Prevention of the death of the rat axotomized hypoglossal nerve and promotion of its regeneration by bovine brain gangliosides.

We have examined the time course of the neuronal death and regeneration of rat axotomized hypoglossal nerve with various conditions of the nerve resection, and established a useful system to measure neurotrophic activities of bioactive substances. In this system, neuronal death can be evaluated by counting surviving neurons in the nucleus of hypoglossal neuron at the brain stem, and the degree of the regeneration can be measured by counting horseradish peroxidase-positive cells at the same region after injection of horseradish peroxidase into tongue. Using this system, the effects of brain gangliosides on rat hypoglossal nerve regeneration following 5 mm transection were examined. The addition of a ganglioside mixture from bovine brain as well as the autograft strongly prevented the death of neurons and promoted the regeneration of the lesioned nerve at 10 weeks after the operation. Further analyses on the dose effects and injection sites of gangliosides were performed. Although the mechanisms of the neurotrophic effects of the gangliosides are unknown, the therapeutic application of gangliosides for neuronal degeneration is a promising approach.     

Specificity of carbohydrate structures of gangliosides in the activity to regenerate the rat axotomized hypoglossal nerve

We previously reported that a ganglioside mixture from bovine brain could prevent neuronal death and promote regeneration in rats with hypoglossal nerve resection. In the present study, we have compared the neurotrophic effects of various glycosphingolipids including lactosyl-ceramide. The findings revealed that GT1b had the activity of neuronal death prevention equivalent to a ganglioside mixture or autograft, while other glycolipids exhibited about 60% activity. However, the capability to promote the regeneration varied among glycolipids, that is, GT1b (86%), GD1b (55%), GD1a (35%), GQ1b (34%), GM1 (20%), lactosyl-ceramide (17%) in the number of horseradish peroxidase-positive neurons as an indicator of regeneration. The experiments with oligosaccharides of GT1b or GD1b and ceramide showed that the carbohydrate moiety mainly exerts neurotrophic effects. These findings suggested that fine structures of carbohydrate moiety in gangliosides are critical in the regenerative activity in this hypoglossal nerve regeneration system.     

Visualization of nerve fibers and taste buds of the tropical catfish, Corydoras aeneus, following treatment by trypsin

Taste buds were removed from the barbels of the tropical catfishCorydoras aeneus following fixation with a 10% solution of formalinand subsequent treatment with a 0.25% solution of trypsin. Individualepithelial cells were dissociated as free cells. The surfaceof the dermis, including dermal papillae, appeared smooth andwavy, but some undissociated epithelial cells remained on thesmooth surface of the dermis. The surface of the dermis withoutfixation appeared fibrous. A few fine bundles of nerve fibersprotruded from the perimeter on the top of dermal papillae.These observations show that fine bundles of nerve fibers penetratethrough the remaining basement membrane at several points alongthe perimeter on the top of the dermal papillae.     

江豚皮肤的超微结构及其与其它豚类的比较

江豚皮肤的超微结构与海洋豚类相同,但在各层表皮细胞中存在大量的膜被颗粒,这很可能与江豚适应从海水进入淡水的生活方式有关。大量成束的张力原纤维的存在,有利于江豚在水中深潜和快速游泳,可适应来自各方面的压力,加强细胞之间的联接。在上下颌皮肤的乳头层基部大量被囊神经末梢和有鞘神经纤维束的分布、与江豚水底觅食方式有关。白鱀豚和条纹原海豚皮肤的超微结构与江豚大致相同,但亦有其各自的特点。     

Collapsin response mediator protein-2 accelerates axon regeneration of nerve-injured motor neurons of rat

The rat collapsin response mediator protein-2 (CRMP-2) is a member of CRMP family (CRMP-1-5). The functional consequence of CRMP-2 during embryonic development, particularly in neurite elongation, is relatively understood; however, the role in nerve regeneration is unclear. Here we examined the role of CRMP-2 during nerve regeneration using rat hypoglossal nerve injury model. Among the members, CRMP-1, CRMP-2, CRMP-5 mRNA expressions increased after nerve injury, whereas CRMP-3 and CRMP-4 mRNA did not show any significant change. In the N1E-115 cells, CRMP-2 has the most potent neurite elongation activity among the CRMP family members. In dorsal root ganglion (DRG) organ culture, CRMP-2 overexpression by adenoviral vector demonstrated substantial neurite elongation. On the other hand, CRMP-2 (DeltaC381), which acts as a dominant negative form of CRMP-2, inhibited neurite formation. Collectively, it would be plausible that CRMP-2 has potent nerve regeneration activity after nerve injury. We therefore examined whether CRMP-2 overexpression in the injured hypoglossal motor neurons accelerates nerve regeneration. A retrograde-tracer, Fluoro-Gold (FG), was used to evaluate the number of reprojecting motor neurons after nerve injury. CRMP-2-overexpressing motor neurons demonstrated the accelerated reprojection. The present study suggests that CRMP-2 has potent neurite elongation activity in nerve regeneration in vivo.     

Fibrin matrices with affinity‐based delivery systems and neurotrophic factors promote functional nerve regeneration

Glial‐derived neurotrophic factor (GDNF) and nerve growth factor (NGF) have both been shown to enhance peripheral nerve regeneration following injury and target different neuronal populations. The delivery of either growth factor at the site of injury may, therefore, result in quantitative differences in motor nerve regeneration and functional recovery. In this study we evaluated the effect of affinity‐based delivery of GDNF or NGF from fibrin‐filled nerve guidance conduits (NGCs) on motor nerve regeneration and functional recovery in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated consisting of GDNF or NGF and the affinity‐based delivery system (DS) within NGCs, control groups excluding the DS and/or growth factor, and nerve isografts. Groups with growth factor in the conduit demonstrated equivalent or superior performance in behavioral tests and relative muscle mass measurements compared to isografts at 12 weeks. Additionally, groups with GDNF demonstrated greater specific twitch and tetanic force production in extensor digitorum longus (EDL) muscle than the isograft control, while groups with NGF produced demonstrated similar force production compared to the isograft control. Assessment of motor axon regeneration by retrograde labeling further revealed that the number of ventral horn neurons regenerating across NGCs containing GDNF and NGF DS was similar to the isograft group and these counts were greater than the groups without growth factor. Overall, the GDNF DS group demonstrated superior functional recovery and equivalent motor nerve regeneration compared to the isograft control, suggesting it has potential as a treatment for motor nerve injury. Biotechnol. Bioeng. 2010;106: 970–979. © 2010 Wiley Periodicals, Inc.     

Protein gene product 9.5-immunoreactive nerve fibres and cells in human skin

Summary Sections of human skin were processed according to the indirect immunofluorescence technique with a rabbit antiserum against human protein gene product 9.5 (PGP 9.5). Immunoreactivity was detected in intraepidermal and dermal nerve fibres and cells. The intraepidermal nerves were varicose or smooth with different diameters, running as single processes or branched, straight or bent, projecting in various directions and terminating in the stratum basale, spinosum or granulosum. The density of the intraepidermal nerves varied between the different skin areas investigated. PGP 9.5-containing axons of the lower dermis were found in large bundles. They separated into smaller axon bundles within the upper dermis, entering this portion of the skin perpendicular to the surface. Then they branched into fibres mainly arranged parallel to the epidermal-dermal junctional zone. However, the fibres en route to the epidermis traversed the upper dermis more or less perpendicularly. Furthermore, immunoreactive dermal nerve fibres were found in the Meissner corpuscles, the arrector pili muscles, hair follicles, around the eccrine and apocrine sweat glands and around certain blood vessels. Such fibres were also observed around most subcutaneous blood vessels, sometimes heavily innervating these structures. Numerous weakly-to-strongly PGP 9.5-immunoreactive cells were found both in the epidermis and in the dermis.     

The adsorption of bovine blood proteins onto the surface of O-(carboxymethyl)chitin

Chitin was found to interact with bovine blood proteins and the affinities of these proteins for chitin tended to be decreased by the introduction of O-carboxymethyl (CM) groups onto the chitin surface, especially with fibrinogen. As the adsorption of blood proteins to the CM-chitin (d.s. 0.35) was assumed to follow an isothermal adsorption-curve, the adsorption coefficients were estimated by applying the Langmuir equation. Bovine serum albumin showed the highest affinity among the proteins applied in this experiment [KBSA (bovine serum albumin); 20.0, KB gamma G (bovine gamma globulin); 1.96, KBF (bovine fibrinogen); 1.20]. The binding site of BSA for CM-chitin was assumed to be regulated not only by the cationic groups of BSA but also by other factors such as the recognition capacity of BSA to bind to GlcNAc residues in CM-chitin.     

Ultrathin chitin films for nanocomposites and biosensors

Chitin is the second most abundant biopolymer and insight into its natural synthesis, enzymatic degradation, and chemical interactions with other biopolymers is important for bioengineering with this renewable resource. This work is the first report of smooth, homogeneous, ultrathin chitin films, opening the door to surface studies of binding interactions, adsorption kinetics, and enzymatic degradation. The chitin films were formed by spincoating trimethylsilyl chitin onto gold or silica substrates, followed by regeneration to a chitin film. Infrared and X-ray photoelectron spectroscopy, X-ray diffraction, ellipsometry, and atomic force microscopy were used to confirm the formation of smooth, homogeneous, and amorphous chitin thin films. Quartz crystal microbalance with dissipation monitoring (QCM-D) solvent exchange experiments showed these films swelled with 49% water by mass. The utility of these chitin films as biosensors was evident from QCM-D and surface plasmon resonance studies that revealed the adsorption of a bovine serum albumin monolayer.     

Morphological changes and expression of protein kinase CK2 β subunit in the microglia after hypoglossal nerve transection

Following hypoglossal nerve transection, the microglia of the rat hypoglossal nucleus expressed protein kinase CK2 β subunit immunoreactivity. CK2 β immunostaining occurred on the operated side from postoperative day 3; on day 5 we observed strong immunoreactivity and the immunopositive microglial cell processes surrounded the injured neurones. Thereafter, the immunoreactivity decreased gradually and on day 10 the immunopositive cells surrounded only a few injured neurones. Electron microscopic observations on the hypoglossal nucleus revealed microglia-neuronal contact within 3 hours of nerve injury, and by day 3 all the injured neurones were in contact with microglial cells. These observations indicated that microglia-neuronal contact occurred earlier than the CK2 β subunit immunoreactivity. CK2 may not be implicated during the initial migration of the microglia to the injured neurones; however, it may enhance the growth and elongation of the microglial cell processes around the injured neurones.     

Changes in localized arrangement into the hypoglossal nucleus after the neurectomy of unilateral hypoglossal nerve (medial branch) in cats

We studied changes in orofacial behavior and the arrangement of bilateral hypoglossal nuclei after the neurectomy of the medial branch of the unilateral hypoglossal nerve in cats. After recovery from surgery in a head holder, the animals were acclimated to take and chew fish paste (1.8 g) from a spoon and lick milk from a wetted paintbrush. Next we performed a neurectomy in the unilateral hypoglossal nerve after training. We firstly recorded behavior during the taking of fish paste and licking of milk, and then performed a neurectomy in the unilateral hypoglossal nerve. After nerve cutting, the cats’ tongue deviated toward the cut side when they licked food, and bilateral activities of EMGs in the genioglossus muscles became stable in about 1 month. After that, we injected two kinds of fluorescent dye (10% Evans blue, EB, and 3% Fast blue, FB) into the bilateral genioglossus muscles using syringes (0.15 ml in each), respectively. Although each injection of FB and EB into the bilateral genioglossus muscles in normal cats revealed cells positively stained with each dye in the hypoglossal nuclei of each injection site, in cats 1 month after nerve cutting, fluorescent dye was only observed in positive cells in the hypoglossal nucleus of the intact side and the dye injected into the neurectomy side showed a mixture into positive cells of the intact side. The findings suggest that muscles in the neurectomy side may be compensated by regeneration of the peripheral nerves on the intact side.     

Innervation and cholinesterase activity in the skin of albino and desert rats

The distribution of nerves and cholinesterase activity in the skin of the desert and albino rats has been studied using both histological and histochemical techniques. In the desert rat, the skin was richly innervated. Specific cholinesterase was concentrated in the nervous network of the dermis and around the hair follicles, in the nerve bundles of the dermis, in perivascular nerves, in fine intra-epithelial nerves and in sensory end organs in the junctional area between the dermis and epidermis. In the albino rat, specific cholinesterase was concentrated in the sebaceous glands. The positive cholinesterase activity that was seen in the desert rat in intra-epithelial nerves, and in dermal and hair follicle networks could not be demonstrated.     

Effect of genipin-crosslinked chitin-chitosan scaffolds with hydroxyapatite modifications on the cultivation of bovine knee chondrocytes

Chitin and chitosan were hybridized in various weight percentages by genipin crosslinkage under various prefreezing temperatures to form tissue-engineering scaffolds via lyophilization. In addition, deposition of hydroxyapatite (HA) on the surface of the porous scaffolds was performed by precipitation method to achieve modified chemical compositions for chondrocyte attachments and growths. The experimental results revealed that a lower prefreezing temperature or a higher weight percentage of chitin in the chitin-chitosan scaffolds would yield a smaller pore diameter, a greater porosity, a larger specific surface area, a higher Young's modulus, and a lower extensibility. Moreover, a higher chitin percentage could also result in a higher content of amine groups after crosslink and a lower onset temperature for the phase transition after thermal treatment. A decrease in the prefreezing temperature from -4 degrees C to -80 degrees C, an increase in the chitin percentage from 20% to 50%, and an increase in the cycle number of alternate immersion for HA deposition from 1 to 5 generated positive effects on the cell number, the content of glycosaminoglycans, and the collagen level over 28-day cultivation of bovine knee chondrocytes.     

SURFACE STRUCTURE OF ISOLATED NEURONS : Detachment of Nerve Terminals during Axon Regeneration

Freehand, isolated neuronal perikarya from the hypoglossal nucleus of the rabbit have been examined with light-and electron-microscopy (transmission and scanning). The surface of the cell bodies was largely covered with spherical particles which were 0.5–2 µ in diameter. Transmission electron microscopy proved that the spherical particles were synaptic nerve terminals. Crush of the hypoglossal nerve which leads to chromatolysis and swelling of the neuronal cell bodies results in a conspicuous reduction in the number of terminals attached to the surface of hypoglossal neurons. This effect was observed both for isolated neurons and in tissue sections. The effect is considered in relation to earlier reported variations in the adherence of neuropil to isolated neuronal perikarya. The functional importance of nerve ending detachment in connection with nerve injury is discussed.     

Characterization of respiratory-modulated activities of hypoglossal motoneurons

In decerebrate, vagotomized, paralyzed, and ventilated cats, activities of the phrenic nerve and single hypoglossal nerve fibers were monitored. The great majority of hypoglossal neuronal activities were inspiratory (I), discharging during a period approximating that of phrenic. Many were not active at normocapnia but were recruited in hypercapnia or hypoxia. Once recruited, discharge frequencies, which rose quickly to near maximal levels in early to midinspiration, significantly increased with further augmentations of drive. Also, the onset of activities became progressively earlier, compared with phrenic discharge, in hypercapnia or hypoxia. Smaller numbers of hypoglossal fiber activities, having inspiratory-expiratory (I-E), expiratory (E), expiratory-inspiratory (E-I), or tonic discharge patterns, were also recorded. Activities of E, I-E, and those I fibers that became I-E in high drive may underlie the early burst of expiratory activity of the hypoglossal nerve. It is concluded that the firing and recruitment patterns of hypoglossal neurons differ from those of phrenic motoneurons. However, responses to chemoreceptor stimuli are similar among the two neuronal groups.     

Highly Fibrous and Porous Raw Material Shaped Chitin Isolated from <Emphasis Type="Italic">Oniscus</Emphasis> sp. (Crustacea)

Chitin was isolated from a crustacean by keeping the original body shape for the first time. The isolation method followed in this study was simple and time and energy saving unlike the labor-intensive classical methods. Chitin samples preserving the original shape were isolated from Isopoda (Oniscus sp.) successfully in a total of 20 min including filtration time. FT-IR, XRD and TGA analysis and chitinase digestion test demonstrated that the chitin was pure, low crystalline (Crystalline index: 51 %) and had low thermal stability (maximum degradation temperature: 328.8 °C). SEM analysis revealed the highly fibrous structure of chitin. The chitin content of the whole body was found significantly higher (29.6?±?4.2 %) than the earlier reports. Interaction of chitin isolates with Bovine Serum Albumin protein were studied at different pH. It was concluded that this three dimensional raw material shaped chitin can be effectively used in any adsorption studies due to 1) highly fibrous and porous nature, 2) low crystallinity and 3) low thermal stability. And also this biodegradable and biocompatible biopolymer can be suggested as a carrier matrix in further studies thanks to its three dimensional shape.     

Striae albae: a morphological study on the human skin

Specimens of abdomen skin, comprising alternate areas of striae albae and healthy skin, were removed during surgical lipectomy from multiparous and obese women between the ages of 24 and 53 years. A flattening and thinning of the striae albae surface and the almost complete disappearance of dermal papillae was observed in paraffin and thin sections. The papillary dermis was found to be almost completely replaced by straight bundles of collagen fibres running parallel to the skin surface. Immunofluorescence data revealed in these bundles high positivity for type I collagen. The underlying reticular dermis was also found to contain large densely packed bundles of collagen fibres running parallel to the skin surface. Both papillary and reticular dermis collagen fibres were mainly arranged orthogonally to the main axis of the stria. Furthermore, the density of the collagen fibre bundles and the diameter of the collagen fibrils was found to be greater than that of the clinically healthy skin. A larger number of elastic fibres, which presented an abnormal ultrastructural appearance, were visible in pathological papillary and reticular dermis.