Efficient gene transfer from innervated muscle into rat peripheral and central nervous systems using a non-viral haemagglutinating virus of Japan (HVJ)-liposome method

We evaluated the feasibility of gene delivery into the peripheral and central nervous systems via retrograde axonal transport following injection of a haemagglutinating virus of Japan (HVJ)-liposome-DNA complex vector into an innervated muscle. Transfection efficiency was assessed by measuring luciferase activity, and was compared statistically with that achieved using a liposome-DNA control vector. High luciferase activity was observed in the injected muscle, the ipsilateral sciatic nerve, and the ipsilateral dorsal root ganglia on day 1 after gene transfer. The spinal cord also showed luciferase activity, although this was lower than in the other tissues. However, no activity was observed in the contralateral sciatic nerve or the contralateral dorsal root ganglia. In addition, we performed gene transfer twice, with a 1-week interval, to evaluate the feasibility of repeated therapeutic gene delivery. Again, a high transfection efficiency was observed immediately, even after the second gene transfer, and transfection efficiency was significantly higher at each defined time-point using the HVJ-liposome complex vector than using a control vector. These results indicate that this method could be used for repeated therapeutic gene delivery into muscle, nerve, dorsal root ganglia, and possibly spinal cord, without the need for a surgical approach, making it well suited to clinical applications.     

Biophysical and functional consequences of receptor-mediated nerve fiber transformation.

Stimulation of the nervous system by substance P, a G protein-coupled receptor, and subsequent receptor internalization causes dendrites to change their shape from homogeneous cylinders to a heterogeneous string of swollen varicosities (beads) connected by thin segments. In this paper we have analyzed this phenomenon and propose quantitative mechanisms to explain this type of physical shape transformation. We developed a mathematical solution to describe the relationship between the initial radius of a cylindrical nerve fiber and the average radii of the subsequently created varicosities and connecting segments, as well as the periodicity of the varicosities along the nerve fiber. Theoretical predictions are in good agreement with our own and published experimental data from dorsal root ganglion neurons, spinal cord, and brain. Modeling the electrical properties of these beaded fibers has led to an understanding of the functional biophysical consequences of nerve fiber transformation. Several hypotheses for how this shape transformation can be used to process information within the nervous system have been put forth.     

Results and current approach for Brachial Plexus reconstruction

We review our experience treating 335 adult patients with supraclavicular brachial plexus injuries over a 7-year period at the University of Southern Santa Catarina, in Brazil. Patients were categorized into 8 groups, according to functional deficits and roots injured: C5-C6, C5-C7, C5-C8 (T1 Hand), C5-T1 (T2 Hand), C8-T1, C7-T1, C6-T1, and total palsy. To restore function, nerve grafts, nerve transfers, and tendon and muscle transfers were employed. Patients with either upper- or lower-type partial injuries experienced considerable functional return. In total palsies, if a root was available for grafting, 90% of patients had elbow flexion restored, whereas this rate dropped to 50% if no roots were grafted and only nerve transfers performed. Pain resolution should be the first priority, and root exploration and grafting helped to decrease or eliminate pain complaints within a short time of surgery.     

Limb congestion enhances the synchronization of sympathetic outflow with muscle contraction

In this report, we examined if the synchronization of muscle sympathetic nerve activity (MSNA) with muscle contraction is enhanced by limb congestion. To explore this relationship, we applied signal-averaging techniques to the MSNA signal obtained during short bouts of forearm contraction (2-s contraction/3-s rest cycle) at 40% maximal voluntary contraction for 5 min. We performed this analysis before and after forearm venous congestion; an intervention that augments the autonomic response to sustained static muscle contractions via a local effect on muscle afferents. There was an increased percentage of the MSNA noted during second 2 of the 5-s contraction/rest cycles. The percentage of total MSNA seen during this particular second increased from minute 1 to 5 of contraction and was increased further by limb congestion (control minute 1 = 25.6 +/- 2.0%, minute 5 = 32.8 +/- 2.2%; limb congestion minute 1 = 29.3 +/- 2.1%, minute 5 = 37.8 +/- 3.9%; exercise main effect <0.005; limb congestion main effect P = 0.054). These changes in the distribution of signal-averaged MSNA were seen despite the fact that the mean number of sympathetic discharges did not increase over baseline. We conclude that synchronization of contraction and MSNA is seen during short repetitive bouts of handgrip. The sensitizing effect of contraction time and limb congestion are apparently due to feedback from muscle afferents within the exercising muscle.     

An anomalous muscle (accessory subscapularis-teres-latissimus muscle) in the axilla penetrating the brachial plexus in man.

An anomalous muscle passing through the brachial plexus was found in 10 cases out of 380 sides of 190 human cadavers in the dissection course. The muscle was designated as 'accessory subscapularis-teres-latissimus muscle'. This muscle arose near the lateral margin of the scapula, either from the surface of the subscapularis muscle or from the border of the quadrangular terminal tendon of the latissimus dorsi or from both of those sources when the muscle was divided into two heads. It ran obliquely upward to fuse with the insertion of the subscapularis. The largest anomaly was 2.5 cm in width and 7 cm in length. This muscle could be classified into three types on the basis of its nerve supply and its relation to the brachial plexus. The type I muscle crossed over the axillary and lower subscapular nerves, behind the radial nerve and was innervated by the lower subscapular nerves. The type II musclepenetrated the brachial plexus separating the radial nerve into two roots; the upper from the posterior division of the upper trunk and the lower from the posterior divisions of the middle and lower trunks. The type II muscle was supplied by a branch of the radial nerve, which originated always at the same level as the origin of the thoracodorsal nerve. The type III muscle passed through the further more ventrocaudal level of the plexus; in one case it divided the radial nerve into an upper root from the posterior divisions of the upper and middle trunks and a lower root from the lower trunk, and, in another case, into an upper main root from all the three trunks and a lower slender root from the lower trunk. The type III muscle was supplied by branches from the radial and in addition from the thoracodorsal nerve in one case. In four out of ten cases, the subscapular or thoracodorsal artery also passed posterior to the anomalous muscle. A discussion was made on the nature of the anomalous muscle.     

Task-dependent inhomogeneous muscle activities within the bi-articular human rectus femoris muscle

The motor nerve of the bi-articular rectus femoris muscle is generally split from the femoral nerve trunk into two sub-branches just before it reaches the distal and proximal regions of the muscle. In this study, we examined whether the regional difference in muscle activities exists within the human rectus femoris muscle during maximal voluntary isometric contractions of knee extension and hip flexion. Surface electromyographic signals were recorded from the distal, middle, and proximal regions. In addition, twitch responses were evoked by stimulating the femoral nerve with supramaximal intensity. The root mean square value of electromyographic amplitude during each voluntary task was normalized to the maximal compound muscle action potential amplitude (M-wave) for each region. The electromyographic amplitudes were significantly smaller during hip flexion than during knee extension task for all regions. There was no significant difference in the normalized electromyographic amplitude during knee extension among regions within the rectus femoris muscle, whereas those were significantly smaller in the distal than in the middle and proximal regions during hip flexion task. These results indicate that the bi-articular rectus femoris muscle is differentially controlled along the longitudinal direction and that in particular the distal region of the muscle cannot be fully activated during hip flexion.     

Dropped head syndrome due to myogenic atrophy --- a case report of surgical treatment

Osteoid osteoma of the spine is a relatively rare bone-forming tumor. Pain that is worse at night and relieved by aspirin and muscle contracture are the most characteristic symptoms of spinal osteoid osteoma. Although radicular pain occasionally occurs in spinal osteoid osteoma, spinal cord and nerve root compression is absent in most cases. Although radicular pain appears to be associated with tumorous inflammation, there have been no presentations of histological findings of inflammation around the nerve root. We present here two rare cases of spinal osteoid osteoma causing radiculopathy and the first histological evidence of tumorous inflammation as a cause of radiculopathy in osteoid osteoma near the intervertebral foramen.     

Identification of genetic factors controlling the efficiency of Agrobacterium rhizogenes-mediated transformation in Brassica oleracea by QTL analysis

We have identified quantitative trait loci (QTL) for transgenic and adventitious root production using an Agrobacterium rhizogenes-mediated co-transformation system in conjunction with a Brassica oleracea double haploid (DH) mapping population. Three QTL for green fluorescent protein (GFP)-fluorescent root production and four QTL for adventitious root production were identified as accounting for 26% and 32% of the genetic variation in the population, respectively. Two of the QTL regions identified were common to both transgenic and adventitious root production. Two different methods of QTL analysis were employed (marker regression and interval mapping) and with the exception of one region on linkage group O7 for transgenic root production, both techniques detected the same regions of the genome. The regions we identified to be associated with the control of transgenic root production following A. rhizogenes-mediated transformation are the first to be detected using a QTL mapping approach. In addition, this is the first study to identify genetic regions that co-regulate both transgenic and adventitious root production within the constraints of an A. rhizogenes-mediated transformation process. We have identified plant genotypes that do not produce any transgenic roots that may be deficient for T-DNA integration via illegitimate recombination and that may also be potentially important for the development of homologous recombination protocols. Conversely, we have also identified plant genotypes with high rates of transgenic root production that will be critical in the development of high throughput transformation systems.     

Adult monkey coronoid process after resection of trigeminal nerve motor root.

A smaller or absent coronoid process has been reported, by some investigators but not by others, in growing animals following resection of the temporalis muscle. The trauma of resection, altered function, hemorrhage, scar tissue, and changes in vascularity may have influenced the results. The purpose of this experiment was to observe in adult Macaca mulatta the fully grown coronoid process after decreasing or eliminating neurofunctional activity of the temporalis muscle unilaterally without the trauma of local resection. In two males and three females the motor root of the trigeminal nerve which innervates the temporalis muscle was resected intracranially. In three control animals of both sexes the same surgical procedure was performed except for resection of the nerve. At postmortem, one year later, the temporalis muscle mass was atrophic on the resected side. There were no significant morphological differences, however, between the right and left sides of the mandible, including the coronoid process, regardless of which motor root of the fifth nerve had been resected, which side had been sham-operated, or sex. An extensive deposit of calculus on the buccal surfaces of the teeth on the operated nerve side was a consistent, conspicuous finding.     

A giant plexiform schwannoma of the brachial plexus: case report

We report the case of a patient who noticed muscle weakness in his left arm 5 years earlier. On examination, a biloculate mass was observed in the left supraclavicular area, and Tinel's sign caused paresthesia in his left arm. Magnetic resonance imaging showed a continuous, multinodular, plexiform tumor from the left C5 to C7 nerve root along the course of the brachial plexus to the left brachia. Tumor excision was attempted. The median and musculocutaneous nerves were extremely enlarged by the tumor, which was approximately 40 cm in length, and showed no response to electric stimulation. We resected a part of the musculocutaneous nerve for biopsy and performed latissimus dorsi muscle transposition in order to repair elbow flexion. Morphologically, the tumor consisted of typical Antoni A areas, and immunohistochemistry revealed a Schwann cell origin of the tumor cells moreover, there was no sign of axon differentiation in the tumor. Therefore, the final diagnosis of plexiform Schwannoma was confirmed.     

Extracellular recordings from the motor nervous system of the nematode,Ascaris suum

1. The close association of muscle and neurons in Ascaris suum makes it difficult to determine whether spikes recorded from nerve cords originate in muscle or neurons. We have developed criteria that distinguish muscle and neuronal activity. There are two categories of extracellular spikes. 2. The first category consists of spikes with a wide range of amplitudes, marked by large spikes. These spikes, which can be recorded over lateral muscle and over the dorsal and ventral nerve cords, are abolished when muscle is disrupted or removed, or when curare is applied. Large spikes are relatively infrequent, are correlated with intracellularly recorded muscle events, and respond to polarizations of motor neurons, implying that they originate in muscle. 3. The second spike category, small amplitude spikes, is exclusive to the ventral nerve cord, occurs more frequently than large spikes and displays patterned firing. Small spikes are not affected by muscle removal or by curare, and are correlated with motor neuronal post-synaptic potentials, but not with intracellularly recorded muscle events. We infer that they originate in neurons. 4. Low level activity recorded extracellularly over nerve cords may represent muscle activity due to tonic motor neuronal synaptic transmission. It responds to motor neuronal polarization and is suppressed by curare or muscle removal.     

Neurovascularized free short head of the biceps femoris muscle transfer for one-stage reanimation of facial paralysis

The single-stage technique for cross-face reanimation of the paralyzed face without nerve graft is an improvement over the two-stage procedure because it results in early reinnervation of the transferred muscle and shortens the period of rehabilitation. On the basis of an anatomic investigation, the short head of the biceps femoris muscle with attached lateral intermuscular septum of the thigh was identified as a new candidate for microneurovascular free muscle transfer. The authors performed one-stage transfer of the short head of the biceps femoris muscle with a long motor nerve for reanimation of established facial paralysis in seven patients. The dominant nutrient vessels of the short head were the profunda perforators (second or third) in six patients and the direct branches from the popliteal vessels in one patient. The recipient vessels were the facial vessels in all cases. The length of the motor nerve of the short head ranged from 10 to 16 cm, and it was sutured directly to several zygomatic and buccal branches of the contralateral facial nerve in six patients. One patient required an interpositional nerve graft of 3 cm to reach the suitable facial nerve branches on the intact side. The period required for initial voluntary movement of the transferred muscles ranged from 4 to 10 months after the procedures. The period of postoperative follow-up ranged from 5 to 42 months. Transfer of the vascularized innervated short head of the biceps femoris muscle is thought to be an alternative for one-stage reconstruction of the paralyzed face because of the reliable vascular anatomy of the muscle and because it allows two teams to operate together without the need to reposition the patient. The nerve to the short head of the biceps femoris enters the side opposite the vascular pedicle of the muscle belly, and this unique relationship between the vascular pedicle and the motor nerve is anatomically suitable for one-stage reconstruction of the paralyzed face. As much as to 16 cm of the nerve can be harvested, and the nerve is long enough to reach the contralateral intact facial nerve in almost all cases. The lateral intermuscular septum, which is attached to the short head, provides "anchor/suture-bearing" tissue, allowing reliable fixations to the zygoma and the upper and lower lips to be achieved. In addition, the scar and deformity of the donor site are acceptable, and loss of this muscle does not result in donor-site dysfunction.     

Morphology and monoaminergic modulation of Crustacean Hyperglycemic Hormone-like immunoreactive neurons in the lobster nervous system

Neuronal somata located near branch points in the second thoracic nerve roots of the lobster are immunoreactive for Crustacean Hyperglycemic Hormone (CHH)-like peptides, a family of putative stress hormones. We have employed intracellular dye injection, immunostaining, and confocal imaging to observe the anatomy of these root neurons, which are morphologically diverse and dye coupled. Some root neurons contribute to neurosecretory structures at the points of exit of the root from the nerve cord. Other CNS-projecting root neurons send projections into the T5-A1 interganglionic connectives. Neurosecretory elements of the serotonin (5HT) and octopamine (OCT) systems, implicated in postural control and aggression, terminate densely in the vicinity of the second thoracic root neurons. We have confirmed by double immunostaining for 5HT and CHH-like peptides that the endings of the 5HT neurons are in close apposition to root neurons in the superficial regions of the root. We have also extended previous studies documenting electrophysiological responses of the root neurons to 5HT or OCT. Bath-applied 5HT and OCT inhibit the spontaneous bursting activity of root neurons at concentrations higher than 100 nM. The root neurons desensitize to the persistent presence of high concentrations of 5HT, but not OCT, in the bath. Nanomolar concentrations of OCT, but not 5HT have an excitatory effect on the spontaneous bursting activity of root neurons. This region of the lobster nervous system is of continuing interest, as identified neurons of three neuromodulatory systems implicated in stress and aggression converge and interact at the level of identified neurons.     

Regeneration studies on a crayfish neuromuscular system. I. Connectivity changes after intersegmental nerve transplants

The superficial flexor muscles of the crayfish are a neuromuscular system of a few muscle cells innervated by six neurons in a precise position-dependent pattern. The neurons are capable of regenerating their normal connectivity patterns within a short span of time when conditions are favorable. The superficial flexor muscles of the second and third segments, despite their similarities in neuronal and muscle cell size and number, have distinctive connectivity patterns; some homologous neurons form similar patterns but other homologous neurons form patterns that are reversed between segments. We transplanted each segment's nerve into each other's muscle in order to observe regeneration of the nerves into a target area that differed in connectivity patterns from their original muscle. During the first weeks of regeneration all neurons formed a connectivity pattern with more connections medially and declining connections laterally, a pattern determined by the medial location of the nerve transplant. This pattern is maintained for most of the neurons, but for some there is an eventual reduction in medial connections as maximum synapse formation shifts to the lateral muscle fibers. Three of the eight neurons studied were able to regenerate connectivity patterns that corresponded to their segment of origin and not to their host muscle. This suggests that intersegmental muscle differences are not influencing the formation of these connectivity patterns, so the neurons will follow their inherent synaptogenesis program.     

Cortical reorganization in training.

Plasticity within the human central motor system occurs and has been studied with transcranial magnetic stimulation in patients with amputations, spinal cord injuries, and ischemic nerve block. These studies have identified a pattern of motor system reorganization that results in enlarged muscle representation areas and large motor evoked potentials (MEPs) for muscles immediately proximal to the lesion. Some of these changes are apparent minutes after ischemic nerve block, weeks after spinal cord injury, and as early as six months after amputation.These studies motivated us to study the cortical motor reorganization after finger movement training in normals and after anastomosis of intercostal nerves to the musculocutaneous nerve in young patients with cervical root avulsions due to a traumatic motorcycle injury.     

Retrograde transport of NGF by early chick embryo spinal cord motoneurons

Neuronal retrograde transport of nerve growth factor (NGF) was examined in chick embryos at 5, 6, and 7 days of incubation. Radiolabeled NGF was injected in the target limb muscle and the retrograde transport was viewed following processing for autoradiography. Silver grains were localized in the peripheral nerve, in the ventral root, in neuronal cell bodies within the dorsal root ganglion, and in motoneurons of the lateral motor column. Comparable injections of 125I-cytochrome c resulted in the presence of label at the peripheral injection site only. The possible developmental significance of these observations is discussed.     

Degeneration of the afferent fibers simulating the effects of motor denervation on skeletal muscle

Degeneration of afferent nerve fibres was induced in rats in order to observe its effects on the properties of the extra-junctional membrane of soleus muscle fibres. In one approach, removal of dorsal root ganglia L4 and L5 was accomplished in preparations with intact or impulse-blocked (with tetrodotoxin containing cuffs around the sciatic nerve) efferent innervation. Spike resistance to tetrodotoxin developed in the inactive deafferented preparations earlier and to a greater extent than in control, that is only impulse-blocked, preparations. In another series of experiments, efferent denervation alone proved to be less effective than the association of efferent and afferent denervation. On the other hand, section of the afferent fibres central to the dorsal root ganglia was without effect. These results are consistent with the interpretation that products of nerve degeneration contribute together with inactivity to the development of the extrajunctional membrane changes observed in skeletal muscle after denervation.     

Functional and morphometric evaluation of end-to-side neurorrhaphy for muscle reinnervation

This study was undertaken to quantify the effect of motor collateral sprouting in an end-to-side repair model allowing end organ contact. Besides documentation of the functional outcome of muscle reinnervation by end-to-side neurorrhaphy, this experimental work was performed to determine possible downgrading effects to the donor nerve at end organ level. In 24 female New Zealand White rabbits, the motor nerve branch to the rectus femoris muscle of the right hindlimb was dissected, cut, and sutured end-to-side to the motor branch to the vastus medialis muscle after creating an epineural window. The 24 rabbits were divided into two groups of 12 each, with the second group receiving additional crush injury of the vastus branch. After a period of 8 months, maximum tetanic tension in the reinnervated rectus femoris and the vastus medialis muscles was determined. The contralateral healthy side served as control. The reinnervated rectus femoris muscle showed an average maximum tetanic force of 24.9 N (control 26.2 N, p = 0.7827), and the donor- vastus medialis muscle 11.0 N (control 7.3 N, p = 0.0223). There were no statistically significant differences between the two experimental groups (p = 0.9914). The average number of regenerated myelinated nerve fibers in the rectus femoris motor branch was 1,185 +/- 342 (control, 806 +/- 166), and the mean diameter was 4.6 +/- 0.6 microm (control, 9.4 +/- 1.0 microm). In the motor branch to the vastus medialis muscle, the mean fiber number proximal to the coaptation site was 1227 (+/-441), and decreased distal to the coaptation site to 795 (+/-270). The average difference of axon counts in the donor nerve proximal to distal regarding the repair site was 483.7 +/- 264.2. In the contralateral motor branch to the vastus medialis muscle, 540 (+/- 175) myelinated nerve fibers were counted. In nearly all cross-section specimens of the motor branch to the vastus medialis muscle, altered nerve fibers could be identified in one fascicle distal and proximal to the repair site. The results show a relevant functional reinnervation by end-to-side neurorrhaphy without functional impairment of the donor muscle. It seems to be evident that most axons in the attached segment were derived from collateral sprouts. Nonetheless, the present study confirms that end-to-side neurorrhaphy is a reliable method of reconstruction for damaged nerves, which should be applied clinically in a more extended manner.