A nanofibrous PHBV tube with Schwann cell as artificial nerve graft contributing to Rat sciatic nerve regeneration across a 30-mm defect bridge

Abstract

A nanofibrous PHBV nerve conduit has been used to evaluate its efficiency based on the promotion of nerve regeneration in rats. The designed conduits were investigated by physical, mechanical and microscopic analyses. The conduits were implanted into a 30-mm gap in the sciatic nerves of the rats. Four months after surgery, the regenerated nerves were evaluated by macroscopic assessments and histology. This polymeric conduit had sufficiently high mechanical properties to serve as a nerve guide. The results demonstrated that in the nanofibrous graft with cells, the sciatic nerve trunk had been reconstructed with restoration of nerve continuity and formatted nerve fibers with myelination. For the grafts especially the nanofibrous conduits with cells, muscle cells of gastrocnemius on the operated side were uniform in their size and structures. This study proves the feasibility of artificial conduit with Schwann cells for nerve regeneration by bridging a longer defect in a rat model.     

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.     

Transient involvement of enkephalins in both the sympathetic and parasympathetic innervations of the submandibular gland of rats

Summary A time course study with enkephalin(Enk)-like immunoreactivity has revealed that nerve fibers intensely immunoreactive for Enk-8 appeared transiently only during the postnatal week 2 and 4 within the acini as well as in the inter- and intralobular connective tissues of the submandibular gland of rats. At these stages numerous nerve fibers immunoreactive for tyrosine hydroxylase (TH) appeared also in the inter- and intralobular connective tissues and within the acini. Coincidently with these postnatal stages, abundant Enk-immunoreactive principal ganglion cells appeared in the superior cervical ganglion. These were not immunoreactive for neuropeptide tyrosine (NPY). A substantial number of Enk-immunoreactive ganglion cells were also present in the submandibular ganglia at these younger postnatal stages. Superior cervical ganglionectomy at these stages resulted in a marked decrease in number of the inter- and intralobular Enk-immunoreactive nerve fibers, a slight decrease in number of the intraacinar Enk-immunoreactive nerve fibers, and almost complete disappearance of intraglandular TH-immunoreactive nerve fibers. Immuno-electron-microscopic analysis revealed that Enk-immunoreactive nerve fibers in the submandibular gland were identified as electron-dense neuronal profiles enclosed by Schwann cells in the inter- and intralobular connective tissues and those directly apposed to secretory cells within the acini. They contained small clear vesicles mixed with some large granular vesicles. After postnatal week 6, no Enk-immunoreactive nerve fibers were detected in the submandibular gland, and no TH-immunoreactive nerve fibers were seen within the acini, while TH-immunoreactive nerve fibers remained numerous in the inter- and intralobular connective tissues. These findings indicate that both sympathetic and parasympathetic nerve fibers exhibit Enk-like immunoreactivity transiently during postnatal weeks 2 and 4. It is further indicated that the inter- and intralobular nerve fibers lose Enk-like immunoreactivity while the intraacinar fibers disappear at the adult stage.     

On the effect of ammonium and lithium ions upon frog nerve deprived of sodium

An analysis has been made of the effect of ammonium and of lithium ions upon frog nerve deprived of sodium. Ammonium ions cannot substitute for sodium ions and restore the excitability of the nerve fibers; nor can they increase the L fraction of the membrane potential and the efficiency of the nerve reaction. Certain observations, however, indicate that the presence of ammonium ions outside the nerve fibers may delay the development of inexcitability in a sodium-free medium of nerve fibers restored by a moderate amount of sodium ions. Lithium ions can substitute for sodium and restore to nerve fibers of the A and C groups the ability to conduct impulses; the effect upon B fibers has not been investigated. Lithium cannot substitute for sodium in the role that sodium plays in the creation of the L fraction and in the establishment of the nerve reaction. In this respect lithium and sodium have opposite effects. This fact establishes an important difference between the two physiological responses that the nerve fibers can produce, the nerve impulse and the nerve reaction. With untreated nerve the depolarization of nerve by lithium ions at high concentrations is preceded by a phase of hyperpolarization; with nerve deprived of sodium the depolarization begins without delay.     

CGRP-immunoreactive sensory nerve fibers in the submandibular gland of the rat

Summary Indirect immunofluorescence technique was used to study the occurrence and distribution of CGRP immunoreactivity in the submandibular gland of normal rats and after unilateral sensory and sympathetic denervations. In normal rats, CGRP-immunoreactive nerve fibers and nerve trunks were seen around or in close contact with interlobular salivary ducts as well as around small blood vessels of the gland. Occasionally, CGRP-immunoreactive nerve fibers were also detected between or around the acini of the gland.The submandibular ganglia contained CGRP-immunoreactive nerve fibers, but the ganglion cells were not immunoreactive for CGRP. The trigeminal ganglion contained a population of CGRP-immunoreactive, mainly small sized ganglion cells and nerve fibers distributed throughout the ganglion. Unilateral electrocoagulation of the trigeminal nerve caused a significant reduction in the number of immunoreactive nerve fibers in the gland, although some fibers still were present in the ipsilateral glandular tissue. Unilateral superior cervical ganglionectomy caused no detectable effect on the number of CGRP-immunoreactive nerve fibers in the gland.The present results suggest that the rat submandibular gland contains CGRP-immunoreactive nerve fibers both around blood vessels and in glandular secretory elements. Denervation experiments support the view that the majority, but perhaps not all of them originate from the trigeminal ganglion.     

Tissue preparation and immunostaining of mouse sensory nerve fibers innervating skin and limb bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.     

Intraepidermal free nerve fiber endings in the hairless skin of the rat as revealed by the zinc iodide-osmium tetroxide technique

The nerve fiber distribution in the epidermis of the hairless rat skin was studied light microscopically by means of zinc iodide-osmium tetroxide staining. Two different morphological types of free nerve fiber endings could be detected: clusters of relatively thick nerve fibers stretched up through the spinous layer up to the granular layer sending off terminal branches. In addition, many solitary thin varicose nerve fibers were seen within the epidermis. The observed discrepancies in nerve fiber diameters appeared to be larger than those reported for human intraepidermal nerve fibers in recent immunohistochemical studies. Moreover, dendritic cells, most probably representing Langerhans cells, could be selectively stained. These cells appeared to be in a close location to thin varicose nerve fibers. Both types of demonstrated free nerve endings have to be functionally connected with different sensoric functions. Possibly, a subpopulation of the thin nerve fibers might possess primarily a nociceptive task, whereas the thick ones have most probably to be regarded as mechanoreceptive. The nerve fibers innervating dendritic cells appear to be identical to the peptidergic ones which may regulate the antigen-presenting capacity of these cells. Due to its selectivity for intraepidermal nerve fibers, the used method might supplement immunohistochemical procedures in a helpful manner.     

Tyrosine-hydroxylase-immunoreactive nerve fibers in the separated capsule of the rat adrenal gland

The present study applied the separated adrenal capsules of rats for wholemount immunocytochemistry and used tyrosine hydroxylase (TH) antibody as a marker for catecholamines. TH-immunoreactive nerve bundles without varicosities and fibers with varicosities were seen to run along or to encircle blood vessels entering the adrenal capsule from the outside, and then to run along a network of blood vessels in the intracapsular region. Also, the TH-immunoreactive nerve bundles and fibers were found to run along blood vessels in the subcapsular region. Some TH-immunoreactive nerve fibers and bundles with varicosities, unassociated with the blood vessels, were seen in the subcapsular region. In this region, TH-immunoreactive nerve fibers with varicosities were often seen to be closely associated with the cortical cells. Some TH-immunoreactive nerve fibers without varicosities were visible within the splanchnic nerve in the subcapsular region. The present study suggests that numerous catecholaminergic nerve fibers are associated with blood vessels forming a network in the superficial region of the rat adrenal gland.     

CGRP-immunoreactive sensory nerve fibers in the submandibular gland of the rat

Indirect immunofluorescence technique was used to study the occurrence and distribution of CGRP immunoreactivity in the submandibular gland of normal rats and after unilateral sensory and sympathetic denervations. In normal rats, CGRP-immunoreactive nerve fibers and nerve trunks were seen around or in close contact with interlobular salivary ducts as well as around small blood vessels of the gland. Occasionally, CGRP-immunoreactive nerve fibers were also detected between or around the acini of the gland. The submandibular ganglia contained CGRP-immunoreactive nerve fibers, but the ganglion cells were not immunoreactive for CGRP. The trigeminal ganglion contained a population of CGRP-immunoreactive, mainly small sized ganglion cells and nerve fibers distributed throughout the ganglion. Unilateral electrocoagulation of the trigeminal nerve caused a significant reduction in the number of immunoreactive nerve fibers in the gland, although some fibers still were present in the ipsilateral glandular tissue. Unilateral superior cervical ganglionectomy caused no detectable effect on the number of CGRP-immunoreactive nerve fibers in the gland. The present results suggest that the rat submandibular gland contains CGRP-immunoreactive nerve fibers both around blood vessels and in glandular secretory elements. Denervation experiments support the view that the majority, but perhaps not all of them originate from the trigeminal ganglion.     

Immunohistochemical localization of calcitonin gene-related peptide and bombesin/gastrin-releasing peptide in nerve fibers of the rat,guinea pig and pig female genital organs

Summary The localization and distribution of calcitonin gene-related peptide (CGRP) and bombesin/gastrin-releasing peptide (GRP) immunoreactivity were studied in the rat, guinea pig and pig female genital organs with indirect immunohistochemical technique. In the rat, guinea pig and pig. CGRP and GRP immunoreactivities were localized in nerve fibers of the uterus, ovary and oviduct. Generally, CGRP-immunoreactive nerve fibers were intensely stained, while GRP-immunoreactive nerve fibers exhibited moderate immunoreactivity. The number of GRP-immunoreactive nerve fibers in these organs was lower in comparison with that of CGRP-immunoreactive nerve fibers. The pattern of distribution of these nerve fibers was very similar in different genital organs of all species studied. In the uterus of rat, guinea pig ang pig, CGRP-and GRP-immunoreactive nerve fibers and nerve bundles were observed in the muscular membrane and around blood vessels. Some delicate CGRP-and GRP-immunoreactive nerve fibers were also present in the submucous layer of the uterus. In the oviduct. CGRP-and GRP-immunoreactive nerve fibers were seen in the muscular membrane, around blood vessels and in the submucous layer. In the ovary, CGRP-and GRP-immunoreactive nerve fibers were distributed in medullary stroma, in close contact with blood vessels and between follicles of different stages of development.     

Ultrastructure of the tentacle nerve plexus and putative neural pathways in sea anemones

Abstract. Neurons of sea anemone tentacles receive stimuli via sensory cells and process and transmit information via a plexus of nerve fibers. The nerve plexus is best revealed by scanning electron microscopy of epidermal peels of the tentacles. The nerve plexus lies above the epidermal muscular layer where it appears as numerous parallel longitudinal and short interconnected nerve fibers in Calliactis parasitica . Bipolar and multipolar neurons are present and neurites form interneuronal and neuromuscular synaptic contacts. Transmission electron microscopy of cross sections of tentacles of small animals, both C. parasitica and Aiptasia pallida , reveals bundles of 50–100 nerve fibers lying above groups of longitudinal muscle fibers separated by intrusions of mesoglea. Smaller groups of 10–50 slender nerve fibers are oriented at right angles to the circular muscle formed by the bases of the digestive cells. The unmyelinated nerve fibers lack any glial wrapping, although some bundles of epidermal fibers are partially enveloped by cytoplasmic extensions of the muscle cells; small gastrodermal nerve bundles lie between digestive epithelial cells above their basal myonemes. A hypothetical model for sensory input and motor output in the epidermal and gastrodermal nerve plexuses of sea anemones is proposed.     

Immunohistochemical localization of calcitonin gene-related peptide and bombesin/gastrin-releasing peptide in nerve fibers of the rat, guinea pig and pig female genital organs

The localization and distribution of calcitonin gene-related peptide (CGRP) and bombesin/gastrin-releasing peptide (GRP) immunoreactivity were studied in the rat, guinea pig and pig female genital organs with indirect immunohistochemical technique. In the rat, guinea pig and pig, CGRP and GRP immunoreactivities were localized in nerve fibers of the uterus, ovary and oviduct. Generally, CGRP-immunoreactive nerve fibers were intensely stained, while GRP-immunoreactive nerve fibers exhibited moderate immunoreactivity. The number of GRP-immunoreactive nerve fibers in these organs was lower in comparison with that of CGRP-immunoreactive nerve fibers. The pattern of distribution of these nerve fibers was very similar in different genital organs of all species studied. In the uterus of rat, guinea pig and pig, CGRP- and GRP-immunoreactive nerve fibers and nerve bundles were observed in the muscular membrane and around blood vessels. Some delicate CGRP- and GRP-immunoreactive nerve fibers were also present in the submucous layer of the uterus. In the oviduct, CGRP- and GRP-immunoreactive nerve fibers were seen in the muscular membrane, around blood vessels and in the submucous layer. In the ovary, CGRP- and GRP-immunoreactive nerve fibers were distributed in medullary stroma, in close contact with blood vessels and between follicles of different stages of development.     

Quantitative studies on the intermediate nerve of the mouse with the electron microscope.

Quantitative counts of the intermediate nerve fibers of the mouse with electron microscope have shown that, on an average, 1,267 (79.3%) and 301 (18.9%) of the total nerve fibers (1,597) were myelinated and unmyelinated fibers, respectively. The number of unmyelinated fibers in the intermediate nerve was less than one-half the number of unmyelinated fibers in the greater petrosal nerve found in previous studies. Therefore, it is postulated that some of the unmyelinated fibers in the greater petrosal nerve may come from the intermediate nerve, while the rest may be derived from another source.     

EXCITATION OF NERVE FIBERS IN THE SQUID (LOLIGO PEALII)

1. Strength-duration data for the giant fiber of the great stellar nerve of the squid (Loligo pealii) can be approximately described by several mathematical formulations. 2. Excitation time constants for isolated giant fibers are essentially the same as constants of the giant fibers in the intact nerve. 3. The strength-duration curves of the fibers in the intact nerve lie higher on the voltage axis than those of the isolated fibers. It is concluded that the principal effect of other fibers upon the excitation of one fiber in a nerve trunk is that of shunting the stimulating current. 4. Deterioration of the nerve shifts the curve upward and to the left, resulting in shorter time constants. 5. Decreasing interelectrode distance also shifts the curve upward and to the left. 6. Excitation time constants of the giant fibers are larger with plate electrodes than with wire or pore electrodes. 7. The strength-duration curves of the smaller fin nerve fibers lie consistently to the right of, and the time constants are longer than those of the giant fibers.     

Peptide-containing nerve fibers in the respiratory tract of the ferret

Summary The ferret is widely used in functional and neuromorphological studies on the respiratory tract. We have examined the occurrence and distribution of peptide-containing and adrenergic nerve fibers (using dopamine--hydroxylase as a marker). Adrenergic nerve fibers and fibers storing vasoactive intestinal peptide have a widespread distribution along the entire respiratory tract. Adrenergic nerve fibers were found in the lamina propria, as well as around blood vessels and glands and in smooth muscle. Nerve fibers storing vasoactive intestinal peptide occurred in the epithelium, the lamina propria, around blood vessels and glands, and among muscle bundles. Substance P-, neurokinin A- and calcitonin gene-related peptide-containing nerve fibers predominated beneath and within the epithelium along the entire respiratory tract. Neuropeptide Y-containing nerve fibers were prominent among smooth muscle bundles and around glands. The blood vessels in the wall of the airways were richly supplied with peptidecontaining nerve fibers and adrenergic fibers. Ganglia located over the outer or dorsal surface of the tracheal wall harbored vasoactive intestinal peptide-containing nerve cell bodies. Substance P and neurokinin A invariably coexisted in the same nerve fibers. Further, coexistence of substance P/neurokinin A and calcitonin gene-related peptide was observed in the nerve fibers associated with the epithelium. Vasoactive intestinal peptide, neuropeptide Y and occasionally also substance P coexisted in the population of nerve fibers associated with blood vessels and smooth muscle. Many adrenergic nerve fibers contained neuropeptide Y.     

Comparative study of the lamina cribrosa and the pial septa in the vertebrate optic nerve and their relationship to the myelinated axons

The optic nerve contains the connective tissues, i.e. the lamina cribrosa and pial septa. This report presents a histological comparison of the lamina cribrosa and pial septa in the five classes (mammals, birds, reptiles, amphibians and teleosts) of vertebrates. Furthermore, the distribution of myelinated fibers was observed from the optic nerve through the retina in the same animals. The lamina cribrosa is found in mammals except for mice, and in birds. Structural complexity of the lamina was different in animals but generally dependent of the optic nerve thickness. The pial septa were present in the optic nerve proper of the mammals except for the mice, in birds and in a part of teleosts. Fasciculation of the optic nerve by the pial septa tended to be more prominent as the optic nerve become thicker. The optic nerve consisted of largely myelinated fibers in vertebrates. The retina contained some myelinated fibers in submammals but was thoroughly devoid of myelinated fibers in mammals. The borderline between myelinated and unmyelinated portions in the optic nerve of different species did not related to the lamina cribrosa. Amphibians had exceptionally only a few myelinated fibers in the optic nerve and no myelinated fibers in the retina.     

Electron-microscopic analysis of the mouse facial nerve near the geniculate ganglion

An electron-microscopic analysis of the mouse facial nerve near the geniculate ganglion shows that there are, on the everage, 603 more nerve fibers in the portion of the nerve distal to the geniculate ganglion than there are in the part proximal to the ganglion. The average distal increase in the number of unmyelinated fibers is 444 and that in the myelinated fibers is 165. The somatic motor nerve fibers and the parasympathetic fibers in the mouse facial nerve may not contribute to the distal excess. It is possible that the increase in the number of unmyelinated fibers distal to the geniculate ganglion is mainly due to the presence of postganglionic sympathetic fibers in the facial trunk distal to the geniculate ganglion and the greater petrosal nerve. The distal increase in the number of myelinated fibers may be mainly contributed by the sensory fibers.     

Distribution of calcitonin gene-related peptide and substance P-immunoreactive nerve fibers and their correlation in the periodontal ligament of the mouse incisor.

The distribution of calcitonin gene-related peptide (CGRP)- and substance P (SP)-immunoreactive (IR) nerve fibers and their correlation in the periodontal ligament of mouse incisors were examined by indirect immunofluorescence. Both CGRP-IR and SP-IR thin nerve fibers were abundant in the apical and middle third of the periodontal ligament. In the lingual portion of the incisal periodontal ligament, these nerve fibers were localized in the alveolar half of the periodontal ligament and were observed as free nerve endings. No CGRP-IR and SP-IR specialized nerve endings, such as Ruffini-like corpuscles, were observed. In the labial periodontal ligament, CGRP-IR and SP-IR nerve fibers ran along the incisal axis. The distribution of CGRP-IR nerve fibers was very similar to that of SP-IR nerve fibers.     

担载神经生长因子(NGF)的乳液法涂层纤维体外缓释研究

目的:目前周围神经修复中,神经导管是研究热点,本文研究乳液法涂层纤维制备的神经导管在神经修复中应用的可能性。方法:本文采用乳液法制备担载NGF的丝素-聚乳酸(PLLA)涂层电纺纤维,观察纤维的形态,测定NGF的体外释放动力学参数,并考察纤维释放液对于PC12细胞增殖的影响。结果:担载NGF的涂层纤维具备类似于细胞外基质(ECM)的三维结构和多孔形态;涂层纤维中NGF体外有效缓释10天;细胞实验中,在含有释放液的培养基中生长的PC12细胞,与空白对照组相比,荧光强度平均多了2000-4000个荧光强度,所以释放液可以更好地促进PC12细胞的增殖。结论:担载NGF的乳液法涂层纺丝纤维具备促进缺损周围神经修复的条件,可以进一步研究在动物体内修复缺损周围神经中的效果,为以后的临床应用打下基础。     

Adrenergic nerve fibers in the human fetal sciatic nerve.

The adrenergic innervation was studied in the human sciatic nerve at the gestational age of 16, 17, 18 and 21 weeks. Formaldehyde-induced catecholamine fluorescence, tyrosine hydroxylase (TH) and neuropeptide Y (NPY) peroxidase-antiperoxidase immunohistochemistry methods were used. At the gestational age of 16, 17 and 18 weeks no adrenergic or NPY-positive nerve fibers were seen. At 21 weeks both fluorescence microscopy and TH immunohistochemistry showed adrenergic nerve fibers around arterioles in the epiperineurium and single nerve fibers in the endoneurium not related to blood vessels. The number of adrenergic nerve fibers appeared to be higher in the sciatic than in the tibial segment of the nerve. At this age, as at earlier stages of gestation, no NPY-positive nerve fibers were seen either in the epiperineurium or in the endoneurium. The results suggest that adrenergic nerve fibers may be associated with the epiperineurial blood vessels in the human sciatic nerve, and that the innervation starts to develop between 18 and 21 weeks of gestational age.