Choice bias from unilateral hippocampal or frontal lesions in the rat.
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Biomedical subjects
S Saporta
Publications and source records attributed to S Saporta.
Reduced ovarian function and lordosis behavior following hypothalamic knife cuts.
Lordosis behavior (LQ) estrous cycles and ovarian status were studied in the same animals following either frontal (FC), frontolateral (FLC) or sham (S) surgical interruption of medial basal hypothalamic (MBH) neural connections made with a modified Halász knife (1.5 mm radius). LQ was studied in adult female rats at 7, 30, 105 and 180 day after FC, FLC or S and estrous cycles were monitored for 28 day after cuts. Vaginal cytology examined before and after S surgery indicated regular 4-5 day cycles, whereas after FC vaginal cytology became predominately cornified and after FLC it was variable. Ovarian weights and appearance at ovariectomy (OVX) supported cycle data: S ovaries had recent corpora lutea (CL); FC gonads were smaller with no recent CL and FLC gonads appeared atrophic. Multiple regression analysis of individual FC and FLC dimensions indicated that cut location and dimensions contributed significantly to the length of postoperative estrus periods. After OVX all animals were tested for LQ after estradiol benzoate (EB) and EB plus progesterone (P) priming. Although there were no group LQ differences after EB alone, S and FC animals showed an increase in LQ after EB plus P, whereas, FLC rats showed no change and this effect persisted for 180 day. Multiple regression analysis of FC and FLC coordinates indicated that the more posterior and lateral extending cuts were associated with lower LQ scores. Collectively, these data suggest a role for similar anterolateral MBH neural connections in (1) basal and cyclic gonadotrophin release and (2) EB plus P effects on lordosis behavior.
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The X-gal caution in neural transplantation studies.
Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme beta-galactosidase (beta-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, "X gal," in an oxidation reaction. LacZ is the "reporter gene" most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully-with adequate optimization and individualization of various parameters (e.g.. pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls--then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial beta-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have empha sized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli beta-gal activity.
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Post-thaw viability and functionality of cryopreserved rat fetal brain cells cocultured with Sertoli cells.
Testis-derived Sertoli cells have been used to create an immune "privileged" site outside of the testis to facilitate cell transplantation protocols for diabetes and neurodegenerative diseases. In addition to secreting immunoprotective factors, Sertoli cells also secrete growth and trophic factors that appear to enhance the posttransplantation viability of isolated cells and, likewise, the postthaw viability of isolated, cryopreserved cells. It would be beneficial if Sertoli cells could be cryopreserved with the transplantable cell type without deleterious effects on the cells. This report describes a protocol for the cocryopreservation of rat Sertoli cells with rat ventral mesencephalic neurons, neurons from the lateral and medial ganglionic eminences and the hNT neuron cell line, and reports on the effects of Sertoli cells on the the postthaw viability of these neurons. Results of trypan blue exclusion analysis indicated that the presence of Sertoli cells did not deleteriously effect cryopreserved neurons and may improve their postthaw recoverability and viability in general. Specifically, results of the tyrosine hydroxylase immunostaining showed that Sertoli cells significantly enhance the postthaw viability of ventral mesencephalic dopaminergic cells in vitro.
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The testis-derived cultured Sertoli cell as a natural Fas-L secreting cell for immunosuppressive cellular therapy.
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Microcarrier enhanced survival of human and rat fetal ventral mesencephalon cells implanted in the rat striatum.
The transplantation of tissue containing dopamine-producing cells into the mammalian central nervous system is an emerging treatment for Parkinson's disease, despite relatively poor survival of implanted tissue. Recent evidence has suggested that Cytodex microcarriers enhance the survival of dopaminergic rat chromaffin cells transplanted into the rat striatum in the absence of immunosuppression. The current study was undertaken to evaluate the survival of rat and human fetal ventral mesencephalic neurons (VM) implanted alone or after attachment to microcarriers in the striatum of rats without immunosuppression. Rat fetal VM neurons demonstrated enhanced survival in the rat striatum when transplanted on microcarriers, compared to their transplantation alone during the 3-mo period examined in the present study. Transplants of human fetal VM neurons on microcarriers also survived remarkably well in the rat striatum without systemic immunosuppression. In contrast, human fetal VM cells transplanted alone into the rat striatum did not survive without systemic immunosuppression. There was no evidence of TH fiber sprouting in the vicinity of any transplant site. These data indicated that Cytodex microcarriers provide enhanced survival of both rat allograft and human xenograft fetal mesencephalic cells in the rat striatum without the necessity of systemic immunosuppression, perhaps by inducing a unique neuron-glia environment.
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Bilateral fetal striatal grafts in the 3-nitropropionic acid-induced hypoactive model of Huntington's disease.
We investigated the 3-nitropropionic acid (3-NP)-induced hypoactive model of Huntington's disease (HD) to demonstrate whether fetal tissue transplantation can ameliorate behavioral deficits associated with a more advanced stage of HD. Twelve-week-old Sprague-Dawley rats were introduced to the 3-NP dosing regimen (10 mg/kg, i.p., once every 4 days for 28 consecutive days), and were then tested for general spontaneous locomotor activity in the Digiscan locomotor apparatus. All rats displayed significant hypoactivity compared to their pre-3-NP injection locomotor activity. Randomly selected rats then received bilateral intrastriatal solid grafts of fetal striatal (lateral ganglionic eminence, LGE) tissues from embryonic day 14 rat fetuses. Approximately 1/3 of each LGE in hibernation medium was infused into each lesioned host striatum. In control rats, medium alone was infused intrastriatally. A 3-mo posttransplant maturation period was allowed prior to locomotor activity testing. Animals receiving fetal LGE grafts exhibited a significant increase in locomotor activity compared to their post-3-NP injection activity or to the controls' posttransplant activity. Surviving striatal grafts were noted in functionally recovered animals. This observation supports the use of fetal striatal transplants to correct the akinetic stage of HD. To the best of our knowledge, this is the first study that has investigated the effects of fetal striatal transplantation in a hypoactive model of HD.
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Trophic effect of porcine Sertoli cells on rat and human ventral mesencephalic cells and hNT neurons in vitro.
The poor survival of embryonic dopaminergic (DA) neurons transplanted into patients with Parkinson's disease (PD) has encouraged researchers to search for new methods to affect the short- as well as long-term survival of these neurons after transplantation. In several previous rodent studies Sertoli cells increased survival of islet cells and chromaffin cells when cotransplanted in vivo. The aims of this study were to investigate whether porcine Sertoli cells had a positive effect on the survival and maturation of rat and human DA neurons, and whether the Sertoli cells had an effect on differentiation of neurons derived from a human teratocarcinoma cell line (hNT neurons). A significant increase of tyrosine hydroxylase (TH)-positive neurons of both rat and human ventral mesencephalic tissue was found when cocultured with Sertoli cells. Furthermore, there was a significantly increased soma size and neurite outgrowth of neurons in the coculture treated group. The Sertoli cell and hNT coculture also revealed an increased number of TH-positive cells. These results demonstrate that the wide variety of proteins and factors secreted by porcine Sertoli cells benefit the survival and maturation of embryonic DA neurons and suggest that cotransplantation of Sertoli cells and embryonic DA neurons may be useful for a cell transplantation therapy in PD.
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CD44 expression and MMP-2 secretion by mouse glioma cells: effect of interferon and anti-CD44 antibody.
We have previously reported that invasiveness of mouse glioma G-26, which expresses CD44 adhesion molecule, was inhibited in vitro following treatment with anti-CD44 antibody or mouse interferon alpha/beta (MuIFN alpha/beta). Here, we evaluated whether the expression of transmembrane CD44 adhesion molecule and/or secretion of extracellular matrix metalloproteinases (MMPs) were affected when glioma cell invasion was inhibited. Flow cytometric evaluation of CD44 adhesion molecule expression in G-26 glioma using anti-CD44 antibody, confirmed that G-26 cells were CD44+. Following 3-day treatment with MuIFN alpha/beta at 8 x 10(2) or 8 x 10(3) IU/ml of glioma cells, the expression of CD44 was not significantly affected as reflected by CD44+ cell number and fluorescence intensity. The pretreatment of glioma cells for 1 day with anti-CD44 antibody resulted in a 30-60% decrease of CD44 expression. This coincided with significantly (p < 0.05) lower cell activity as judged by MTT assay for mitochondrial activity. The zymographic evaluation of MMP activity in the G-26 glioma cell culture showed a high level of the active form of MMP-2. This level of MMP-2 was decreased following 3 day treatment of G-26 glioma cells with either 8 x 10(2) or 8 x 10(3) IU/ml of MuIFN alpha/beta but only the latter concentration produced statistically significant 55% decrease. However, following a 1 day treatment of G-26 glioma cells with anti-CD44 antibody, the level of active MMP-2 form was not significantly affected. These findings indicate that while the inhibitory effect of IFN on glioma invasion was accompanied by a decreased level of the active form of MMP-2 released extracellularly, the expression of the transmembrane CD44 adhesion molecule was not affected. Conversely, anti-CD44 antibody pretreatment of G-26 glioma, which led to the inhibition of glioma invasion, resulted in decreased CD44 expression and lower cell activity but had no effect on the MMP-2.
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Interferon entry through the blood-brain barrier in glioma and its effect on lipoxygenase activity.
This study evaluates cerebral entry of mouse interferon alpha/beta (MuIFN alpha/beta) or mouse interferon gamma (MuIFN-gamma) following continuous (3 day), subcutaneous infusion of normal or glioma bearing mice. The intracerebral C57BL/6 mouse glioma-26 (G-26) model was used at days 10-14 post tumor implant, the advanced stage of glioma progression as defined by histology and the median survival time (27 +/- 3.8 days). The infusion of horseradish peroxidase (HRP) in vivo at day 10 or 11 post glioma implant showed strong staining in the tumor bed indicating compromised blood-brain barrier (BBB). In addition, histochemistry with Bandeiraea simplicifolia isolectin B4 demonstrated the accumulation and/or activation of macrophage/microglia. The 3 day infusion of mice (day 11-14 post tumor implant) via subcutaneous (sc) osmotic micro-pumps with MuIFN alpha/beta (8x10(5) - 1.7x10(6) international units [IU]/ml) or with recombinant mouse interferon gamma (rMuIFN-gamma) (1x10(6) IU/ml) resulted in a low but detectable (1-5 IU/ml) cerebral level of IFN. The IFN levels in the blood (20-40 IU/ml) and brain, measured by assay of inhibition of viral cytopathic effect (CPE) or ELISA assay for MuIFN-gamma, showed no difference between normal and glioma bearing mice. The lipoxygenase (LO) activity (dioxygenase) of glioma tissue and contralateral control was evaluated in non-treated and MuIFN alpha/beta continuously (3 day) treated mice. The LO activity in glioma tissue was significantly higher (p < 0.05) than the contralateral control in non-treated mice. However, following sc MuIFN alpha/beta infusion the LO activity of glioma decreased to control level.
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The effect of interferon and anti-CD44 antibody on mouse glioma invasiveness in vitro.
In this study the effect of interferon and anti-CD44 antibody on the invasiveness of mouse glioma G-26 cells was evaluated. We confirmed the glial nature of G-26 glioma cells (G-26) in vitro and in vivo using immunohistochemistry: G-26 stained strongly for S-100 and stained weakly for glial fibrillary acidic protein (GFAP). Immunohistochemical evaluation for CD44 adhesion molecule showed that G-26 was positive both in vitro and in vivo. Weakly positive punctate staining for CD44 was seen in the cytoplasm of all viable glioma cells and focally strong staining was observed in a membranous pattern in the invading glioma cells. Evaluation of untreated G-26 cells using an in vitro invasion assay showed that they were able to digest a Matrigel matrix and to invade through an 8 microns microporous membrane. Treatment of the G-26 glioma cells for 3-4 days with mouse interferon alpha/beta at 8 x 10(2) or 8 x 10(3) mu/ml resulted in a significant decrease of invasiveness: 68.8% (p < 0.05) and 32.8% (p < 0.001) of cells, respectively, remained invasive when compared to control. Treatment of G-26 with antibody to the CD44 adhesion molecule significantly decreased invasiveness with 39.4% (p < 0.001) of cells remaining invasive when compared to control. We feel that both of these approaches, each of which produced significant inhibition of G-26 glioma cell invasion should be further evaluated for their usefulness in antiglioma therapy.
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