With its abilities of trans-synaptic tracing and self-replication and wide host range, pseudorabies virus (PRV) has been applied in the field of neuroanatomy since the 1970s. Four decades of PRV application have made many advances in researches on neuronal tracing with PRV. Mechanism studies focused on investigating infection of primary neurons and tracing direction in secondary neurons, while application studies focused on development of new pathological strains and innovation of tracing techniques. To date, the mechanism and application of viral tracing are not completely figured out yet. Integration of molecular biology technology will improve the efficiency in related researches.
Animals ; Cell Tracking ; Herpesvirus 1, Suid ; genetics ; physiology ; Humans ; Neurons ; virology ; Pseudorabies ; virology

BACKGROUNDTo establish a neurons model for herpes simplex virus type 1 (HSV-1) infection using cultures of mouse embryo.

METHODSThe cortical neuron of 14-16 day old mouse embryo were cultured and infected with HSV-1. Microscopic examination, immunofluorescent test, MTT colorimetric analysis and flowcytometric assay were used to detect the immunofluorescent reaction of normal and infected neurons and astrocytes, respectively. The percentage of the two kinds of cells were measured.

RESULTSThe mouse neurons could proliferate well in vitro. The neurons amount met the requirement of cytological experiment after treatment with Ara-C in the tissue culture. HSV-1 could infect the neurons directly, the activity of the infected neurons was obviously reduced.

CONCLUSIONHSV-1 could successfully infect the primary cultured neurons. The model may provide a useful approach for studying the pathogenesis of herpes simplex virus encephalitis.

Animals ; Cell Culture Techniques ; methods ; Cells, Cultured ; Embryo, Mammalian ; virology ; Female ; Herpes Simplex ; virology ; Herpesvirus 1, Human ; physiology ; Humans ; Male ; Mice ; Neurons ; virology

The delivery of transgenes to the central nervous system (CNS) can be a valuable tool to treat CNS diseases. Various systems for the delivery to the CNS have been developed; vascular delivery of viral vectors being most recent. Here, we investigated gene transfer to the CNS by intravenous injection of recombinant adenoviral vectors, containing green fluorescence protein (GFP) as a reporter gene. Expression of GFP was first observed 6 days after the gene transfer, peaked at 14 days, and almost diminished after 28 days. The observed expression of GFP in the CNS was highly localized to hippocampal CA regions of cerebral neocortex, inferior colliculus of midbrain, and granular cell and Purkinje cell layers of cerebellum. It is concluded that intravenous delivery of adenoviral vectors can be used for gene delivery to the CNS, and hence the technique could be beneficial to gene therapy.
Adenoviruses, Human/isolation & purification* ; Animals ; Blood-Brain Barrier ; Brain/virology* ; Cerebellum/cytology ; Cerebellum/virology ; Comparative Study ; Female ; Genes, Reporter ; Genetic Vectors/administration & dosage ; Genetic Vectors/isolation & purification ; Genetic Vectors/pharmacokinetics* ; Hippocampus/virology ; Inferior Colliculus/virology ; Injections, Intravenous ; Luminescent Proteins/analysis ; Luminescent Proteins/biosynthesis ; Luminescent Proteins/genetics ; Mice ; Mice, Inbred BALB C ; Neuroglia/virology ; Neurons/virology* ; Purkinje Cells/virology ; Pyramidal Cells/virology ; Recombinant Fusion Proteins/analysis ; Recombinant Fusion Proteins/biosynthesis ; Recombinant Fusion Proteins/genetics ; Tail/blood supply ; Tissue Distribution

OBJECTIVETo observe the effect of HSV-1 infection on the viability of the cultured chicken embryo telencephalon neurons in vitro.

METHODSPrimary culture model of chicken embryo of telencephalon neurons was established. Herpes simplex virus type 1 (HSV-1) was propagated in Vero cells and viral titer was measured by plaque forming method. Inverted microscope, transmission electron microscope, MTT cell viability and DNA agarose gels electrophoresis were used to evaluate the effect of HSV-1 infection on the neurons.

RESULTSAt 24, 48, 72 hours post infection the viability of the neurons decreased by 25%, 56%, 97% as compared with the control group. Morphological changes and DNA agarose gel electrophoresis showed a necrotic effect.

CONCLUSIONSHSV-1 infection induced a remarkable decrease on the viability of neurons by necrosis rather than apoptosis.

Animals ; Cercopithecus aethiops ; Chick Embryo ; Herpes Simplex ; pathology ; virology ; Herpesvirus 1, Human ; In Vitro Techniques ; Necrosis ; Neurons ; pathology ; Telencephalon ; cytology ; Vero Cells ; Virus Cultivation

PURPOSE: c-fos expression in spinal neurons that are activated by lower urinary tract stimulation are not organ specific. In this experiment, we demonstrated changes of c-fos expression in bladder-specific preganglionic neurons (PGNs) and interneurons using pseudorabies virus (PRV). MATERIALS AND METHODS: Forty Sprague-Dawley rats were used. We identified the neuronal pathway associated with the bladder by injecting PRV into the detrusor. An immunohistochemical method was used to stain Fos-protein encoded by the c-fos gene. Immunofluorescent staining for PRV was performed to evaluate changes in bladder-specific spinal neurons. RESULTS: Immunofluorescent staining with choline acetyltransferase (ChAT) revealed that the sacral parasympathetic nucleus (SPN) regions contained 9.8 PGNs/ section. In rats with chronic spinal cord injury by intravesical saline instillation, 82.4+/-10.3% of PGNs in SPN exhibited Fos-immunoreactive (IR). Two and a half days after PRV infection, PRV-IR PGNs were observed at 5.4 PGNs/ section, and 2.7+/-1.6% of them exhibited Fos-IR. Unlike ChAT-IR PGNs, PRV-IR PGNs are bladder-specific neurons and PRV-IR and Fos-IR cells found in the back of PRV-IR PGNs are bladder- specific interneurons. Three days after PRV infection, we observed many PRV-IR and Fos-IR cells in the dorsal commissure. These neurons are interneurons distributed in the bladder. CONCLUSION: We confirmed that in chronic spinal cord injury, the patterns of c-fos expression in bladder-specific spinal neurons were similar to those in voiding-reflex related spinal neurons, which had already been demonstrated earlier. We believe that our methodology can be applied to study interactions between voiding and other organs as well, such as the urethra and prostate.
Animals ; Female ; Herpesvirus 1, Suid/*physiology ; Immunohistochemistry ; Interneurons/cytology/metabolism ; Neurons/cytology/*metabolism ; Proto-Oncogene Proteins c-fos/*metabolism ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries/*physiopathology ; Urinary Bladder/cytology/*metabolism/virology

OBJECTIVETo study the apoptosis of facial motor neurons and the expression of apoptosis-related genes, Bcl-2 and Bax, in the animal model of viral facial paralysis.

METHODSTotal of 84 Balb/c mice were divided into viral inoculation group and nerve transaction group. The animals were executed 1, 3, 7, 10, 15, 20 and 30 days after being operated respectively. The histopathological features of facial neurons in brain stem were observed by HE and Nissl stain. The changes of facial neuronal apoptosis were observed by TUNEL. The changes of expression of Bcl-2 and Bax genes in facial neurons were observed by immunohistochemistry staining.

RESULTSAfter nerve transection, increased apoptotic cells were found in homolateral facial motor nucleus and the peak appeared at 10 and 15 days. The level of Bcl-2 expression in neurons declined while the expression of Bax increased gradually. Correspondingly, the ratio of Bcl-2/Bax declined. In the viral inoculation group, no visible change of apoptosis and Bax expression, but the level of Bcl-2 and the ratio of Bcl-2/Bax increased gradually.

CONCLUSIONSComparing to axotomy, facial motor nucleus in HSV-1 infective animal model are free of apoptosis. Both the mild form of lesion and the ability to block apoptosis of HSV-1 are likely to be involved into the phenomenon. Bcl-2 and Bax might interfere with the apoptotic response.

Animals ; Apoptosis ; Facial Paralysis ; pathology ; virology ; Female ; Herpesvirus 1, Human ; pathogenicity ; Mice ; Mice, Inbred BALB C ; Neurons ; pathology ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; bcl-2-Associated X Protein ; metabolism

The objective of present study is to investigate the effect of human cytomegalovirus (HCMV) infection on human hippocampus neural stem cells NSCs differentiation in vitro, Fetal hippocampus tissue was dissociated mechanically and then cultured in proliferation medium with EGF and bFGF. Immunofluorescence method was used to detect the expression of NSCs marker-Nestin within these cells. Cultured in 10% FBS, NSCs began to differentiate. On the onset of the differentiation, HCMV AD169 (MOI=5) was added into the differentiation medium. After 7 days differentiation, the effect of HCMV infection on NSCs differentiation was observed by detecting the rate of nestin, GFAP and HCMV immediate-early (IE) positive cells with confocal microscopy and immunofluorescence method. The resucts showed most of the cells (passage 4-6 ) were Nestin positive and could differentiate into NSE-positive neurons and GFAP-positive astrocytes. On day 7 postinfection, 86% +/- 12% of infected cells were IE positive. The percentage of Nestin-positive cells was 50% +/- 19% and 93% +/- 10% (t= 6.03, P<0.01)and those of GFAP-positive cells was 81% +/- 11% and 55 +/- 17% (t=3.77, P<0.01) in uninfected and infected cells respectively. These findings indicated that NSCs were HCMV permissive cell and HCMV AD 169 infection suppressed the differentiation of Hippocampus-genetic human neural stem cells into astrocytes.
Astrocytes ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Cytomegalovirus ; growth & development ; physiology ; Epidermal Growth Factor ; pharmacology ; Fibroblast Growth Factor 2 ; pharmacology ; Hippocampus ; cytology ; Humans ; Intermediate Filament Proteins ; metabolism ; Microscopy, Fluorescence ; Multipotent Stem Cells ; cytology ; drug effects ; metabolism ; virology ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neurons ; cytology

OBJECTIVECytomegalovirus (CMV) is the leading infectious cause of congenital anomalies of the central nervous system caused by intrauterine infection. However, the exact pathogenesis of these brain abnormalities has not been fully elucidated. It has been reported that periependymitis, periventricular necrosis and calcification are the most frequent findings in the brains of congenital CMV infection. Because a number of multipotential neural stem cells (NSCs) have been identified from ventricular zone, it is possible that NSCs in this area are primary targets for viral infection, which seems to be primarily responsible for the generation of the brain abnormalities. Therefore, the objective of the present study was to investigate the effect and mechanism of murine cytomegalovirus (MCMV) infection on neural stem cells' differentiation in vitro and its role in the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection.

METHODSNSCs were prepared from fetal BALB/c mouse and were infected with recombinant MCMV RM461 inserted with a report gene LacZ at 1 multiplicity of infection (MOI = 1). The effect of MCMV infection on neural stem cells' differentiation was observed by detecting the ratio of nestin, GFAP and NSE positive cells with immunohistochemistry and flow cytometry on day 2 postinfection. The effects of MCMV infection on gene expression of Wnt-1 and neurogenin 1 (Ngn1) related to neural differentiation were detected by RT-PCR.

RESULTSNSCs isolated from embryonic mouse brains strongly expressed nestin, a specific marker of NSCs and had the capacity to differentiate into NF-200 and NSE positive neurons or GFAP positive astrocytes. At MOI = 1, the results of flow cytometry assay showed that nestin positive cells' proportion in the infection group [(62.2 +/- 1.8)%] was higher than that in the normal group [(37.2 +/- 2.4)%] (t = 4.62, P < 0.01). At the same time, the rates of GFAP and NSE positive cells' in the infection group were significantly lower than those in the normal group (P < 0.01). The scanning densities of Wnt-1 was 0.14 +/- 0.03 in the infection group while 0.32 +/- 0.04 in the control group (t = 7.21, P < 0.01). The scanning densities of Ngn1 were 0.09 +/- 0.01 and 0.21 +/- 0.02 in the two groups (t = 10.7, P < 0.01).

CONCLUSIONSThese results suggest that MCMV infection could inhibit neuronal differentiation, which may be primary causes of disorders of brain development in congenital CMV infection. The decreased expression of Wnt-1 and Ngn1 may be involved in the inhibitory effect of murine cytomegalovirus infection on neural stem cells' differentiation, which may lead to a new strategy for preventing and treating brain abnormalities caused by CMV infection through regulating these two signal pathways.

Animals ; Astrocytes ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Brain ; cytology ; Carrier Proteins ; metabolism ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Cytomegalovirus Infections ; congenital ; Embryo, Mammalian ; cytology ; Female ; Glial Fibrillary Acidic Protein ; metabolism ; Immunohistochemistry ; Intermediate Filament Proteins ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Multipotent Stem Cells ; metabolism ; virology ; Muromegalovirus ; Nerve Tissue Proteins ; genetics ; metabolism ; Nestin ; Neurons ; Pregnancy ; Reverse Transcriptase Polymerase Chain Reaction ; Wnt1 Protein ; genetics ; metabolism