YANG Lan-ying, LIU Zai-qun, ZOU Xue-mei

(College of Life Sciences, Anhui Normal University, Wuhu 241000, China)

Introduction

Pachyhynobiusshangchengensisbelongs to Urodela HynobiidaePhchyhynobius[1], which is endemic to the Dabie Mountain in China and is an endangered stream salamander. The distributions of theP.shangchengensisinvolved are five small districts such as Shangcheng, Jinzhai, Huoshan, Yuexi and Yingshan, with an extremely narrow distribution habitat[2].

The researches about theP.shangchengensiswere focused on the histologic and immunohistochemical studies in skin, digestive tract, blood, muscles, and genetic material, etc. Wu et al[3]used HE staining to observe the histology of shin onP.shangchengensis. They found that the thickness of skin in different parts was different and the distribution of granular and mucous glands was regular. Lv et al[4]used HE staining to study the form, length, gross structure and micro-structure of the digestive tract of theP.shangchengensis. They found that the digestive tract was different in mucoso and muscularis among the different segments such as esophagus, stomach and intestine. Wang et al[5]used Giemsa staining to observe the morphology of blood cell and tested the blood ofP.shangchengensis. They identified erythrocytes, monocytes, lymphocytes, thrombocytes, eosinophilic granulocytes, basophilic granulocytes and neutrophilic granulocytes from the blood smear ofP.shangchengensis. Wang et al[6]used the FLASCO (fast isolation by AFLP of sequences containing repeats) approach to isolate di-nucleotide/tetra-nucleotide repeat microsatellite loci and to analyze the sequences for the purpose of learning how microsatellite structure impacted on the isolation efficiency. They found that most di-nucleotide repeat microsatellite loci were involved in the microsatellite DNA family, which resulted in low isolation efficiency. Chen et al[7]used the acrylamide gel electrophoresis to study the esterase (EST) and peroxidase (POD) isozyme patterns in skin, muscle, spleen, intestine, liver, lung ofP.shangchengensis. The results showed that there were great differences in patterns tissues of interspecies. He et al[8]used different fixatives and staining methods to observe mast cells (MCs) from digestive system, some of other organs inP.shangchengensis, then statistically counted and analyzed the cell numbers in digestive tract and tongue. They found that improved toluidine blue staining (MTB) could be used to display MCs in all organs except for the fallopian tube and kidney in 3 fixatives，but Alcian blue-Safranin O staining (AB-S) displayed AB+MCs and S+MCs in digestive tract and only AB+MCs in other organs．

Brain-derived neurotrophic factor (BDNF) mainly distributed in central nervous system and had the most extensive distribution in different parts of brain[9]. The positive of BDNF was not only found in cell body, but extended into fiber of the neuron in cerebral cortex, hippocampus, striatum et al. BDNF had a widely effect on the peripheral nervous system and central nervous system. It could help to promote survival, differentiation, regeneration, metabolism of the various neurons and had the function of nutrition, support and protection. At the same time, BDNF also could improve the survival of sensory neurons and motor neurons whose axon were cut in postoperative, promote the repair and regeneration of neurons and reduce the normal death of motor neurons. In addition, BDNF could strengthen the release of neurotransmitter, the synaptic connections between synapse, stimulate the budding of axon and dendrites in adult neuron, regulate anabolism and increase the size of the neuron cell body. At the same time, BDNF could regulate the secretion of neurotransmitters such as acetylcholine and dopamine, promote the decline and fall of nerve cells and against the traumatic stimulus, and had an important influence on the learning and memory behavior′s change under the condition of stressing[10].

Interleukin-1 (IL-1) played an important role in the development of neurons system within the embryo development of rats by contacting with Interleukin-1 receptor (IL-1R), promoted the synthesis of neurons and the release of neurotransmitter. IL-1 included two subtypes, IL-1α and IL-1β[11-14]. IL-1α could induce neural stem cells differentiate into neurons and glia cells in the midbrain of rats[16], produced reactive oxygen species (ROS) in the glial cells of the cerebellum of Embryonic rat, which played an important role in the inflammatory response[16].

In this present, the reports about the neurons system ofP.shangchengensiswere generally lacking. We used the histologic and immunohistochemical methods to research the spinal cord and the spinal ganglia ofP.shangchengensisto provide a theoretical basis for the nervous system′s research ofP.shangchengensis.

Material and methods

The adult specimens ofP.shangchengensiswere used. Their tissues were fixed in 4% formaldehyde solution for 4 hours, routinely processed for paraffin embedding and tissue sections mounted onto 0.02% APES-coated slides. Sections were stained by HE-Staining, Nissl-Staining, and Homes-Silver-Staining.

Immunocytochemistry

SABC-Staining sections were treated sequentially in the following processes: immersed in 3% H2O2for 20 minutes at 37℃; rinsed in PBS for three times and incubated during 5 minutes at 37℃; incubated in 5% bovine serum albumin (BSA) for 20 minutes at 37℃； then incubated anti-serum for 10% BDNF and IL-1α at 37℃ for 2 h; rinsed in PBS for three times and incubated during 2 minutes at 37℃； incubated in goat anti-rabbit IgG at 37℃ for 20 minutes； rinsed in PBS for three times and incubated during 2 minutes at 37℃； incubated in SABC at 37℃ for 20 minutes； rinsed in PBS for four times and incubated during 5 minutes at 37℃； then incubated in DAB (A: 50 μL and B:50 μL pusl 1 mL 1×HPR buffer ) for 5-20 minutes under room temperature (25℃). BDNF and IL-1α were replaced by PBS in the control experiment and the other procedures were the same as above.

Result

Thehistologicalstructureofthespinalcord

The spinal cord ofP.shangchengensisstorecould be divided into white matter and gray matter after stained by HE-staining and Nissl-staining (Plate 1). The white matter located in the periphery, and the gray matter located in the center, whose staining was deeper than the white matter (Plate 1: Wh). There were several grooves in the surface of spinal cord. We could see the hallers line and dorsal median suleus clearly. Hallers line was wider than dorsal median suleus (Plate 1: Vm, Dc).

Thegraymatterofthespinalcord

The gray matter was butterfly shape, which had two wide anterior horn (Plate 1: Vh) and two narrow posterior horn on both sides (Plate 1: Dh). In the centre of the gray matter, there was an irregular circle which was surrounded by ependymal cells, we called it central canal (Plate 1, 8, 9: Cc). The ependymal cells of spinal central canals were surrounded by pseudostratified columnar epithelium cells (Plate 8: Ep). IL-1α expressed in them (Plate 9: Ep).

Thespinalmater

There were three maters out the spinal cord, which were the dura mater, the arachnoid mater and the pia mater from outside to inside (Plate 1,10： c, b, a). The dura mater was harder and connected with spinal nerve through the foramen intervertebrale. There was an epidural space between the dura mater and spinal canal or periosteum which contains blood vessels and fat. The thin and transparent arachnoid mater was loose reticulate structure (Plate 10: b). The pia mater closed to the surface of the spinal cord which was rich in blood vessels and went deeply into the ditch crack (Plate 11).

Thestructureofthespinalganglia

Histologicstaining

The irregular ovoid spinal ganglia located in the posterior root of spinal nerve, was covered by the capsule with HE-Staining and Homes-silver-staining (Plate 5: d), which were irregular circle (Plate 2). There were two kinds of ganglion cells in the spinal ganglias. One was the ganglion cell, whose body was roundness or ovoid (Plate 3, 4, 5). It could be divided into big spinal ganglion cell (Plate 3, 4, 5 :↓) and small spinal ganglion cell (Plate 3, 4, 5 :←) according to the different size of their cell bodies(P<0.05). Their bodies were covered by a layer of satellite cells (Plate 4,5:↑). There was a nucleus in the center or eccentric of the spinal ganglion cells place which was more deep stained by Homes-silver-staining.

BDNFandIL-1αImmunohistochemicalstaining

The spinal ganglion cells were positive but the satellite cells were negative through the BDNF and IL-1α staining (Plate 6, 7). However, the big ganglion cell’s positive (Plate 6: ↓) was significantly stronger than the small ganglion cell’s positive (Plate 6: ←) by the BDNF staining (P<0.05), but there was no significant difference between them through the IL-1α staining (Tab. 1).

Table 1 The spinal ganglion cells and the optical densities(OD) BDNF and IL-1α-immunoreactive cells in spinal ganglion (mean±SD)

In the same line, the superscripts with different letters means significant difference (P<0.05), whereas, with same letters mean insignificant difference (P>0.05).

plate 1 HE staining, the overall concept of spinal cord and spinal ganglia. WH: white matter, Vh: anterior horn, Dh: posterior horn, Cc: central canal, Vm: Hallers line, Dc: dorsal median suleus, a: the pia mater, b: the arachnoid mater, c: the dura mater. 4；plate 2 HE staining, the position of the spinal ganglion. 4；plate 3 Nissl-staining, the structure of the spinal ganglion. spinal ganglion cell (↓), ×20; plate 4 HE staining, the structure of the spinal ganglion.spinal ganglion cell (↓), satellite cells (↑), ×20; plate 5 Homes-silver-staining, the structure of the spinal ganglion. Big spinal ganglion cells(↓), small spinal ganglion cells (←), satellite cells(↑), d: capsule, ×20; plate 6 BDNF experession in the the spinal ganglion. big spinal ganglion cells(↓), small spinal ganglion cells (←), satellite cells(↑), d: capsule, ×20; plate 7 IL-1α experession in the the spinal ganglion. big spinal ganglion cells(↓), small spinal ganglion cells (←), ×20; plate 8 Homes-silver-staining, Cc: central canal, Ep: the ependymal cells, ×20; plate 9 IL-1α experession in the spinal ganglion. Cc: central canal, Ep: the ependymal cells, ×20; plate 10 Nissl-staining, a: the pia mater, b: the arachnoid mater, c: the dura mater, ×20; plate 11 Nissl-staining, a: the pia mater, ×20; plate 12 Negative control, ×20.

Disscussion

Thecentralcanal

The central canal ofP.shangchengensisstorewas irregular circle, which was different withRanacatesbeiana, whose central canal was irregular triangle, and its diameter was about 369.33 μm, which was bigger thanRanacatesbeiana’s, but the ependymal cells of them were all pseudostratified columnar epithelium cells[17]by Homes-silver-staining and IL-1α staining.

It was proven that the IL-1α was expressed in the ependymal cells by IL-1α staining. IL-1 could promote the prolifer-ation and differentiation of glial cells. So the ependymal cells of the spinal cord had the potential ability of proliferation differentiation[18].

Thespinalganglia

It was showed that the structure of the spinal ganglia between the other mammals andPachyhynobiusshangchengensisstorewas similar by HE-staining, Nissl-staining and Homes-silver-staining. They were all composed with capsule, satellite and ganglion cells. The ganglion cells could be divided into two subtypes based on the size of their body. The positive of big the ganglion cell was deeper than that of the small ganglion cell. However, the nucleus of small ganglion cells were extremely obvious, while the ganglion cells′ were not.

The family of neurotrophic factors was mainly target-derived neurotrophic factors. They were produced by the target tissue, mediated by axon terminal receptors, reverse transported to the cell body by the neuronal axons so as to promote the expression of neural cell survival, growth, differentiation[19]. The recent researchers also found that these neurotrophic factors played an important role in the biological regulation of embryonic development, cell differentiation, wound healing, immune regulation and tumorigenesis etc. These neurons, as the effect cells of the neurotrophic factors, also had the ability to secrete the neurotrophic factors. We called this phenomenon as autocrine effect of the neurotrophic factors. It had been reported that the central nervous cells and peripheral ganglion cells had the expression of neurotrophic factors not in embryonic development, but in adult animals[20, 21]. BDNF had a wide effect on the peripheral nervous system and central nervous system. It helped to promote survival, differentiation, regeneration, metabolism of the various neurons and had the function of nutrition, support, protection. Wang[22]et al had reported the expression of BDNF in the developing of spinal cord in chicken embryo. Our researcher also found that BDNF was expressed in the spinal cord and spinal ganglia ofP.shangchengensis. The difference positive between the big and small ganglion cells had proved that BDNF played an important role in the regulation of the size of the cell body.

IL-1α was positive expression in the spinal ganglia cells, but the positive intensity had no significant difference between the big and small ganglion cells, which told us that there was no significant correlation between the size of cell body and IL-1α.

The role of BDNF and IL-1α in spinal ganglion cells remained to be further study by using molecular means.

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