Expression of DRD1 mRNA after Spinal Cord Injury Induced Spasticity in Rats

[Objectives]To investigate the spasticity of rat tail and the expression of dopamine receptor-1(DRD1)mRNA in the spinal cord after spinal cord injury(SCI)induced tail spasticity in rats.[Methods]Adult male Wistar rats were randomly divided into Sham group and SCI group.The second sacral spinal cord(S2)segment of SCI rats was completely transected.60 d after operation,the rat tail spasticity was scored,and then the spinal cord tissues below the level of S2 spinal cord transection were taken.The expression of DRD1 mRNA in the sacrococcygeal spinal cord was detected by qPCR.In addition,3 normal rats were used for DAR/neuronal nuclei(NeuN)and DRD1/choline acetyltransferase(ChAT)immunofluorescence staining to study the distribution of DRD1 in spinal cord and the properties of DRD1 positive cells.[Results]60 d after operation in SCI group,the tail spasticity of rats developed fully,and the symptoms of spasticity were typical.qPCR results showed that the expression of DRD1 mRNA in SCI group was significantly lower than that in Sham group(P<0.05).DRD1 was widely distributed in the dorsal horn,intermediate zone and ventral horn at the sacrococcygeal end of the rat spinal cord.[Conclusions]The decrease of DRD1 mRNA expression after SCI may be related to the occurrence and development of spasticity.

Expression pattern of neuregulin-1 type Ⅲ during the development of the peripheral nervous system

Neuregulin-1 type Ⅲ is a key regulator in Schwann cell proliferation, committing to a myelinat- ing fate and regulating myelin sheath thickness. However, the expression pattern of neuregulin- 1 type III in the peripheral nervous system during developmental periods （such as the premyelin- ating stage, myelinating stage and postmyelinating stage） has rarely been studied. In this study, dorsal root ganglia were isolated from rats between postnatal day 1 and postnatal day 56. The expression pattern of neuregulin-1 type III in dorsal root ganglia neurons at various develop- mental stages were compared by quantitative real-time polymerase chain reaction, western blot assay and immunofluorescent staining. The expression of neuregulin-I type Ⅲ mRNA reached its peak at postnatal day 3 and then stabilized at a relative high expression level from postnatal day 3 to postnatal day 56. The expression of neuregulin-1 type III protein increased gradually from postnatal day 1, reached a peak at postnatal day 28, and then decreased at postnatal day 56. Immunofluorescent staining results showed a similar tendency to western blot assay results. Experimental findings indicate that the expression of neuregulin-1 type III in rat dorsal root ganglion was increased during the premyelinating （from postnatal day 2 to postnatal day 5） and myelinating stage （from postnatal day 5 to postnatal day 10）, but remained at a high level in the postmyelinating stage （after postnatal day 10）.

Human chromosome pellicle antibody recognizing centromere protein-C(CENP-C),the main component of the kinetochore

Recently the antichromosome antisera from several scleroderma patients have been found to recognize the pellicle of metaphase and anaphase chromosomes. In order to identify the pellicle components, we used these antichromosome antisera to screen a human embryonic cDNA library. The sequences of the positive clones are identical to the cDNA gene sequence of CENP-C (centromere protein C), a human centromere autoantigen. This result suggusts that CENP-C is a component of the pellicle of human metaphase and anaphase chromosomes.

CDH1, a Novel Surface Marker of Spermatogonial Stem Cells in Sheep Testis

Spermatogonial stem cells（SSCs） are unique stem cells in adult body that can transmit genetic information to the next generation. They have self-renewal potential and can continuously support spermatogenesis throughout life of a male animal. However, the SSC population is extremely small, isolation and purification of the SSCs is challenging, especially for livestock animals. It has been confirmed that CDH1（cadherin-1, also known as E-cadherin） can be expressed in undifferentiated SSCs of mouse and rats, but it has not been verified in sheep. Here, CDH1 was found as a novel surface marker for sheep SSCs. In this paper, sheep antiCDH1 polyclonal antibodies were prepared and its activity was checked. Using the obtained antibodies and immunohistochemistry analysis, we confirmed that CDH1 can be expressed by SSCs in sheep testis.

Calpain mediated cisplatin-induced ototoxicity in mice

Ototoxic drug-induced apoptosis of inner ear cells has been shown to be associated with calpain expression. Cisplatin has severe ototoxicity, and can induce cochlear cell apoptosis. This study assumed that cisplatin activated calpain expression in apoptotic cochlear cells. A mouse model of cisplatin-induced ototoxicity was established by intraperitoneal injection with cisplatin （2.5, 3.5, 4.5, 5.5 mg/kg）. Immunofluorescence staining, image analysis and western blotting were used to detect the expression of calpain 1 and calpain 2 in the mouse cochlea. At the same time, the auditory brainstem response was measured to observe the change in hearing. Results revealed that after intraperitoneal injection with cisplatin for 5 days, the auditory brainstem response threshold shifts increased in mice. Calpain 1 and calpain 2 expression significantly increased in outer hair cells, the spiral ganglion and stria vascularis. Calpain 2 protein expression markedly increased with an increased dose of cisplatin. Results suggested that calpain 1 and calpain 2 mediated cisplatin-induced ototoxicity in BALB/c mice. During this process, calpain 2 plays a leading role.

The distribution and cell uptake of ApoA1 modified lipid carriers of siRNA in mouse liver in vivo

Fluorescence labeled small interfering RNAs(siRNAs)were loaded into lipopolyplexes modified with ApoA1(named as rHDL)and administered by intravenous injection.The biodistribution with time of these lipopolyplexes inside the liver and among various cell types was followed using tissue sections by Confocal fluorescence microscopy.At about 0.5 h after tail vein injection at a dose of 0.408 mg/kg,very few fluorescence signals were found in the liver.But then the signals could be seen to accumulate inside hepatocytes as discrete spots and diffused signals at around 2e4 h after injection.Such a distribution and uptake pattern was significantly different from what were observed using the commercial agent Invivofectamine
2.0 or DOTAP lipoplexes as the carriers.The differences indicated different mechanisms concerning the in vivo behavior of these carriers.The rHDL carrier system we developed was able to deliver siRNA specifically into hepatocytes while avoiding the uptake by REM cells especially the Kupffer cells.With it’s low toxicity and off target effect,it may be suitable to be developed as a hepatocyte targeting delivery system for siRNA.

Antigen unmasking does not improve the visualization of phospholipase C zeta in human spermatozoa

Phospholipase C zeta(PLCζ)is a sperm-specific protein that triggers oocyte activation.The analysis of PLCζexpression in human spermatozoa can be used as a diagnostic marker for oocyte activation deficiency.Our laboratory has previously optimized a standard"in-house"assay to determine PLCζexpression in human spermatozoa.However,one study has suggested that an antigen unmasking method(AUM)would be more efficient in visualizing PLCζin human sperm.This study aimed to compare our established assay and AUM(involving HCl,acidic Tyrode's solution[AT],and heat).The mean relative fluorescence(RF)intensity of PLCζin frozen-thawed spermatozoa from fourteen fertile donors stained with the in-house method was significantly higher than three other AUM groups(in-house[mean±standard error of mean]:18.87±2.39 arbitrary units[a.u.]vs non-AUM:11.44±1.61 a.u.,AT-AUM:12.38±1.89 a.u.,and HCl-AUM:12.51±2.16 a.u.,P<0.05,one-way analysis of variance).The mean RF intensity of PLCζin AT-and HCl-treated spermatozoa from 12 infertile males was not significantly different from that of the non-AUM group.However,the in-house method resulted in the highest RF intensity(12.11±1.36 a.u.,P<0.01).Furthermore,specificity testing of antibody-antigen binding indicated that the in-house method showed more specific binding than spermatozoa treated by the AUM.In conclusion,our in-house method showed superior visualization and reliability than the AUM,thus supporting the continued use of our in-house assay for clinical research screening.

Vitrification of In vitro-matured Oocytes:Effects of Meiotic Spindle Morphology on Clinical Outcome

Objective:The meiotic spindle controls chromosome movement and mediates various functions essential for fertilization and early postfertilization events.This study aimed to examine whether vitrification causes meiotic damage in vitro-matured metaphase II(MII)human oocytes,and whether the meiotic spindle morphology influences the subsequent developmental outcomes.Methods:The spindle characteristics of MII human oocytes in vitro matured were studied before and after vitrification using PolScope imaging and immunofluorescence staining.The developmental competence of oocytes was also examined.Results:A total of 419 human MII oocytes were obtained from 593 intracytoplasmic sperm injection cycles at our hospital.Of these oocytes,54 were used for immunofluorescence staining,whereas the other oocytes were examined by PolScope imaging and classified into three groups according to the meiotic spindle morphology:(A)normal morphology,(B)weak refraction and short meiotic spindle,and(C)no detectable meiotic spindle.The three groups demonstrated statistically significant differences in terms of survival after vitrification.However,differences were not found in terms of oocyte chromosome structure and meiotic spindle morphology on immunofluorescence staining performed before and after vitrification.Oocyte survival,fertilization,and early embryonic development rates were significantly higher in Group A than in Groups B and C with or without vitrification.While vitrification had no effect on these metrics in Group A,Groups B and C demonstrated significantly lower fertilization and cleavage rates after vitrification/warming.Conclusions:Screening for normal meiotic spindle morphology and chromosome configuration before vitrification may increase the yield of healthy viable oocytes for various assisted reproductive technologies.