Generation of pure cultures of autologous Schwann cells by use of biopsy specimens of the dorsal cutaneous branches of the cervical nerves of young adult dogs.

OBJECTIVE To identify an optimal technique for isolation, purification, and amplification of Schwann cells (SCs) from biopsy specimens of the dorsal cutaneous branches of the cervical nerves of dogs. SAMPLE Biopsy specimens of dorsal cervical cutaneous nerves from the cadavers of three 1- to 2-year-old dogs. PROCEDURES Nerve specimens were dissected, predegenerated, and dissociated to isolate single cells. After culture to enhance SC growth, cells were immunopurified by use of magnetic beads. Cell purity was evaluated by assessing expression of cell surface antigens p75 (to detect SCs) and CD90 (to detect fibroblasts). Effects of various concentrations of recombinant human glial growth factor 2 (rhGGF2) on SC proliferation were tested. Cell doubling time was assessed in SC cultures with selected concentrations of rhGGF2. RESULTS Mean ± SD wet weight of nerve fascicles obtained from the biopsy specimens was 16.8 ± 2.8 mg. A mean predegeneration period of 8.6 days yielded approximately 6,000 cells/mg of nerve tissue, and primary culture yielded 43,000 cells/mg of nerve tissue in a mean of 11 days, of which 39.9 ± 9.1% expressed p75. Immunopurification with magnetic beads yielded a mean of 85.4 ± 1.9% p75-positive cells. Two passages of subculture with 10µM cytosine arabinoside further enhanced SC purity to a mean of 97.8 ± 1.2% p75-positive cells. Finally, rhGGF2 supplementation at a range of 40 to 100 ng/mL increased the SC proliferation rate up to 3-fold. CONCLUSIONS AND CLINICAL RELEVANCE SCs could be cultured from biopsy specimens of dorsal cervical cutaneous nerves and purified and expanded to generate adequate numbers for autologous transplants to treat dogs with spinal cord and peripheral nerve injuries.

Histological description of the interaction between muscle fibers and connective tissue of the fascia of the human trapezius muscle / Descripción histológica de la interacción entre las fibras musculares y tejido conectivo de la fascia del músculo trapecio humano

The skeletal muscle fascia corresponds to a condensation of connective tissue. Fascias are highly innervated and sensitive, and can cover non-expandable structures as well as musculature. It is suggested that fascias have a pivotal role in functions such as postural regulation, peripheral motor coordination and proprioception. Also, the presence of inflammation and microcalcification in fascia of patients with localized muscle pain has been described, suggesting a pathogenic role in pain. The aim was to describe the histological structure of the external deep fascia of the trapezius muscle, with emphasis on the content and arrangement of muscle fibers, type I collagen, and adipose tissue. Sample material was obtained from a male cadaver (60-70 years old), by dissection of the posterior cervical region of the superficial fascia of the trapezius muscle and fixed in buffered formalin. Samples were processed by routine histological techniques and embedded in paraffin, obtaining 5 µm-thick sections that were stained according to the van Gieson technique. The trapezius fascia is composed of type I collagen, organized into high-density collagen bundles and oriented in different directions, and by adipocytes disposed in longitudinal groups on the main axis of the fascia. Muscle fibers are organized into bundles that are inserted laterally on the thickness of the fascia. It is possible that lateral transmission of tensional forces between the fibers might be present.

La fascia del músculo esquelético corresponde a una condensación de tejido conectivo. Las fascias están inervadas y son sensibles y pueden cubrir estructuras no distensibles, así como las fibras musculares esqueléticas. Tienen un rol importante en la regulación de la postura, la coordinación motora periférica y la propiocepción. Además, se ha descrito la presencia de inflamación y microcalcificaciones en la fascia de los pacientes con dolor muscular localizado, lo que sugiere un rol patogénico en el dolor. El objetivo del trabajo fue describir la estructura histológica de la fascia externa profunda del músculo trapecio, con énfasis en el contenido y la disposición de las fibras musculares, colágeno tipo I y el tejido adiposo. Material y métodos: El material de la muestra fue obtenido de un cadáver de sexo masculino (60-70 años), por la disección de la región cervical posterior de la fascia superficial del músculo trapecio e inmediatamente fijado en formalina tamponada (pH 7,2) durante 48 horas. La muestra fue procesada por técnicas histológicas e impregnada en parafina (punto de fusión 56-58 C). Secciones de 5 µm de espesor fueron montadas en láminas silanizada para el desarrollo del protocolo de la técnica de van Gieson. Resultados y discusión: Se observa que la fascia del trapecio está compuesta por tejido conectivo denso irregular con abundante colágeno tipo I, organizado en paquetes grandes como verdaderos haces de colágeno de alta densidad orientada en diferentes direcciones; y por adipocitos dispuestos en grupos longitudinales en el eje principal de la fascia. Las fibras musculares estriadas están organizadas en paquetes que se insertan lateralmente en el espesor de la fascia. Es posible que la transmisión lateral de la tensión entre las fibras esté presente.

Nerve communication between the glossopharyngeal nerve, external carotid plexus and the superficial cervical ansa: human autopsy case.

The authors encountered a very rare human autopsy case in which the supernumerary branch of the glossopharyngeal nerve and a nerve branch arising from the external carotid plexus communicated with the superficial cervical ansa. This anomaly was observed on the left side of a 71-year-old male cadaver during the gross anatomical seminar at Niigata University in 2004. The nerve fascicle and fiber analyses indicated that the supernumerary branch of the glossopharyngeal nerve separated cranial to the branches to the pharyngeal constrictor muscles, carotid sinus and stylopharyngeal muscle and sent the nerve fibers to the muscular branches to the platysma and the cutaneous branches to the cervical region. Additionally, it was shown that the branch arising from the external carotid plexus sent the nerve fibers to the cutaneous branch to the cervical region. Although the external carotid plexus is primarily postganglionic sympathetic fibers originating from the superior cervical ganglion, the vagus and glossopharyngeal nerves gave off branches connecting to the plexus, and therefore it was not possible to determine the origins of this branch of the external carotid plexus. The present nerve fascicle analysis demonstrates that the supernumerary branch of the glossopharyngeal nerve, which innervated the platysma, did not share any nerve components with the branches to the pharyngeal constrictor muscles, carotid sinus and stylopharyngeal muscle, suggesting that this supernumerary branch may be categorized into the different group from these well-known branches.

Modulation of human cervical premotoneurons during bilateral voluntary contraction of upper-limb muscles.

A greater proportion of the voluntary command to the affected upper limb of well-recovered stroke patients descends via the putative C3/4 propriospinal system compared with the unaffected limb or the limbs of control subjects. We have examined whether there are differences in the pattern and extent of the modulation in intact subjects when the upper-limb muscles are bilaterally activated compared with unilateral activation. Motor evoked potentials (MEPs) recorded from flexor carpi radialis (FCR) in response to transcranial magnetic stimulation (TMS) were assessed for an effect of cocontraction with biceps brachii (BB) and subthreshold electrical conditioning of the musculocutaneous (MC) nerve. No between-hand differences were revealed, but there was less facilitation during bilateral than unilateral contraction. The findings suggest that the C3/4 propriospinal system is more active during unilateral muscle contraction and less active during bilateral contraction of upper-limb muscles. Therefore, bimanual rehabilitation strategies for patients with upper-limb hemiparesis should be developed with caution.

GABA-, glycine-, and glutamate-immunoreactive bouton profiles in apposition to neurons of the central cervical nucleus in the rat.

The neurons of the central cervical nucleus (CCN) convey information about the position and movements of the head, and receive excitatory input from dorsal neck muscles and the labyrinth. Both of these afferent sources form glutamatergic synaptic contacts with CCN neurons. However, these sensory afferent sources can also inhibit CCN neurons. To further elucidate the synaptic organization, we made an electron microscopic investigation, identifying and evaluating the relative frequency of bouton profiles containing the inhibitory transmitters GABA and glycine in apposition to identified CCN neurons. In addition, labeling for glutamate was performed. The identification of the CCN neurons was made possible by injections of retrograde tracer substances into the cerebellum. These substances were made visible by preembedding immunocytochemistry or postembedding immunogold staining. Such staining was also used to detect the three amino acids that were found in boutons apposed to the identified neurons (cf. Ornung et al., J. Comp. Neurol. 1996;365:413-426; Lindå et al., J. Comp. Neurol. 2000;425:10-23). Due to the relatively poor transport of the tracer substances into dendrites of the CCN neurons, the analysis was restricted to the cell body and included bouton profiles in direct apposition to the soma membrane. Data from 10 CCN neurons revealed that about 50% of the apposing bouton profiles were immunoreactive for GABA, and about 34% for glycine. In four neurons, the degree of colocalization of GABA and glycine was determined to be close to 30%. Thus, the vast majority of glycine-labeled profiles also contained GABA, while a considerable fraction of the profiles were immunoreactive for only GABA. The values for glycine immunoreactive bouton profiles presented here may represent somewhat low estimates, depending on the method used. Data from four neurons showed that about 18% of the profiles were labeled for glutamate. The large fraction of purely GABA immunoreactive profiles, or at least a substantial group of them, is suggestive of their derivation from axons descending from the brainstem.

Distribution of sensory neurons of ventral and dorsal cervical cutaneous nerves in dorsal root ganglia of adult rat--a double-label study using DiO and DiI.

To examine distribution of sensory neurons of ventral and dorsal cervical cutaneous nerves in dorsal root ganglia (DRGs), DiO and DiI tracers were applied at the proximal section of nerves (transverse superficial cervical and anterior supraclavicular nerves were selected as ventral cervical cutaneous nerves; dorsal cutaneous branches of second, third and fourth cervical nerves were selected as dorsal cervical cutaneous nerves). Located distributions were observed in DRGs of C2, C3, and C4 (25/46 DRGs). Sensory neurons of the ventral cervical cutaneous nerves were distributed in dorso-lateral or dorso-medial portions; neurons of dorsal cervical cutaneous nerves were distributed in ventro-medial or ventro-lateral portions of DRGs. Moreover, sensory neurons of transverse superficial cervical and anterior supraclavicular nerves were mainly distributed from the caudal half of C2 to whole part of C4 DRGs. Results show that there is a tendency for located distribution in two group sensory neurons; also, sensory neurons of ventral cervical cutaneous nerves have a segmental distribution, which has been verified in the brachial and lumbar plexus.

Ultrastructural study of external cuneothalamic neurons and their synaptic relationships with primary afferents in the gerbil.

The present study examined the synaptic organization of external cuneothalamic neurons and their relationships with primary afferents in the gerbil external cuneate nucleus (ECN) following an injection of horseradish peroxidase (HRP) into the anterodorsal cap of the ventrobasal thalamus in conjunction with a simultaneous injection of HRP into the contralateral brachial and cervical nerve plexuses. The thalamus-projecting neurons have been shown to be confined to the intermediate portion of the caudal half of the ECN at the light microscopic level (Lan et al., 1994c). In this study, HRP-labelled external cuneothalamic neurons were ultrastructurally characterized by their relatively small-sized soma bearing a variable number of somal spines. Their nucleus had a slightly indented contour with an eccentric nucleolus. The HRP-labelled somata were postsynaptic to many axon terminals, which were classified into round (Rs type; 53.0%), pleomorphic (Ps type; 32.7%), and flattened (Fs type; 14.3%) vesicle-containing boutons. The HRP-labelled dendritic elements were postsynaptic to a greater number of axon terminals, which were also classified into the round (Rd; 64.7%), pleomorphic (Pd; 25.2%), and flattened (Fd; 10.1%) type boutons. These presynaptic axonal boutons tended to synapse on distal and secondary dendrites of external cuneothalamic neurons. In the present simultaneous HRP labelling study, some of the primary afferent terminals made direct synaptic contacts with the dendrites of the external cuneothalamic neurons. In view of the multiple inputs onto the external cuneothalamic neurons, impinging particularly on their somata and secondary dendrites, it is suggested that the proprioceptive information reaching these neurons is intensively modulated and integrated before transmission ultimately to the cerebral sensorimotor cortex.

[Development of neurons in dissociated culture of the cranial cervical ganglion]. / Razvitie neironov v dissotsiirovannoi kul'ture kranial'nogo sheinogo gangliia.

Development of the sympathetic neurons of the dissociated cranial cervical ganglia taken from newborn rats has been studied in tissue culture. Vital microscopical, histological, histochemical and electrophysiological methods have been used, as well as an automatic morpho- and cytospectrometrical computer analysis. Active processes of the neurocyte regeneration and development begin on the 3d-4th day of cultivation. Average areas of the cellular body optic cross section increase more than two times, and an average size of nuclei-1.8 times. Some processes appear. An active synthesis of catecholamines takes place, its location coinciding with the forming chromatophilic substance zones. The end of the first and the beginning of the second week of cultivation is characterized with an active growth of processes, with formation of a complex neuropil, with differentiation of the neurite terminal parts. Catecholamines are displaced towards the processes. A spike activity to the intracellular electric stimulation appears. The first intraneuronal contacts are established. At the end of the second and during the third week the cells reach their maximal size. The ratio of the cytoplasm cross section area to the nuclear cross section reaches its constant level. The catecholamines concentration in the neuronal soma corresponds to its maximal values. Chromatophilic substance and the neurofibrillar apparatus are formed. On the neuronal soma pericellulars are formed. There are not any essential changes after the third week of incubation.

[Information analysis of neurocytes of various peripheral ganglia, and critical periods of their differentiation]. / Informatsionnyi analiz neirotsitov nekotorykh perifericheskikh gangliev i kriticheskie periody ikh differentsirovki.

Calculation of the informatory parameters of entropy and redundancy performed, taking as a base nuclear-cytoplasmic relations of neurocytes in the inferior ganglion of the vagus nerve, in the superior ganglion and in the interorganic ganglia of the respiratory tube, demonstrated certain interconnection of the integral characteristics obtained with some stages of the neuronal morphological differentiation. Periods of an abrupt reduction in the redundant information up to the minimal values which goes in parallel with an increasing entropy on the 2d-4th months of embryogenesis (taking into account heterochronia in the development of various ganglia) are considered as some critical periods in the neuron development when it passes to the stage of growth (the first critical period) and to the stage of maturation (the second critical period). Their existence is regarded in connection with the hierarchical joining of the vascular and neural trophic types of the vegetative ganglia. A supposition is made on a dependence of the specific neuronal differentiation on the epigenetic factors of morphogenesis.

SIF cells, cyclic AMP responses, and catecholamines of the guinea pig superior cervical ganglion.

The superior cervical ganglion (SCG) of the guinea pig has been investigated by a multidisciplinary approach. Dopamine (50 micron) produced no increase in cyclic AMP levels above control values of 27.9 pmole/mg protein, but 50 micron isoproterenol produced cyclic AMP levels of 210 pmole/mg protein, indicating the existence of a beta-adrenergic receptor-adenylate cyclase complex. The SIF cells were studied by fluorescence histochemistry, which indicated that two morphological types were present. A few Type I cells of the guinea pig SCG were solitary, but most were present in clusters containing many Type II cells. Immunohistochemical localization of antibodies to dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) demonstrated that types of SIF cell localize antibodies to DBH but not PNMT, providing strong evidence that norepinephrine is the neurotransmitter for all the SIF cells of the guinea pig SCG. Determination of the ratio of norepinephrine to dopamine confirmed that no other dopamine pools exist in the guinea pig SCG.

Monoamine storage sites in the rat superior cervical ganglion following synthesis inhibition.

Monoamine storage sites in paraganglionic (PG-) cells of the rat superior cervical ganglion were investigated by electron and fluorescence microscopy following treatment with p-chlorophenylalanine (pCPA), disulfiram or guanethidine respectively. Dense core vesicles in PG-cells are significantly decreased (p less than 0.001) in number following pCPA, and in the majority of these cells following disulfiram and guanethidine. However in a minor portion of PG-cells the latter agents cause an increase in number and in size of dense core vesicles, in parallel with structural alterations. In agreement with these electron microscopic findings fluorescence microscopic and cytophotometric evaluations reveal a general decrease in catecholamine content with few cells showing an increase. The findings provide a morphological basis for the assumption, that monoamine storage sites in PG-cells can be decreased by inhibition of monoamine synthesis, following administration of pCPA, disulfiram and guanethidine. However the two types of responses of PG-cells which occur after disulfiram and guanethidine demonstrate a functional heterogeneity of this cell system in the rat superior cervical ganglion which is discussed.