Formation Of Nerve Fibers Research Articles

Dual effects of TNFα on nerve fiber formation from ventral mesencephalic organotypic tissue cultures

Tumor necrosis factor alpha (TNFα) is toxic to dopamine neurons and increased levels of TNFα are observed in Parkinson's disease. Dopamine nerve fiber outgrowth in organotypic cultures of fetal ventral mesencephalon occurs in two waves. The early appearing nerve fibers are formed in the absence of astroglia, while migrating astrocytes guide the late appearing dopamine nerve fibers. TNFα (40 ng/ml) was added to the medium of organotypic ventral mesencephalic tissue cultures between days 4–7 and 11–14. The cultures were evaluated at days 7 or 19 to study the effects of TNFα on both types of nerve fiber formation. Tyrosine hydroxylase (TH)-immunohistochemistry demonstrated that the number of cultures showing non-glial-guided TH-positive outgrowth was reduced compared to controls, when TNFα was added at day 4. By contrast, the glial-guided TH-positive nerve fiber outgrowth and the astrocytic migration reached significantly longer distances by early TNFα treatment. Ki67-immunohistochemistry revealed that TNFα did not affect proliferation of astrocytes. Treatment with TNFα and antibodies against TNFα receptor 1 between days 4 and 7 revealed that the non-glial-guided TH-positive outgrowth reappeared. TNFα treatment between days 11 and 14 triggered neither the TH-positive glial-guided outgrowth, nor promoted the astrocytic migration to reach longer distances. The number of microglia was significantly increased after the late but not early TNFα treatment. In conclusion, TNFα is toxic for the non-glial dopaminergic nerve fiber outgrowth but stimulates the glial-guided outgrowth and the migration of astrocytes at an early time point. TNFα increased the number of microglia in VM tissue cultures after late but not after early treatment.

Signal functions of NCAM

The neuronal protein of cell adhesion belongs to the immunoglobulin superfamily of cell adhesion proteins. It consists of an extracellular domain providing homo-and heterophilic interactions with surrounding molecules that are located on the cell surface or are components of the extracellular matrix, a transmembrane part, and intracellular domains (NCAM140 and NCAM180). In addition to its role in cell adhesion, NCAMs act as a signal receptor molecule. Adhesion and initiation of signal cascades induced by binding to the NCAM extracellular domains occur interdependently but influence each other. The homo-and heterophilic binding of NCAM can activate a number of intracellular signal cascades resulting in neurite growth, axone guidance, axone myelinization, and formation of nerve fibers. It has been established that the intracellular signal is initiated by the interactions between NCAMs and fibroblast growth factor receptors (FGFR), non-receptor tyrosine kinases (Fyn and FAK), glia-derived neurotrophic factor (GDNF), ATP, prion proteins, and several other molecules. The review discusses possible mechanisms of functioning of these signal cascades.

Studies of the protective effect of the hypothalamic peptide PRP-3 on spinal cord neurons at different periods after lateral hemisection

The protective effect of a new neurosecretory cytokine, proline-rich peptide (PRP-3) that is produced by the cells of the hypothalamic paraventricular and supraoptical nuclei was studied after lateral hemisection of the spinal cord. Experiments were performed at different postoperative periods (3, 4, and 5 weeks) under conditions of daily administration of PRP-3 in order to study dynamics of the development of rehabilitation states. Flow of single impulse activity induced by stimulation of the mixed (N. ischiadicus), extensor (N. peroneus communis), and flexor (N. gastrocnemius) nerves, at the level of the transection site and lower than it, on the damaged and symmetrical intact sides of the spinal cord was observed in comparison with the control in the interneurons and motoneurons of the spinal cord after daily administration of PRP-3 for 3–4 weeks. PRP-3 was shown to restore activity of the neurons of the lumbar part of the spinal cord and to cause more pronounced early and late postimpulse manifestations of the activity of spinal cord neurons. Morphological studies demonstrated that PRP-3 exhibited a powerful effect on the migration of motoneurons to the transection site and on intensification of proliferation of glial elements. Moreover, PRP-3 stimulated the migration of motoneurons to more distant regions along with penetration to the white matter at the level of the symmetrical undamaged side of the spinal cord. Glial and neurite penetration into places without symphysis, predominantly in the grey matter of the spinal cord, and the prevention of scar formation in the regions of damage were demonstrated. PRP-3 facilitated active ingrowth of vessels in the damaged regions. This process, in turn, favors regeneration with the restoration of locomotor activity at the rehabilitation stage. PRP-3 prevented scar formation and favored ingrowth of nerve fibers of the white matter in the damaged area and thus prevented degeneration of neuroglial elements. This fact was confirmed by recording activity at the level of the injury and by restoration of locomotor activity on the damaged side. This properties of PRP-3 may allow its use in clinical practice for the prevention of neurodegeneration induced by craniocerebral and spinal trauma.

Enhancement of Peripheral Nerve Regeneration Using Bioabsorbable Polymer Tubes Packed With Fibrin Gel

Nerve conduit tubes were developed using bioabsorbable polymer membranes, and the effects of tube shape--straight or bellows-shaped tubes--and the fibrin gel filling were investigated. The mechanical properties of the tubes were examined by in vitro tests, and their effectiveness for peripheral nerve regeneration was determined by grafting into experimentally transected sciatic nerves of rats. The bellows tube showed mechanically superior properties, and when used with the fibrin gel, it induced superior tissue formation of myelinated nerve fibers as compared to other tube types. The total area of myelinated axons regenerated in the fibrin-filled bellows tube was comparable to that of an isograft control, whereas those of the other tubes demonstrated inferior regeneration. This result suggests that the mechanically superior bellows tube filled with fibrin gel is an effective graft alternative for peripheral nerve regeneration.

Glial influence on nerve fiber formation from rat ventral mesencephalic organotypic tissue cultures

Rat fetal ventral mesencephalic organotypic cultures have demonstrated two morphologically different dopamine nerve fiber growth patterns, in which the initial nerve fibers are formed in the absence of astrocytes and the second wave is guided by astrocytes. In this study, the presence of subpopulations of dopamine neurons, other neuronal populations, and glial cells was determined. We used "roller-drum" organotypic cultures, and the results revealed that beta-tubulin-positive/tyrosine hydroxylase (TH)-negative nerve fibers were present as early as 1 day in vitro (DIV). A similar growth pattern produced by TH-positive neurons was present from 2 DIV. These neurites grew to reach distances over 4 mm and over time appeared to be degenerating. Thin, vimentin-positive processes were found among these nerve fibers. As the first growth was retracted, a second outgrowth was initiated and formed on migrating astrocytes. TH- and aldehyde dehydrogenase-1 (ALDH1)-positive nerve fibers formed both the nonglia-associated and the glia-associated outgrowth. In cultures with membrane inserts, only the glia-associated outgrowth was found. Vimentin-positive cells preceded migration of NG2-positive oligodendrocytes and Iba-1-positive microglia. Oligodendrocytes appeared not to be involved in guiding neuritic growth, but microglia was absent over areas dense with TH-positive neurons. In conclusion, in "roller-drum" cultures, nerve fibers are generally formed in two sequences. The early-formed nerve fibers grow in the presence of thin, vimentin-positive processes. The second nerve fiber outgrowth is formed on astroglia, with no correlation to the presence of oligodendrocytes or microglia. ALDH1-positive nerve fibers, presumably derived from A9 dopamine neurons, participate in formation of both sequences of outgrowth.

Inhibition of proteoglycan synthesis affects neuronal outgrowth and astrocytic migration in organotypic cultures of fetal ventral mesencephalon

Grafting fetal ventral mesencephalon has been utilized to alleviate the symptoms of Parkinson's disease. One obstacle in using this approach is the limited outgrowth from the transplanted dopamine neurons. Thus, it is important to evaluate factors that promote outgrowth from fetal dopamine neurons. Proteoglycans (PGs) are extracellular matrix molecules that modulate neuritic growth. This study was performed to evaluate the role of PGs in dopamine nerve fiber formation in organotypic slice cultures of fetal ventral mesencephalon. Cultures were treated with the PG synthesis inhibitor methyl-umbelliferyl-beta-D-xyloside (beta-xyloside) and analyzed using antibodies against tyrosine hydroxylase (TH) to visualize dopamine neurons, S100beta to visualize astrocytes, and neurocan to detect PGs. Two growth patterns of TH-positive outgrowth were observed: nerve fibers formed in the presence of astrocytes and nerve fibers formed in the absence of astrocytes. Treatment with beta-xyloside significantly reduced the distance of glial-associated TH-positive nerve fiber outgrowth but did not affect the length of the non-glial-associated nerve fibers. The addition of beta-xyloside shifted the nerve fiber growth pattern from being mostly glial-guided to being non-glial-associated, whereas the total amount of TH protein was not affected. Further, astrocytic migration and proliferation were impaired after beta-xyloside treatment, and levels of non-intact PG increased. beta-Xyloside treatment changed the distribution of neurocan in astrocytes, from being localized in vesicles to being diffusely immunoreactive in the processes. To conclude, inhibition of PG synthesis affects glial-associated TH-positive nerve fiber formation in ventral mesencephalic cultures, which might be an indirect effect of impaired astrocytic migration.

Effects of glial cell line-derived neurotrophic factor deletion on ventral mesencephalic organotypic tissue cultures

Glial cell line-derived neurotrophic factor (GDNF) is potent for survival and promotion of nerve fibers from midbrain dopamine neurons. It is also known to exert different effects on specific subpopulations of dopamine neurons. In organotypic tissue cultures, dopamine neurons form two diverse nerve fiber growth patterns, targeting the striatum differently. The aim of this study was to investigate the effect of GDNF on the formation of dopamine nerve fibers. Organotypic tissue cultures of ventral mesencephalon of gdnf gene-deleted mice were studied. The results revealed that dopamine neurons survive in the absence of GDNF. Tyrosine hydroxylase immunoreactivity demonstrated, in gdnf knockout and wildtype cultures, nerve fiber formation with two separate morphologies occurring either in the absence or the presence of astrocytes. The outgrowth that occurred in the absence of astrocytes was unaffected by gdnf deletion, whereas nerve fibers guided by the presence of astrocytes were affected in that they reached significantly shorter distances from the gdnf gene-deleted tissue slice, compared to those measured in wildtype cultures. Treatment with GDNF reversed this effect and increased nerve fiber density independent of genotype. Furthermore, migration of astrocytes reached significantly shorter distances from the tissue slice in GDNF knockout compared to wildtype cultures. Exogenous GDNF increased astrocytic migration in gdnf gene-deleted tissue cultures, comparable to lengths observed in wildtype tissue cultures. In conclusion, cultured midbrain dopamine neurons survive in the absence of GDNF, and the addition of GDNF improved dopamine nerve fiber formation — possibly as an indirect effect of astrocytic stimulation.

Glial-cell-line-derived neurotrophic factor induces nerve fibre formation in primary cultures of adrenal chromaffin cells.

Neurotrophic factors, such as nerve growth factor (NGF), have been shown to promote the differentiation of neural crest neuroblasts into sympathetic neurons, whereas glucocorticoids promote the endocrine phenotype of adrenal medullary chromaffin cells. This pluripotency is preserved to some extent in adult chromaffin cells, with NGF and other neurotrophic factors influencing the differentiation of these cells. In this study, the effects of glial cell line-derived neurotrophic factor (GDNF) on explanted chromaffin tissue have been investigated. The localization of mRNAs corresponding to the two components of the GDNF receptor, GDNF family receptor alpha 1 (GFRalpha1) and Ret, were demonstrated in adult adrenal medullary ganglion cells. GFRalpha1 mRNA was expressed in explanted chromaffin tissue at levels dependent on the presence of serum in the medium but decreased on the addition of blocking antibodies against transforming growth factor beta (TGFbeta). However, TGFbeta1 (1 ng/ml) did not upregulate GFRalpha1 mRNA expression when added to serum-free medium. GDNF induced neurite formation from chromaffin cells, as measured by the ratio of neurite-bearing versus total number of chromaffin cells in primary cultures of adult adrenal medulla. The most potent dose inducing neurites from chromaffin cells was 100 ng/ml GDNF. However, this dose was not as efficient as that seen when chromaffin cells were stimulated with NGF (100 ng/ml). Thus, adrenal medullary cells express mRNAs for the GDNF receptor components Ret and GFRalpha1, increase their expression upon being cultured in serum-containing medium and respond to GDNF treatment with an increase in the number of cells that develop nerve processes.

Mutual Induction of TGFβ1 and NGF after Treatment with NGF or TGFβ1 in Grafted Chromaffin Cells of the Adrenal Medulla

Chromaffin cells have been recognized for their ability to transform into sympathetic ganglion-like cells in response to nerve growth factor (NGF) or to stimulation of other neurotrophic factors. Transforming growth factor β (TGFβ) family members have been shown to potentiate the effect of different trophic factors. The aim of this study was to investigate if TGFβ may influence NGF-induced neuronal transformation and regulation of NGF, TGFβ1, and their receptors in the adult rat chromaffin tissue after grafting. Intraocular transplantation of adult chromaffin tissue was employed and grafts were treated with TGFβ1 and/or NGF. Graft survival time was 18 days after which the grafts were processed for TGFβ luciferase detection assay, NGF enzyme immunoassay, or in situ hybridization. In grafts stimulated with NGF, increased levels of TGFβ1 and TGFβ1 mRNA were detected. When grafts instead were treated with TGFβ1, enhanced levels of NGF protein were found. Furthermore, a positive mRNA signal corresponding to the transforming growth factor II receptor (TβRII) was found in the chromaffin cells of the normal adrenal medulla as well as after grafting. No increase of TβRII mRNA levels was detected after transplantation or after TGFβ1 treatment. Instead a reduction of TβRII mRNA expression was noted after NGF treatment. NGF stimulation of grafts increased the message for NGF receptors p75 and trkA in the chromaffin transplants. Grafts processed for evaluations of neurite outgrowth were allowed to survive for 28 days and were injected weekly with NGF and/or TGFβ1. NGF treatment resulted in a robust innervation of the host irides. TGFβ1 had no additive effect on nerve fiber formation when combined with NGF. Combined treatment of NGF and anti-TGFβ1 resulted in a significantly larger area of reinnervation. In conclusion, it was found that NGF and TGFβ1 may regulate the expression of each other's protein in adult chromaffin grafts. Furthermore, TβRII mRNA was present in the adult rat chromaffin cells and became downregulated as a result of NGF stimulation. Although no synergistic effects of TGFβ1 were found on NGF-induced neurite outgrowth, it was found that TGFβ1 and NGF signaling are closely linked in the chromaffin cells of the adrenal medulla.

Lazaroid-enhanced survival of grafted dopamine neurons does not increase target innervation.

The lazaroid U-74006F enhances survival of grafted ventral mesencephalic neurons. In this study the intraocular grafting model was used and survival and outgrowth from fetal ventral mesencephalic grafts treated with U-74006F was evaluated in nigrostriatal co-grafts. Fetal lateral ganglionic eminence was implanted into the anterior eye chamber and left to mature. Fetal ventral mesencephalon was then implanted and the eyes were treated with U-74006F. The lazaroid treatment enhanced survival of tyrosine hydroxylase (TH)-positive neurons, but did not enhance TH-positive nerve fiber growth into the striatal portions of the co-grafts. However, a marked increase in nerve fiber formation was found within the ventral mesencephalic grafts. In conclusion, increased cell survival enhanced nerve fiber formation within the ventral mesencephalic portion of the co-graft and not, as expected, in the striatal part.

Tirilazad mesylate increases dopaminergic neuronal survival in the in Oculo grafting model.

Grafting of fetal ventral mesencephalon in Parkinson's disease has been extensively studied. A crucial draw back of this technique is the low survival rate of the dopaminergic neurons. It has been documented that only 5-20% of the grafted neurons survive, and to enhance graft efficacy to a satisfying level, increased cell survival is of utmost desire. In this study we have used the antioxidant tiriliazad mesylate (U-74006F) to study the effect on the survival of dopaminergic neurons after grafting. The in oculo grafting model was used and ventral mesencephalon was dissected from E14-E15 rat fetuses in Hanks' balanced salt solution (HBSS), in Dulbecco's modified Eagle medium (DMEM), or in 0.3, 3.0, or 30 microM U-74006F diluted in DMEM. The tissue was then inserted into the anterior chamber of the eye. Some of the transplants were further treated with intraocular injections of 3 or 30 microM U-74006F (5 microliters) weekly for 2 weeks. Quantification of tyrosine hydroxylase (TH)-immunoreactive profiles revealed that in transplants treated with U-74006F at dissection only, no change in the number of TH-positive neurons was found. Pretreatment of 0.3 microM U-74006F during dissection combined with intraocular injections of U-74006F after grafting, on the other hand, resulted in a dose-dependent enhancement of survival of TH-positive neurons. Dissection in, and intraocular treatment with, 3 microM U-74006F resulted in a significantly enhanced survival of TH-positive neurons whereas using U-74006F at a concentration of 30 microM did not change the cell survival compared to solely DMEM-treated grafts. Thus, 30 microM was interpreted to be an overdose. Comparing cell survival when dissected in DMEM with that dissected in HBSS showed that DMEM was clearly superior. Nerve fiber formation was most pronounced in grafts treated with 3 microM U-74006F. In conclusion, survival of TH-positive neurons is enhanced by U-74006F, which is readily available for clinical use and thus could be employed to enhance graft survival when transplanting patients suffering from Parkinson's disease.

Directed expression of the growth-associated protein B-50/GAP-43 to olfactory neurons in transgenic mice results in changes in axon morphology and extraglomerular fiber growth

B-50/GAP-43, a neural growth-associated phosphoprotein, is thought to play a role in neuronal plasticity and nerve fiber formation since it is expressed at high levels in developing and regenerating neurons and in growth cones. Using a construct containing the coding sequence of B-50/GAP-43 under the control of regulatory elements of the olfactory marker protein (OMP) gene, transgenic mice were generated to study the effect of directed expression of B-50/GAP-43 in a class of neurons that does not normally express B-50/GAP-43, namely, mature OMP-positive olfactory neurons. Olfactory neurons have a limited lifespan and are replaced throughout adulthood by new neurons that migrate into the upper compartment of the epithelium following their formation from stem cells in the basal portion of this neuroepithelium. Thus, the primary olfactory pathway is exquisitely suited to examine a role of B-50/GAP-43 in neuronal migration, lifespan, and nerve fiber growth. We find that B-50/GAP-43 expression in adult olfactory neurons results in numerous primary olfactory axons with enlarged endings preferentially located at the rim of individual glomeruli. Furthermore, ectopic olfactory nerve fibers in between the juxtaglomerular neurons or in close approximation to blood vessels were frequently observed. This suggests that expression of B-50/GAP-43 in mature olfactory neurons alters their response to signals in the bulb. Other parameters examined, that is, migration and lifespan of olfactory neurons are normal in B-50/GAP-43 transgenic mice. These observations provide direct in vivo evidence for a role of B-50/GAP-43 in nerve fiber formation and in the determination of the morphology of axons.

Effects of Glial Cell Line-Derived Neurotrophic Factor on Developing and Mature Ventral Mesencephalic Grafts in Oculo

The search for trophic factors that can support injured dopaminergic neurons and can enhance dopaminergic graft survival and outgrowth for therapeutic uses in Parkinson's disease has lately focused on members of the transforming growth factor (TGF) β superfamily. In this paper we have studied the effects of a member of the TGBβ family, glial cell line-derived neurotrophic factor (GDNF), on immature and mature ventral mesencephalic tissue grafted to the anterior chamber of the eye. The results confirm that GDNF increases survival of TH-positive neurons and enhances TH-immunoreactive nerve fiber formation when the grafts are treated during their development. The distribution of nerve terminals is densest within the area of TH-immunoreactive neurons and at the surface of the grafts. However, there is no change in the number of calcium-binding protein (CaBP)-positive neurons, suggesting that the subpopulation of TH-positive neurons that is increased are the CaBP-negative neurons of the ventral tier of pars compacta. Terminals from those neurons form the striatal patches during normal development. When the grafts are treated with GDNF after maturation, no change in TH-positive cell survival is seen but an increase of nerve terminals is still found within the cell dense area of the graft. Potassium-evoked dopamine release, measured using in vivo chronoamperometry, revealed significantly increased extracellular overflow in transplants treated with GDNF during development. The dopamine uptake blocker nomifensine significantly increased the time for clearance of the released dopamine. These data suggest that GDNF treatment of immature grafts enhances survival of TH-positive neurons, which would have innervated the striatal patches, and also increases TH-immunoreactive nerve fiber formation and dopamine release. Furthermore, GDNF treatment of mature grafts also increases dopamine fiber formation within the TH-positive neuronal area, indicating that adult dopaminergic neurons are also responsive to this agent.

Nerve fiber production by intraocular adrenal medullary grafts: stimulation by nerve growth factor or sympathetic denervation of the host iris.

This study evaluates the production of adrenergic nerve fibers by adrenal medullary tissue of the adult rat grafted to the anterior chamber of the eye of adult recipients. The chromaffin grafts attach to and become vascularized by the host iris. They decrease in size intraocularly during the first 3 weeks. This decrease is somewhat counteracted by sympathetic denervation of the host iris, and better counteracted by sympathetic denervation and addition of nerve growth factor (NGF, given at grafting and 1 and 2 weeks after grafting). Outgrowth of adrenergic nerve fibers from the grafts into the host iris was studied in wholemount preparations by use of the Falck-Hillarp technique 3 weeks after grafting. The innervated area of the host iris was approximately doubled in the chronically sympathectomized group and doubled again in the chronically sympathectomized NGF-supplemented group. Chronic sympathetic denervation had no effect on density of outgrowing nerves, whereas addition of NGF more than doubled nerve density. Since sympathetic denervation causes a slight elevation of NGF activity in the iris, the present experiments are taken as evidence that the level of NGF in the iris regulates formation of nerve fibers by adrenal medullary tissue grafts from adult rats.

A method to separate spatially the temporal sequence of axon-Schwann cell interaction during nerve regeneration.

This paper describes a surgical method of manipulating feline peripheral nerve regeneration to separate spatially the temporal sequence of events of axon-Schwann cell interaction during nerve fibre formation. The method allows regenerating axons from the peroneal nerve to reinnervate the distal stump of a axon- and myelin-free, Schwann cell-enriched, chronically denervated tibial nerve distal stump. Three zones can be morphologically delineated in the tibial nerve stump after three weeks of reinnervation: (1) a proximal myelinated zone, (2) a more distal, non-myelinated, axon-Schwann cell contact zone, and (3) a distal axon-free Schwann cell non-contact zone. The distal limit of the second zone can be determined accurately by the front of an axonally transported label. The large volume of available tissue makes this method suitable for interdisciplinary studies to elucidate the control of axon elongation, axon growth, and axon-Schwann cell inter-relationships.

Colchicine inhibition of nerve fiber formation in vitro.

Inhibition of nerve fiber (neurite) formation by colchicine and Colcemid was studied in monolayer cultures of dissociated spinal ganglia of the chick. Replica cultures were fixed after appropriate incubation and alkaloid treatment. Quantitative estimates of the mean total neurite length per neuron (MNL) were made by use of camera lucida tracing. MNL values plotted against time of incubation gave control curves with an initial lag period, a phase of rapid increase, and a final phase in which MNL increase was retarded. Colchicine at 0.01-0.05 microg/ml (2.4 x 10(-8)-1.2 x 10(-7)M) caused reversible, concentration dependent, inhibition of the increase in MNL when applied during the lag period or phase of rapid increase. At the highest concentration there was a net decrease in MNL. The effect of Colcemid at 0.05 microg/ml was similar to that of colchicine, but more rapidly reversible. In most experiments there was no loss of neurons during the period of inhibition of MNL increase by colchicine or Colcemid. Therefore selective destruction of neurons was not involved in the inhibition of neurite growth. Prolonged incubation after treatment with the highest concentration used resulted in a 50% loss of neurons, in part through detachment of viable cells. Quantitative radioautography of the alkaloid-treated neurons with leucine-(14)C indicated little or no inhibition of incorporation into protein during inhibition of MNL increase. The results strongly suggest that inhibition of neurite growth involves a specific effect of colchicine, presumably the disruption of microtubules. They are thus consistent with the hypothesis that the polymerization of microtubules is essential to the formation of nerve fibers.

Early formation of nerve fibers in the human otocyst

Early formation of nerve fibers in the human otocyst

STUDIES ON THE PERIPHERAL NERVE ROUTE REACHING THE LIP OF THE GUINEA PIG, AND, DISTRIBUTION OF, NERVES, ESPECIALLY OF THE AUTONOMIC NERVES. IN THE. LIP

Peripheral nerve routes reaching to the lip and palate were studied in the guinea pig sacrificed by suboccipital puncture, and distribution of the nerve fibers, especially that of autonomic nerve fibers was studied systematically. The following is the findings obtained in these studies:1) The trigeminal nerve which supplies the fibers to the lip and palate divide into three main brances, i. e., Nn. ophthalmicus, maxillaris and mandibulais, after emerging from the side of the pons and passing through the semilunar ganglion. The lip and palate are innervated by fine branches of the anterior superior alveolar nerve which branches off from the maxillary nerve, passes through Foramen lacerum anterior, reaches Fossa pterygopalatina and passes through the suborbital nerve canal with zygomatico-temporal branch and zygomatico-facial brach.2) The sympathetic nerve fibers are contained in the maxillary nerve, and are communicating, in their course, with perivascular plexuses and are distributed abundantly in various areas along with small vessels. Parasympathetic nerve fibers are difficult to distinguish histologically from the sympathetic nerve fibers and it is difficult to pursue, their course in detail.3) The lip is covered with cornifid squamous epithelium, with well-developed dermal papillae, and its cutis consists of loose connective tissue with a good supply of capillaries. Many striated muscle fiders running in an irregular fashion are seen in the subcutaneous adipose tissue. Skin appendages can also be seen, demonstrating the dermal nature of the lip.4) With the aid of silvar stains, it is demonstrable that the nerve bundles which are the mixture of various nerve fibers, divide into smaller bundles after reaching the periosteum, traverse the muscle layer and reach the subcutaneous tissue where they from loose nerve plexuses. The fibers branching off from these plexuses form compact plexuses within the cutis. Smaller nerve. pixeuss can also be seen within the papillae and adjacent to the basal membrane.Autonomic nerve fibers ere present mainly around the vessels in abundance, forming there fine vegetative terminal reticulum. Sensory nerve fibers form undividing or simply dividing terminal endings within the papillae. Intradermal fibers ars also demonstrable relatively in abnddance.The palate belongs histologically to the mucous membrane with few papillae formation and poorer supply of nerve fibers compared to the human palate.On the basis of these findings, histological studies of these regions of the guinea pig were made from the view-point of the comparative anatomy. Clinical significance of the anatomy of the neck, which was pointed out by H. Stolze, was discussed.A numbers of studies on Reilly's phenomen have been under-taken in our department by Suzuki, Iida, Fukazawa, Takasaki and Suzuki. Inoue, also from our department, has performed a series of experimental studies in which he demonstrated Iesions in the cervical and abdominal ganglia in the animal receiving irritating stimuli to its pharyngeal mucous membrane.The purpose of my studies has been to give support from anatoinieal-histolagical view-point to the results of these studies as well as to the concept that the autonomic nerve plays an impor-tant role in above mentioned studies.