest that the entire length of a regenerating nerve terminal becomes functional at early stages of reinnervation and matures at essentially the same rate.

Loss of extrasynaptic acetylcholine sensitivity upon reinnervation of parasympathetic ganglion cells.

1. The extrasynaptic acetylcholine sensitivity of frog cardiac ganglion cells was measured both after denervation and during the early stages of reinnervation by preganglionic axons. Sensitivity was measured by ionophoretic application of acetylcholine (ACh) to randomly chosen sites on ganglion cell bodies. 2. Extrasynaptic sensitivity rose gradually following denervation and after 3 weeks reached a mean value of approximately 1000 mV/nC. 3. Reinnervation of the cardiac ganglion began about 3 weeks after nerve crush. The ACh sensitivity of ganglion cells fell markedly during the 23--31 day period, to a mean of 184 mV/nC. None of forty-three neurones studied during that period received synaptic inputs sufficient to generate action potentials. 4. Twenty-nine of the forty-three neurones examined 23--31 days after nerve crush had not yet received detectable synaptic inputs, yet even these cells had markedly reduced ACh sensitivity. 5. When reinnervation of cardiac ganglia was delayed by resecting the preganglionic nerves, ACh sensitivity was reduced slightly (43%) between 14--21 and 23--31 days after surgery. Thus, most of the sixfold reduction in sensitivity that occurs during this time after nerve crush is a specific effect of reinnervation. 6. We conclude that loss of extrasynaptic receptors coincides with, or may even precede, the earliest physiological signs of synapse formation. Restoration of action potential activity in the ganglion cells is not essential to initiate this loss.

Specificity of initial synaptic contacts made on guinea-pig superior cervical ganglion cells during regeneration of the cervical sympathetic trunk.

1. Largely appropriate synaptic connexions are formed with neurones in the superior cervical ganglion at long intervals after interruption of the preganglionic nerve. In the present study we have assessed the accuracy of connexions during the early stages of re-innervation by observing end-organ responses to ventral root stimulation in vivo, and by recording intracellularly from ganglion cells during ventral root stimulation in isolated preparations. 2. Appropriate, but weak, end-organ responses were elicited by stimulation of the first and fourth thoracic ventral roots (T1 and T4) 15--30 days after freezing the cervical sympathetic trunk. 3. Intracellular recordings from ganglion cells during stimulation of the ventral roots C8--T7 in vitro showed that synaptic contacts are first re-established 8--11 days after freezing the preganglionic nerve. The proportion of re-innervated cells, and the strength of innervation of individual neurones, increased rapidly for up to about 3 months after nerve injury, but showed little change thereafter. Innervation remained weaker than normal even after 6 months. 4. Patterns of segmental innervation recorded intracellularly during the early stages of regeneration were similar to, but more restricted than normal. Even 13--19 days after interruption of the preganglionic nerve, neurones re-innervated by more than one spinal cord segment tended to be innervated by a contiguous subset of the spinal segments which contribute innervation to the ganglion. The incidence of neurones receiving innervation from a discontinuous segmental subset was about the same at early and late stages or re-innervation. 5. Throughout the course of nerve regeneration, re-innervated neurones tended to receive dominant synaptic input from axons arising at a particular spinal level, as do normal cells, with adjacent segments contributing a synaptic influence that diminished as a function of distance from the dominant segment. 6. The results of these experiments argue against the initial formation of imprecise connexions with subsequent retention of appropriate contacts and a loss of inappropriate ones. Rather our findings suggest that the re-innervation of ganglion cells proceeds by a gradual accumulation of synaptic connexions which are, from the outset, appropriate.

Polyneuronal innervation of skeletal muscle in new‐born rats and its elimination during maturation.

1. The events taking place during the elimination of polyneuronal innervation in the soleus muscle of new-born rats have been studied using a combination of electrophysiological and anatomical techniques. 2. Each immature muscle fibre is supplied by two or more motor axons which converge on to a single end-plate. There was no sign of electrical coupling between muscle fibres receiving multiple synaptic inputs. By the end of the second week after birth virtually all muscle fibres are innervated by only a single motor axon. 3. The average tension produced by individual motor units, measured in terms of the percentage of the total muscle twitch tension, declined dramatically during the first 2 weeks after birth. During this period there was no significant change in the number of motor neurones innervating the soleus muscle. Thus, the disappearance of polyneuronal innervation reflects a decrease in the number of peripheral synapses made by each motor neurone. 4. The decline in motor unit size was delayed, but not ultimately prevented, by the early surgical removal of all but a few motor axons to the soleus muscle. This procedure also caused a delay in the removal of polyneuronal innervation involving the remaining motor units. 5. Following a crush of the soleus nerve in neonatal animals, regenerating axons usually returned to the original end-plates. Polyneuronal innervation was extensive at early stages of re-innervation and it disappeared during the second week after birth just as in normal muscles. 6. Cross-innervation of neonatal muscles by an implanted foreign nerve caused a rapid disappearance of cholinesterase at denervated original end-plates and in most fibres prevented re-innervation by the original nerve. In the small proportion of fibres that did become innervated through both the foreign and original nerves the end-plates were more than 1 mm apart, and both foreign and original nerve end-plates could persist indefinitely. 7. Many cross-innervated fibres received multiple inputs through the foreign nerve. Some foreign end-plates were separated by distances ranging up to 1 mm. Polyneuronal innervation through the foreign nerve was completely eliminated during maturation but over a slightly longer period than in normal muscles. Apparently the elimination process can act over a distance up to but not much more than 1 mm. 8. These observations suggest that there are several factors influencing the elimination of redundant inputs in immature muscles. Individual motor neurones appear to have an inherent tendency to withdraw the majority of their original complement of peripheral terminals. The determination of which particular synapses are to survive, however, seems to be made in the periphery by a selection among all the synapses that innervate a limited region of each muscle fibre. There may be a competitive interaction among synapses in which those belonging to smaller motor units are less likely to be eliminated, thereby leading to a relatively uniform size of the motor units in the soleus.

Complex end-plate potentials at the regenerating neuromuscular junction of the rat

Intracellular recordings of end-plate potentials (epps) were made in cut fiber preparations of reinnervating extensor digitorum longus muscles at various times after crushing the deep peroneal nerve. Beginning at 9 days after nerve crush, reinnervation was associated with the presence of multi-peaked epps. The percentage of fibers having such complex epps increased from 9% at 10 days to a maximum of 31% at 18 days. The complex epps could be fractionated into distinct peaks of different latencies by varying the intensity of nerve stimulation. Complex epps were also found in nerve muscle preparations that had been blocked with MgCl 2 or d-tubocurarine. In the presence of elevated MgCl 2 and lowered CaCl 2, the component peaks of complex epps failed separately. Similar complex epps were not found in control muscles. It is suggested that mammalian muscle fibers are multi-innervated during the early stages of reinnervation.

A study of the increased sensitivity of denervated and re‐innervated muscle to depolarizing drugs

1. The response of re-innervated muscles to depolarizing blocking drugs was studied.2. During the early stages of re-innervation the ;new' neuromuscular junctions are more sensitive to decamethonium and suxamethonium. The recovery from the block and the repolarization of the end-plate are markedly slowed.3. This increased sensitivity does not appear to be characteristic of all newly formed neuromuscular junctions. It was found that in new-born kittens the muscles are very insensitive to neuromuscular blocking drugs.4. It is concluded that the increased sensitivity of re-innervated muscles to depolarizing blocking drugs and the slow repolarization are due to a persistence of changes which developed during denervation.