Cells and viruses
The generation and characterization of the neuro-attenuated pseudorabies virus (Prv-rrep0lacgfp) used in our experiments has already been described [10]. Briefly, the virus strain contains two mutations: a frame shift mutation in the small subunit of the ribonucleotide reductase gene and a large deletion in the early protein 0 gene. The gfp and lacZ reporter genes were inserted in place of the early protein 0 gene. Prv was propagated in porcine kidney (PK-15) cells. The GFP gene was placed under the control of a chimeric promoter composed of latency-associated transcript promoter (LAP) and human cytomegalovirus major IE promoter [10].
Infection of the donor embryos
The human embryonic spinal cords derived from 8-12 weeks old embryos were obtained from abortions and used with the permission of the Human Ethical Committee of University of Szeged. The human tissues were used according to the guidelines of Helsinki Declaration of Human Rights. The lumbar enlargement of the spinal cord was dissected and the ventral portion was chopped into 1-2 mm pieces. The pieces of tissue were incubated with the virus for 6 and 12 hours at 37°C in a Sanyo CO2 thermostat. In each experiment 1 ml virus suspension containing 108 plaque-forming units (p.f.u./ml) of virus particles was used for the infection. Following incubation the samples were washed three times in Hanks' solution to remove excess virions. We separated 8 infected spinal cord pieces and allowed in Hanks' solution for 13 hours then processed for either Trypan Blue staining or immunohistochemistry. However, infected tissues marked for transplantation were used up immediately. Mock-infection was performed by keeping embryonic spinal cords tissues in Hanks' solution for 24 hours.
Surgery
Adult homozygous BALB/c rnu-/rnu- nude mice (Charles River Laboratories, Inc., Gödöllő, Budapest, n = 15) were used as hosts for transplantation. The mice were athymic and therefore had no T-cell-based immune response. All surgical interventions were performed under deep anaesthesia achieved with a combination of Hypnorm (0.0045 ml/10 g body weight, Hypnorm, Janssen) and Diazepam (0.0075 ml/10 g body weight, from 5 mg/ml stock solution). On postnatal day 3, the left sciatic nerve of the mice was crushed to bring about motoneuron depletion in the spinal segments L4-L5. The animals were allowed to recover and used for grafting when they reached full maturity (18-20 g). After sciatic nerve crush, the animals exhibited permanent partial paralysis in the affected hindlimb. At the age of 8 weeks, the mice were anaesthetized again as described above and, after skin incision and muscle dissection, L1 laminectomy was performed corresponding to segment L4 of the spinal cord. The dura was opened to expose the cord and 0.5 μl of solid embryonic cord pieces were pressure-injected into the segment L4 via a Hamilton syringe equipped with a glass micropipette. Finally, the muscles were closed over the laminectomy site and the skin was sutured. After survival time of 7 days or 3 weeks, the animals were perfused transcardially with 4% phosphate-buffered paraformaldehyde (pH = 7.4).
Histochemistry
The Trypan Blue exclusion test was performed on the Prv-treated human embryonic spinal cord cells after Prv incubation for 6 h or 12 h. The tissues were first trypsinated with a mixture of trypsin and EDTA, and the dissociated cells were then incubated with Trypan Blue solution (Sigma Aldrich, Budapest, Hungary) for 20 min, followed by a brief wash in physiological saline. The numbers of labelled and unlabelled cells were determined by using a Bürker chamber. The above procedure was carried out on untreated human embryonic spinal cord tissues, too. The proportions of viable cells were determined in the Prv-treated and and in the control samples.
The embryonic tissues incubated with the Prv and the grafted spinal cords were postfixed in 4% paraformaldehyde and then cryoprotected in 30% sucrose in phosphate-buffered saline overnight. Serial cryostat sections (25 μm thick) were cut in a cryostat (Leica CM1850, Bensheim, Germany) and mounted onto gelatine-coated microscopy slides.
The following antibodies were used to detect antigens: polyclonal rabbit antibody against glial fibrillary acidic protein (GFAP, 1:1000, Dakopatts, Denmark) labelling astrocytes and radial glial cells which are typical glial cells in the foetal CNS, polyclonal rabbit antibody against the whole virion (1:10.000, generous gift of Lynn Enquist, Princeton University, Princeton, NJ, USA), monoclonal mouse antibody against β-galactosidase (β-gal; 1:200, Boehringer Mannheim, Germany) and polyclonal chicken antibody against GFP (AB160901, 1:4000; Chemicon International, Temecula, CA, USA). For clarity, only the results of GFP immunohistochemistry are shown. The human neurons were detected by monoclonal mouse antibody against the human neural cell adhesion molecule (hu-NCAM, clone CD56, 1:400; IQ Products, The Netherlands). The antibody recognizes hu-NCAMs expressed on human postmitotic neurons [18]. The primary antibodies were visualized with appropriate biotinylated secondary antibodies and various fluorescent chromophores. Some immunohistochemical reactions were tyramide-amplified by using TSA-Cy3 and TSA-FITC (Perkin Elmer Life Sciences, Boston, MA, USA).
The sections were investigated in an Olympus BX50 fluorescent microscope and photographs were taken with a DP70 digital camera (Olympus Ltd., Tokyo, Japan). Some sections were investigated in a Zeiss LSM510 Meta confocal microscope, too. The labelled cells in the embryonic cords and in the grafts were identified and counted: hu-NCAM or GFP-positive cells were counted only if the cell profile was not present in the consecutive section.
The subcellular localization of virions and GFP was investigated in some Prv-infected embryonic tissues by using electron microscopic immunohistochemistry. Vibratome sections (40 μm thick) were processed for immunohistochemistry. In the immunohistochemical procedure after the ABC step, 3,3'-diaminobenzidine tetrahydrochloride (Sigma Aldrich Co. Ltd., Budapest, Hungary) was added and the oxidized coloured end-product was silver-intensified [19]. Sections were postfixed with 1% aqueous OsO4 and then embedded in Durcupan (Fluka, Buchs, Switzerland). Ultrathin sections (50-60 nm) were cut on an ultramicrotome (Leica Ultracut R, Bensheim, Germany) and the sections were treated with uranyl acetate and investigated in an electron microscope (JEOL JEM 1010, JEOL Ltd, Tokyo, Japan).
Statistical analysis
Whenever needed, the paired t-test was used for the statistical analysis of data.