Browsing by Author "Jortner, Bernard S."

Now showing 1 - 20 of 22
  • Thumbnail Image
    Changes in the Murine Nigrostriatal Pathway Following Pyrethroid and Organophosphate Insecticide Exposure: An Immunohistochemical Study
    Pittman, Julian Thomas (Virginia Tech, 2002-08-22)
    Parkinson's disease (PD) is a debilitating motor disorder that primarily afflicts older individuals (> 50yrs). Although its cause is unknown, many factors are thought to contribute to the disease. There is growing epidemiological evidence supporting a link between pesticide exposure and PD. The present immunohistochemical study was undertaken to characterize the role of insecticide exposure in the etiology of idiopathic PD. The insecticides selected for study were the pyrethroid permethrin (PE) and the organophosphate chlorpyrifos (CP), both of which possess properties that could damage or disrupt the nigrostriatal pathway, which is the principal neurodegenerative target in PD. The present study examined possible alteration of the amount of dopamine re-uptake transporter protein (DAT), within the striatum of the C57BL/6 mouse, using DAT antibodies, following low (0.8, 1.5 & 3.0 mg/kg) and high (200 mg/kg) doses of PE, respectively. Possible nigrostriatal terminal degeneration was examined using antibodies to tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, following treatment with 50 mg/kg of CP alone or in combination with the high dose of PE. For both the high dose of PE alone and for the combined PE/CP treatment, glial fibrillary acidic protein (GFAP) antibodies were used to examine the possibility of non-degenerative tissue injury. Groups of matched treated/vehicle-control mice received three IP injections of the insecticide/dose of interest over a 2-week period. Counts of immunoreactive (IR) neuropil in the dorsolateral striatum were made from four pre-selected fields per striatal tissue section. Counts were compared between matched sections, processed on the same slide, from a treated mouse and its vehicle control. A mean difference score, across slides, for each treated/vehicle control pair was determined. All low dose PE groups showed a trend of decreased DAT IR neuropil, but only the 3.0mg/kg group showed a statistically significant reduction (p<.0078). The 200 mg/kg PE group showed a trend toward reduced TH IR neuropil that was not statistically significant, but a significant increase in GFAP IR (p = .048) was observed. No significant change in TH IR neuropil was observed for CP (50mg/kg) alone. A significant increase was observed for GFAP IR neuropil for the PE/CP (200/50 mg/kg) combination dose (p = .033). The combined insecticide treatment failed, however, to produce a significant change in TH IR within the striatum, compared to vehicle controls. These data suggest that the significant increases in GFAP IR neuropil, in the striatum, reflect some form of tissue insult, following exposure to a high dose of PE, or PE/CP in combination, that is insufficient to induce degeneration of dopaminergic terminals within the temporal interval investigated. Although such damage may be sufficient to account for previously reported decreases in maximal dopamine uptake observed with high doses of these compounds, the DAT IR data appear to indicate that this damage is unlikely to be a change in the amount of DAT in these high dose conditions. The decreases in striatal DAT IR neuropil observed for low doses of PE suggest an alteration in the normal integrity of the nigrostriatal pathway and in the route by which environmental toxins may enter dopaminergic neurons.
  • Thumbnail Image
    Comparative evolution of mipafox-induced delayed neuropathy in the rat and hen
    Carboni, Deborah Ann (Virginia Tech, 1993-08-09)
    The group of chemicals designated organophosphorus compounds have had a significant impact on modern life, including use as pesticides, industrial plasticizers and chemical warfare agents. Exposure to certain organophosphates produces a delayed degeneration of the longest and largest nerve fibers, including those of the ascending and descending tracts of the spinal cord, a condition termed organophosphorus ester-induced delayed neuropathy (OPIDN). Recorded incidents of such an effect in humans have led to research regarding this neurological disease. Among the OPIDN-inducing agents is mipafox, an organophosphate insecticide, the compound we chose to employ in our studies. Although the hen is the primary experimental model in the safety assessment of organophosphates, current research has suggested that the rat may have some validity as an experimental model. We examined the sequential neuropathic effects of a single dose of mipafox (30mg/kg) in rats and hens on a comparative basis to determine the better experimental model.
  • Thumbnail Image
    Development of a model cell culture system in which to study early effects of neuropathy-inducting organophosphates
    Nostrandt, Amy Carol (Virginia Tech, 1991)
    Certain organophosphorus (OP) compounds produce a delayed neuropathy in man and susceptible animal species after early inhibition and aging of the enzyme, neurotoxic esterase (neuropathy target esterase, or NTE). In this study, the human neuroblastoma cell line, SY-5Y, was examined for its potential to serve as a nonanimal model for the study of the early effects of neuropathy-inducing OPs. For these investigations, the time course of inhibition and aging of NTE after toxicant treatment in neuroblastoma cells was compared to that in brain tissue from the adult chicken, which is the recognized animal model for organophosphorus ester-induced delayed neuropathy (OPIDN). Concentrations of toxicants to be used for treating neuroblastoma cells were determined after observing viability of treated differentiated SY-SY cells incubated for 24 hours with a range of concentrations of an OP (mipafox) that induces neuropathy, an OP (paraoxon) that does not induce neuropathy, a carbamate (aldicarb), a neurotoxicant (β, β’-iminodipropionitrile, or IDPN) that acts by a different mechanism than the OPs, and a cholinergic agonist (carbachol). Treatment concentrations were chosen that caused less than 30% loss of viability over the 24 hour period. The time course and extent of detrimental effects of mipafox, which induces OPIDN in hens, and the carbamate, aldicarb, on NTE were similar in the SY-5Y cells to those observed in homogenized chicken brain tissue after the same treatments. Mipafox produced rapid inhibition and aging of NTE, with maximal effects occurring within 10 minutes of exposure. Aldicarb inhibited NTE but did not age the enzyme. Instead, spontaneous reactivation was observed both in SY-5Y cells and in brain tissue. None of the other negative control compounds (paraoxon, IDPN, carbachol) affected NTE activity in either SY-5Y cells or chicken brain tissue. To determine if the neuroblastoma cells could be used to study early events that could lead to modification of OPIDN, NTE inhibition and aging were determined in the differentiated SY-5Y cell line after mipafox was removed. Removal of mipafox from the cell culture medium at 5 minutes after exposure, or earlier, resulted in essentially no NTE inhibition or aging. NTE inhibition and aging were also determined after treatment of the SY-5Y cells with the neuropathy-inducing OP, mipafox, and representatives of 2 classes of compounds (carbamates and calcium channel blockers) previously demonstrated to modify OPIDN in hens. The modifiers (aldicarb and verapamil) were used as a 5 minute pre-treatment, simultaneous treatment, and a 2 minute post-treatment. Significant prevention of most of mipafox-induced NTE inhibition and aging was observed. Effects on NTE inhibition and aging in differentiated SY-5Y cells after each mipafox-aldicarb combination and mipafox-verapamil combination of treatments were similar to those in chicken brain homogenate. These results indicated that both aldicarb and verapamil protected NTE against the early biochemical effects of mipafox that are thought to initiate OPIDN in vivo. The temporal relationship of NTE inhibition and aging to other detrimental effects on neuroblastoma cells was assessed by the capability of mipafox to cause changes in free intracellular calcium ion concentration, measured using fluorescent calcium probes, and by its capability to alter cell morphology as assessed by phase contrast microscopy. NTE inhibition and aging preceded these changes. Capability to inhibit activity of the neural enzyme, acetylcholinesterase, was also determined. The results of these studies indicate that the SY-5Y model system shows promise for use in the determination of initial mechanisms contributing to the development of organophosphorus-induced delayed neuropathy.
  • Thumbnail Image
    Early Effects of Organophosphate Compounds on In Vitro Intracellular Signaling and Levels of Active Neurotrophin Receptors, and on In Vivo Neurotrophin Concentrations
    Pomeroy-Black, Melinda J. (Virginia Tech, 2005-10-14)
    Organophosphorus (OP) compounds are found in household pest control products, plastics, and petroleum. Due to the neurotoxic nature of OP compounds, exposure can cause both acute and delayed symptoms, including organophosphate-induced delayed neuropathy (OPIDN). This syndrome is characterized by Wallerian-like degeneration of nerves in the central and peripheral nervous system after exposure to neuropathic OP compounds. There are many questions surrounding the mechanisms of the onset of OPIDN, including possible alterations in proteins associated with neuronal maintenance and repair. This dissertation investigated the changes in levels of neurotrophins in vivo and how in vitro levels of neurotrophin receptors and their downstream signaling cascades are affected after exposure to OP compounds. We also characterized the molecular weight of a soluble factor responsible for inducing neurite outgrowth in vitro after in vivo exposure to a neuropathic OP compound. We evaluated in vivo endpoints using enzyme-linked immunosorbant assays. Results indicated that nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are found in chicken spinal cord but do not increase as a result of exposure to neuropathic OP compounds. This study also noted that NGF, BDNF, and NT-3 concentrations were not altered after exposure to a non-neuropathic OP compound. We evaluated in vitro endpoints using Western blots, ultrafiltration, and digital morphometry. These studies revealed that activated forms of high-affinity and low-affinity neurotrophin receptors are present after OP compound exposure, that the ratio of these two receptors to each other is stable after OP compound exposure, and that the activated form of the low-affinity receptor, which can lead to apoptosis, was present in greater levels than the activated form of the high-affinity receptor. Furthermore, OP compound exposure resulted in time-dependent changes of protein levels central to the mitogen-activated kinase and phosopholipase C-gamma intracellular pathways. Changes in a third pathway, the protein kinase C pathway, were dependent on the concentration and type of OP compound. Finally, in vitro neurite length was not affected by the type of OP compound administered in vivo or when a whole protein fraction was separated by molecular weight. This research has revealed in vivo consequences and early effects on intracellular protein and activated neurotrophin receptor levels after OP compound exposure. These early effects may contribute to the delayed development of neurotoxic effects associated with OP compound exposure.
  • Thumbnail Image
    Effects of Blood Contamination on Cerebrospinal Fluid Cell Counts, Protein, and D-dimer Concentrations
    Rossmeisl, John H. Jr. (Virginia Tech, 2003-01-20)
    Cerebrospinal fluid analysis (CSF) is commonly performed in clinical neurology, and is a sensitive, but non-specific indicator of central nervous system (CNS) pathology. Blood contaminated CSF samples have the potential to adversely affect results of cytologic, serologic, microbiologic, and molecular biologic diagnostics. A clear consensus of the effects of blood contamination on CSF analysis could not be drawn following a review of the existing veterinary literature. Based on data from earlier reports, it was hypothesized that iatrogenic blood contamination of CSF would result in significant increases in both the CSF total protein (TP) concentration and nucleated cell count (WBC). As hypothesized, in vitro CSF blood contamination resulted in statistically significant (p < 0.01) linear increases in both the CSF TP and WBC with increasing RBC concentration in CSF from sixteen normal dogs. Although increases in TP and WBC are statistically significant, their clinical impact is negligible. Results of this study demonstrate that in normal dogs, the mean CSF TP concentration collected from the cerebellomedullary cistern, is lower than previously reported. D-dimers are plasminolytic cleavage products formed by the cross-linkage of fibrin by Factor XIIIa. In humans, D-dimer analysis can be used to differentiate iatrogenic from pathologic CNS hemorrhage. An additional objective of this study was to determine if canine D-dimers could be assayed using commercially available latex agglutination (LA) and enzymatic immunoassay (EIA) kits in normal and diseased subjects. It was hypothesized that qualitative and quantitative determinations of blood and CSF D-dimer activities could be aid in the diagnosis of dogs with altered CNS and/or systemic coagulation. D-dimers were able to be assayed in all subjects studied. D-dimer concentrations in CSF samples, when analyzed using a qualitative LA assay system, from healthy dogs with iatrogenically blood contaminated CSF were consistently negative. Quantitation of CSF D-dimer concentrations in normal dogs using an EIA assay resulted in lower values (mean 16.2 + 4.3 ng/ml; range, 0 to 54 ng/ml) than detected in the peripheral blood of dogs and humans (normal cutoff value < 250 ng/ml). These findings suggest that D-dimer formation does not occur in canine CSF freshly contaminated with blood. Significantly (p < 0.001) higher mean blood D-dimer concentrations were present in dogs with systemic coagulation disorders (1,093.4 + 172.3 ng/ml; range, 0 to > 2,000 ng/ml) when compared to normal dogs (54.6 + 19.8 ng/ml; range, 0 to 190 ng/ml), when assayed with the EIA. When used as an adjunct in the diagnosis of systemic coagulation abnormalities, the EIA assay had an overall sensitivity of 92%, specificity of 100%, positive predictive value (PPV) of 100%, and negative predictive value (NPV) of 94%. When applied to the same dogs, the LA D-dimer was less sensitive and specific (sensitivity of 73%, specificity of 100%, PPV of 100%, and NPV of 80%) than the EIA. Evidence of intrathecal fibrinolysis in the absence of systemic abnormalities was also demonstrated using CSF LA and EIA D-dimer assays in some dogs with a variety of infectious (Rocky mountain spotted fever), non-infectious inflammatory (granulomatous meningoencephalitis, steroid-responsive meningitis), traumatic (intervertebral disc disease, spinal fracture), and neoplastic (meningioma) diseases. When all dogs with CNS diseases were examined together, the mean EIA D-dimer concentration was significantly (p = 0.03) higher (511.6 + 279.8 ng/ml) than normal dogs (mean 16.2 + 4.3 ng/ml). Future studies will be required before the definitive role of D-dimer analysis can be defined in veterinary medicine.
  • Thumbnail Image
    Evaluation of the Role of Astrocyte Glutamate Transport and of Synaptic NMDA Receptor Subtype Representation in the Pathogenesis of PTSD
    Cotrone, Thomas Steven (Virginia Tech, 2017-06-22)
    Post-traumatic stress disorder (PTSD) is a psychological disorder that can cause great social/economic hardship. Progress towards treating PTSD has been slow due to a lack of understanding of its pathogenesis. This dissertation aimed to address this issue by investigating the involvement of the astrocytic glutamate reuptake transporter, GLT-1, and regional differences in expression of NMDA receptor subtypes in the development of a rat model of PTSD. We hypothesized that impaired astrocytic glutamate reuptake inhibits long-term memory processes, and that concurrent presence of glucocorticoids (GCs) during situational trauma selectively inhibits fear extinction memory processes in the prefrontal cortex, but not of conditioned fear memory processes in the amygdala, due to differences between these brain regions in expression of NMDA receptor subtypes. The effect of GLT-1 manipulation was studied in vivo. Utilizing the Single Prolonged Stress (SPS) model of PTSD, rats were either exposed to SPS or not. Within these groups, rats were administered a saline sham, a GLT-1 facilitator (ceftriaxone (CEF)), or a GLT-1 inhibitor (dihydrokainic acid (DHK)). Using Classical Fear Conditioning (CFC) and Fear Extinction (EXT) paradigms, retention of fear extinction memories was measured to determine the effect of GLT-1 manipulation on SPS-induced behavior (i.e., impaired fear extinction retention). From the brain of each rat, the amygdala, hippocampus, and prefrontal cortex (PFC) were collected and expression of GLT-1, p-CREB (a molecular indicator of long-term memory), and glucocorticoid receptor (GR, a molecular indicator of a PTSD-like state) were quantified. Analysis of the behavioral data showed that SPS exposure alone reduced the retention of extinction memories, but CEF and DHK both eliminated this effect. Analysis of the brain tissues revealed that SPS induced an increase in GR expression in the hippocampus. SPS also increased GLT-1 expression, but not p-CREB, in the PFC and amygdala. To evaluate the involvement of regional differences in NMDA receptor subtype expression ex vivo, tissue sections of amygdala, hippocampus, and PFC were taken from SPS and non-SPS exposed rats. Synaptic transmission was stimulated in these tissues using bicuculline in the presence of glucocorticoids, NVP-AA077 (a NR2A NMDA receptor subtype inhibitor), or Ro-25 (a NR2B NMDA receptor subtype inhibitor). P-CREB was measured in the tissues treated with GCs to determine if GCs exert greater inhibition of long-term memory in the PFC (a region reported to express high NR2A) than in the amygdala (a region reported to express high NR2B). P-CREB was also measured in the tissues treated with NVP or Ro-25 to determine if these reported receptor profile differences could be demonstrated, and if they changed following SPS exposure. Contrary to the stated hypothesis, analysis of non-SPS exposed rats revealed that GCs, NVP, and Ro-25 decreased p-CREB in all three regions with no differences between regions. However, in the SPS exposed group, p-CREB was not decreased in PFC and hippocampal tissues treated with GCs, amygdalar and PFC tissues treated with NVP, and PFC tissue treated with Ro-25. Overall, the results of the in vivo experiment did not convincingly demonstrate a role of glutamate spill-over in the pathogenesis of PTSD, but did show that modulation of glutamate reuptake can mitigate some of the behavioral consequences of exposure to situational trauma. The results of the ex vivo experiment did not reveal evidence that regional differences in NMDA receptor profiles exist across the three regions analyzed, nor did they show that GCs exert a region specific inhibition of long-term memory formation. However, it was demonstrated that SPS may affect long-term memory by altering expression of synaptic NMDA receptors. This study provides evidence that glial cells may play a role in the pathogenesis of PTSD, and thus may serve as targets for future therapy.
  • Thumbnail Image
    Involvement of calcium in organophosphorus-induced delayed neuropathy: a functional morphological, and biochemical study
    El-Fawal, Hassan Ahmed Naguib (Virginia Polytechnic Institute and State University, 1989)
    Organophosphorus compounds are widely used in agriculture as pesticides and in industry as petroleum additives and modifiers of plastics. Some of these compounds are capable of inducing an irreversible neuropathy developing weeks to months after exposure, yet there is no effective treatment. This may be due in part to the lack of knowledge of how this neuropathy develops. In this dissertation, it is proposed that as a consequence of a triggering event, peripheral nerves may be predisposed to an increase in calcium (Ca⁺⁺) mobilization and the neuronal accumulation of this cation. This increase in Ca could thereby initiate a cascade of events, in both nerve and muscle, that may account for some of the detrimental changes occurring during organophosphorus-induced delayed neuropathy (OPIDN). The involvement of Ca⁺⁺ in the pathogenesis of OPIDN was tested using functional, morphological, and biochemical techniques in the domestic hen, the recognized animal model of OPIDN. The isolated biventer cervicis nerve-muscle preparation was developed for quick assessment of the time course of OPIDN deficits and validated by comparison to in vivo preparations. This preparation proved more sensitive by functional and morphological evaluation indicating early damage at 4 days following exposure and before appearance of clinical signs. Regeneration was detected after 21 days. OPIDN was modified by using Ca⁺⁺ channel blockers, nifedipine, and verapamil, in the presence of phenyl saligenin phosphate, an active neurotoxicant. Attenuation of OPIDN by these compounds was revealed by clinical assessment, by changes in nerve excitability denoted by strength-duration relationships in response to electrical stimulation, by denervation hypersensitivity to neurotransmitter, and by morphology. These modifiers attenuated all degenerative responses. Furthermore, it was revealed that the activity of Ca⁺⁺-activated neutral protease (CANP), an enzyme responsible for neurofilament degradation, was increased in OPIDN. Such increases were ameliorated by modifiers of Ca movement. This study strongly suggests that Ca⁺⁺, possibly through activation of CANP, may contribute to functional and morphological deficits of OPIDN.
  • Thumbnail Image
    The Molecular Mechanisms of Organophosphorus Compound-induced Cytotoxicity
    Carlson, Kent Richard (Virginia Tech, 2000-05-19)
    Certain organophosphorus compounds have the ability to induce a delayed neuropathic condition in sensitive species termed organophosphorus compound-induced delayed neurotoxicity (OPIDN). Esteratic changes associated with OPIDN have been successfully modeled in vitro. The physical characteristics of lesion development in OPIDN including the mode of nerve cell death, cytotoxic initiator and effector molecules, and cytoskeletal involvement have received little in vitro investigation. This dissertation evaluated the mode of cell death (apoptosis versus oncotic-necrosis), and cell cycle, cytoskeletal, nuclear, and mitochondrial alterations induced by OP compounds in SH-SY5Y cultures, an in vitro human neuroblastoma model. The distribution of in vivo neural degeneration in white Leghorn hen models was also assessed as a prelude to validating the mode of OP compound-induced in vivo neural cell death. These endpoints were evaluated by utilizing flow cytometry, spectrophotometry, gel electrophoresis, immunohistochemistry, light, and electron microscopy. Initial data gathered on culture parameters revealed that the mitotic status, basal rates of cell death, and total culture density were dependent on the condition of the media and the initial seeding density. Subsequent in vitro investigations used standardized culture conditions with OP compounds (diisopropylphosphorofluoridate (DFP), paraoxon, parathion, phenyl saligenin phosphate (PSP), tri-ortho-tolyl phosphate (TOTP), and triphenyl phosphite (TPPi); 1uM - 1mM). These studies revealed that OP compounds altered the cell cycle phase, decreased the amount of intracellular f-actin, altered the mitochondrial membrane potential, and induced caspase-3 activation and nuclear partitioning characteristic of apoptosis. The amount of change in these parameters was strongly dependent on the OP compound, the length of incubation time, and the presence of modulators of cytotoxicity such as phenylmethylsulfonyl fluoride (PMSF), carbachol, Ac-DEVD-CHO, Ac-IETD-CHO, and cyclosporin A. Preliminary in vivo experiments designed to validate in vitro results revealed neural degeneration involving fibers, terminals, and cell soma in spinal cord and brain tissue of PSP- and TPPi- exposed hens. The distribution and magnitude of these changes were contingent on the OP compound and length of time post-exposure. Subsequent experiments designed to evaluate the mode of cell death in these tissues revealed little evidence of either necrosis or apoptosis. These results, therefore, did not support or refute in vitro observations. Many OP compound-induced subcellular alterations have been demonstrated in our in vitro SH-SY5Y neuroblastoma model. Even though the mode of cell death observed in SH-SY5Y cells was not validated in in vivo experiments, in vitro observations nonetheless provide stimulating areas to further research the mechanisms of OPIDN and OP compound-induced cell death.
  • Thumbnail Image
    Morphological and Immunocytochemical Investigation of Canine Oligodendrogliomas
    Higgins, Michael Anthony (Virginia Tech, 2006-10-06)
    Previous studies of human oligodendroglial neoplasms have demonstrated the diagnostic and prognostic values of histomorphologic features and immunocytochemical markers. Primary spontaneous canine intracranial tumors share many of the biologic behaviors and pathologic features of their human counterparts. The objectives of this study were to determine if associations existed between five histomorphologic features (mitoses, cellular atypia, necrosis, vascular hypertrophy, and vascular proliferation), and three immunocytochemical markers (GFAP, EGFR, and Ki-67 labeling index) and the degree of malignancy, as defined by WHO grading criteria, of 15 canine oligodendroglial tumors. Of the histomorphologic variables examined, mitoses and cellular atypia were significantly greater in Grade III oligodendrogliomas than in Grade II oligodendrogliomas (p = 0.002, and p = 0.004, respectively), but no differences were noted between these features and Grade II oligoastrocytomas and Grade II or Grade III oligodendrogliomas. No significant associations were found between GFAP or EGFR immunoreactivity and tumor type or grade. The median percentage of Ki-67 immunoreactivity was significantly different between all tumor types and grades (p < 0.05), and was significantly higher in Grade III oligodendrogliomas than in both oligoastrocytomas (p = 0.014) and Grade II oligodendrogliomas (p = 0.006). Results of this study indicate that although mitoses and cellular atypia are useful histomorphologic features for the differentiation of tumors with oligodendroglial phenotypes, none of the variables examined reliably distinguished mixed gliomas from oligodendrogliomas. The presence of GFAP immunoreactivity in all tumor types suggests that oligodendroglial tumors may arise from a common multipotential cellular lineage. Similar to what has been demonstrated in humans, the Ki-67 labeling index correlated well with the degree of malignancy in the tumors studied.
  • Thumbnail Image
    A neonatal gnotobiotic pig model of human enterovirus 71 infection and associated immune responses
    Yang, Xingdong; Li, Guohua; Wen, Ke; Bui, Tammy; Liu, Fangning; Kocher, Jacob; Jortner, Bernard S.; Vonck, Marlice; Pelzer, Kevin D.; Deng, Jie; Zhu, Runan; Li, Yuyun; Qian, Yuan; Yuan, Lijuan (Nature Publishing Group, 2014-05-21)
    Vaccine development and pathogenesis studies for human enterovirus 71 are limited by a lack of suitable animal models. Here, we report the development of a novel neonatal gnotobiotic pig model using the non-pig-adapted neurovirulent human enterovirus 71 strain BJ110, which has a C4 genotype. Porcine small intestinal epithelial cells, peripheral blood mononuclear cells and neural cells were infected in vitro. Oral and combined oral–nasal infection of 5-day-old neonatal gnotobiotic pigs with 53108 fluorescence forming units (FFU) resulted in shedding up to 18 days post-infection, with viral titers in rectal swab samples peaking at 2.223108 viral RNA copies/mL. Viral capsid proteins were detected in enterocytes within the small intestines on post-infection days (PIDs) 7 and 14. Additionally, viral RNA was detected in intestinal and extra-intestinal tissues, including the central nervous system, the lung and cardiac muscle. The infected neonatal gnotobiotic pigs developed fever, forelimb weakness, rapid breathing and some hand, foot and mouth disease symptoms. Flow cytometry analysis revealed increased frequencies of both CD41 and CD81 IFN-c-producing T cells in the brain and the blood on PID 14, but reduced frequencies were observed in the lung. Furthermore, high titers of serum virus-neutralizing antibodies were generated in both orally and combined oral–nasally infected pigs on PIDs 7, 14, 21 and 28. Together, these results demonstrate that neonatal gnotobiotic pigs represent a novel animal model for evaluating vaccines for human enterovirus 71 and for understanding the pathogenesis of this virus and the associated immune responses.
  • Thumbnail Image
    Neurochemical Effects of Concurrent Exposure to Repeated Stress and Chlorpyrifos on the Central Nervous System
    Pung, Thitiya (Virginia Tech, 2004-09-14)
    Repeated stress has been reported to cause reversible impairment to the hippocampus. Glutamatergic and cholinergic systems were proposed to be involved in responses seen after exposure to stress and cholinesterase inhibitors. Effects of concurrent exposure to repeated stress and chlorpyrifos (CPF) on concentrations of excitatory amino acids, activities of cholinergic enzymes, and maximum binding density (Bmax) and equilibrium dissociation rate constant (Kd) of NMDA and total muscarinic receptors were studied in Long-Evans rats. The study was divided into 4 experiments. The first experiment was to find the dose of CPF to use for studies on the interaction of stress and CPF. From the results obtained, 60% of the maximum tolerated dose was chosen. An experiment to determine effects of repeated stress and CPF on cholinergic enzymes and glutamate included groups of rats (n=7-8) that were handled 5 days/week; restrained 1 hour/day for 5 days/week; swum 30 minutes for 1 day/week; or restrained 4 days/week and swum for 1 day/week, for 28 days. On day 24, each group was injected either with corn oil or CPF 160 mg/kg sc 4 hours after restraint. On day 28, blood samples were collected for acetylcholinesterase (AChE) activity. Brains were dissected into hippocampus (HP) and cerebral cortex (CC) to determine activities of acetylcholinesterase (AChE), carboxylesterase (Cbxy), and choline acetyltransferase (ChAT), and glutamate and aspartate concentrations. CPF inhibited AChE activity in blood, CC and HP, but stress did not affect AChE activity. Repeated restraint with swim reduced Cbxy and CPF inhibited Cbxy. Restraint with swim had a statistical trend to increase concentrations of glutamate in the HP more than swim alone (p = 0.064); but CPF had no effect on glutamate in the HP. CPF decreased concentrations of elevated aspartate in the HP of rats that were restrained and swum. The results suggested that restraint with swim indirectly elevated acetylcholine in the CC, and tended to increase glutamate in the HP. The experiment designed to study the effects of concurrent exposure to stress and CPF on NMDA and total muscarinic receptors was designed similar to the previous study, except that endpoints were Bmax and Kd of NMDA and total muscarinic receptors in the HP and CC, and NMDA receptors in the hypothalamus (HT). Restrained rats had higher Kd of NMDA receptors in the HP than control and restrained with swim rats; however, Bmax was similar. CPF deceased Bmax and Kd of total muscarinic receptors in the CC of swum rats (237.64 ± 17.36 fmol/mg protein, 0.216 ± 0.023 nM) and CPF also decreased Bmax of total muscarinic receptors in the CC of restrained rats (229.08 ± 17.36 fmol/mg protein). There were no effects of stress, CPF, or interactions of stress and CPF on NMDA receptors in the CC or on total muscarinic receptors in the HP. In summary, CPF was capable of modulation of total muscarinic receptors of swum and restrained rats, suggesting that cholinergic transmission in the CC for cognition, sensory and motor activity may be modified. Furthermore, we examined effects of stress and CPF on concentrations of monoamines. Swim stress and CPF individually decreased concentrations of norepinephrine in the HP, whereas swim and restraint with swim decreased concentrations of norepinephrine but increased concentrations of DOPAC in the HT. Swim stress increased concentrations of dopamine in the HT more than control or restraint. CPF did not alter concentrations of norepinephrine, dopamine, or DOPAC in the HT. The interactions of repeated stress and CPF on serotonin approached significance in the HP (p = 0.06) and HT (p = 0.08). CPF increased serotonin concentrations in rats that were handled and restrained but not swum. CPF reduced the elevated concentrations of serotonin in restrained rats and restrained with swim rats (p < 0.05). Swim and restraint with swim were potential stress models that altered noradrenergic, dopaminergic, and serotonergic responses in the HT. In summary, repeated stressors had effects on glutamatergic, cholinergic, and monoamine systems. CPF had effects on cholinergic and monoamine systems but the interactions between stress and CPF were few.
  • Thumbnail Image
    Neuropathologic Effects of Phenylmethylsulfonyl Fluoride (PMSF)-Induced Promotion and Protection inn Organophosphorus Ester-Induced Delayed Neuropathy(Opidn) in Hens
    Massicotte, Christiane II (Virginia Tech, 2000-05-12)
    The serine/cysteine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) has been used both to promote and to protect against neuropathic events of organophosphorus-induced delayed neuropathy (OPIDN) in hens (Lotti et al., 1991; Veronesi et al., 1985; Pope and Padilla, 1990; Pope et al., 1993). This study expands upon this work by correlating clinical and neuropathological findings in these modifications of OPIDN. To provide appropriate models of OPIDN, single phenyl saligenin phosphate (PSP) dosages of 0.5, 1.0, or 2.5 mg/kg were administered to adult hens. PMSF (90 mg/kg) was given either 4 hours after or 12 hours prior to PSP administration. Clinical signs and pathologic changes in the biventer cervicis nerve (El-Fawal et al., 1988) were monitored. PSP alone, 2.5 mg/kg, ellicitated severe OPIDN (terminal clinical score 7.5 & ± 1.0 [0-8 scale]; neuropathology score 2.7 ± 0.3 [0-4 scale, based on myelinated fiber degeneration]). PMSF given 12 hours prior to PSP gave complete protection (clinical and neuropathology scores of 0; p<0.0001). Signs and lesions of OPIDN were absent following 0.5 mg/kg PSP alone, but PMSF given 4 hours after PSP potentiated its neurotoxic effects (clinical score 4.0 ± 0.0; neuropathology score 3.5 ± 0.3; p<0.0001). At the time of sacrifice, there was a correlation (r = 0.61) between the clinical score on the last day of observation and the neuropathology scores (p<0.0001). This study demonstrates that the intensity of peripheral nerve myelinated fiber degeneration correlates with clinical deficits in PMSF-induced potentiation and protection in OPIDN.
  • Thumbnail Image
    Neurotoxic effects of malathion and lead acetate on the blood-brain barrier: Disruptive effects caused by different mechanisms examined with an in vitro blood-brain barrier system
    Balbuena, Pergentino (Virginia Tech, 2010-06-14)
    Organophosphates (OP) such as malathion are organic derivatives of phosphoric acid with broad use in everyday life throughout the world, especially as insecticides. Lead particles can accumulate in soil and from there leach into our water supplies. Interaction with the environment offers opportunities for multiple exposures to combinations of different toxicants (such as lead and malathion). Thus, it is important to assess effects that these compounds exert not only on the nervous system, but also on the blood-brain barrier (BBB). The BBB consists of specialized endothelial cells that form the vasculature of the brain; it regulates passage of nutrients, while preventing potentially damaging substances from entering the brain. The main feature of the BBB is the presence of tight junctions between cells, which provide the BBB with its low permeability. The work presented in this dissertation tests the hypothesis that lead and malathion disrupt BBB integrity by affecting tight junctions of the BBB. The hypothesis suggests that disruptions involve changes in protein levels and gene expression as well as activation of transient receptor potential canonical channels (TRPC) that in turn increase intracellular calcium levels affecting tight junction structure. The hypothesis was tested by assessing lead-malathion interactions in an in vitro BBB model. This model was constructed with rat astrocytes and rat brain endothelial cells (RBE4). Assessments of cell toxicity in response to different concentrations of the neurotoxicants tested showed that concentrations between 10-5 µM and 10-6 µM were ideal to assess combinations of neurotoxicants. In general, protein levels of occludin, claudin 5, ZO1, and ZO2 decreased at all times, however, qPCR analysis of gene expression for all the proteins did not correlate with the assessments on protein levels. TRPC channel protein levels increased in response to neurotoxicant insult, which correlated with results for gene expression. This study suggests that at least one of the mechanisms that neurotoxicants lead and malathion utilize to disrupt permeability of the BBB is by affecting tight junction structure. This effect could be regulated by increases in gene expression of TRPC1 and TRPC4 that are associated with increases in the number of TRPC channels on the membrane of endothelial cells of the cerebral microvasculature.
  • Thumbnail Image
    Optic nerve astrocytoma in a dog
    Rozov, Orr; Piñyero, Pablo E.; Zimmerman, Kurt L.; Herring, Ian P.; Matusow, Rachel; Rossmeisl, John H. Jr.; Jortner, Bernard S.; Dreyfus, Jennifer (Wiley, 2016)
    Intraocular neoplasms in dogs are uncommon in comparison with other anatomic locations [1–4]. Over 75% of these cases are attributed to melanocytic neoplasia [1]. A majority of the remaining cases (~22%) are comprised of lymphoma, metastatic neoplasia, iridociliary epithelial tumors, optic nerve meningiomas, and histiocytic sarcomas in decreasing order of frequency [1, 2]. Rarely, optic nerve astrocytomas have been reported in humans, dogs, and horses [5–7]. In humans, astrocytomas involving the optic nerve are uncommon accounting for only about 1% of neoplasms at this site in comparison with 25% of neoplasms occurring in the brain [5, 8–14]. Most of these neoplasms occur unilaterally, are benign, and arise in children under 10 years of age; those involving the cerebellum have a more favorable prognosis [8, 14]. These younger patients commonly have an underlying familiar disorder such as neurofibromatosis. In older patients in their fourth and fifth decade of life, astrocytomas more commonly involve the cerebrum with a less favorable prognosis and can be associated with familiar disorders such as Li–Fraumeni syndrome [13, 15–17]. Most canine ocular astrocytoma cases are sporadic and not associated with any familial disorder [1, 13, 18–21]. However, Thomas et al. demonstrated a genomic risk factor associated with frequency of chromosome copy number aberrations within canine brain astrocytomas and tumor grade [22]. Similar to humans, canine astrocytomas account for less than 1% of ocular and optic nerve neoplasms and 10–36% of primary intracranial neoplasms [1, 4, 16–18, 20, 21, 23]. There appears to be a breed predisposition for development of intracranial astrocytomas in English Toy Spaniels, Boston Terriers, French Bulldogs, Boxers, and English Bulldog with a peak prevalence at 7– 8 years and 1.5 odds ratio in favor of larger versus smaller breeds [13, 23]. These risk factors have not been shown in association with canine ocular forms of this neoplasm [1, 16, 17]. Gender as a risk factor has not been described for either anatomic location [1, 13, 16–18, 23]. Case reports related to ocular astrocytomas are still rare in veterinary literature. The purpose of this report is to add to this sparse body of information. This report presents the clinical, histological, and immunohistochemical features of an optic nerve astrocytoma in a dog.
  • Thumbnail Image
    Paradoxical Effects of All-Trans-Retinoic Acid on Lupus-Like Disease in the MRL/lpr Mouse Model
    Liao, Xiaofeng; Ren, Jingjing; Wei, Cheng-Hsin; Ross, A. Catharine; Cecere, Thomas E.; Jortner, Bernard S.; Ahmed, Sattar Ansar; Luo, Xin M. (PLOS, 2015-03-16)
    Roles of all-trans-retinoic acid (tRA), a metabolite of vitamin A (VA), in both tolerogenic and immunogenic responses are documented. However, how tRA affects the development of systemic autoimmunity is poorly understood. Here we demonstrate that tRA have paradoxical effects on the development of autoimmune lupus in the MRL/lpr mouse model. We administered, orally, tRA or VA mixed with 10% of tRA (referred to as VARA) to female mice starting from 6 weeks of age. At this age, the mice do not exhibit overt clinical signs of lupus. However, the immunogenic environment preceding disease onset has been established as evidenced by an increase of total IgM/IgG in the plasma and expansion of lymphocytes and dendritic cells in secondary lymphoid organs. After 8 weeks of tRA, but not VARA treatment, significantly higher pathological scores in the skin, brain and lung were observed. These were accompanied by a marked increase in B-cell responses that included autoantibody production and enhanced expression of plasma cell-promoting cytokines. Paradoxically, the number of lymphocytes in the mesenteric lymph node decreased with tRA that led to significantly reduced lymphadenopathy. In addition, tRA differentially affected renal pathology, increasing leukocyte infiltration of renal tubulointerstitium while restoring the size of glomeruli in the kidney cortex. In contrast, minimal induction of inflammation with tRA in the absence of an immunogenic environment in the control mice was observed. Altogether, our results suggest that under a predisposed immunogenic environment in autoimmune lupus, tRA may decrease inflammation in some organs while generating more severe disease in others.
  • Thumbnail Image
    Role of entF Gene in Iron Acquisition by Brucella abortus 2308
    Jain, Neeta (Virginia Tech, 2009-05-11)
    Brucella causes undulant fever in humans and uterine and systemic infection leading to abortions in domestic animals and wild life. For the acquisition of iron in mammalian hosts, species of Brucella are known to produce two siderophores, 2, 3-dihydroxy benzoic acid (2, 3-DHBA) and brucebactin. Inability to synthesize of 2, 3-DHBA affects the ability of pathogen to metabolize erythritol, replicate in trophoblast cells and cause abortion in pregnant ruminant host. The entF gene has been implicated in the unresolved pathway allowing brucebactin biosynthesis in Brucella. The research effort presented in this thesis tries to relate the role of entF in iron acquisition and potential relation with erythritol metabolism by wild type B. abortus 2308. An entF deletion mutant (BAN1) of B. abortus 2308, generated using cre-lox methodology was found to be growth inhibited in iron minimal media compared to wild type strain. Growth inhibition was further enhanced with the addition of an iron chelator or 0.1% erythritol. Compared to wild type strain, no growth inhibition of BAN1 mutant was found in murine J774A.1 macrophages, which suggests that Brucella could acquire iron inside mammalian cells. The entF gene complemented mutant strains of BAN1 (BAN2A and BAN2B) were found to be intermediate in their ability to grow in iron minimal media supplemented with 0.0.05% erythritol, when compared to wild type and BAN1 strain. The results from the present thesis demonstrate that entF gene plays an important role in iron acquisition and erythritol metabolism by B. abortus 2308 under iron limiting conditions.
  • Thumbnail Image
    The role of potassium buffering and apoptosis of trigeminal satellite glial cells in the induction and maintenance of orofacial neuropathic pain in rats
    Bustamante Diaz, Hedie A. (Virginia Tech, 2011-05-31)
    Satellite glial cells (SGC) are laminar cells that wrap completely around the sensory neuron and are responsible for buffering extracellular K+ after neuronal excitation. A decrease in the potassium buffering capacity of SGC has been associated with neuropathic pain (NP) behavior and apoptosis. This dissertation investigated the role of the potassium buffering capacity and apoptosis of trigeminal satellite glial cells (SGC) in the maintenance and development of orofacial NP in rats using in vivo and in vitro methodologies. In vivo endpoints were evaluated after performing chronic constriction injury (CCI) of the infraorbital nerve (IoN). NP signs and behavior were evaluated at 5, 10, 20 40 and 80 hours after injury. We evaluated the potassium buffering capacity of SGC by measuring the intracellular potassium concentration and protein levels and gene expression of the Kir4.1 and the SK3 potassium channels and gap junction protein connexin 43 (Cx43). We evaluated apoptosis endpoints including protein levels and gene expression of apoptotic related proteins bcl-2, caspase 9, caspase 3 and p53. Results indicate that NP signs developed as early as 5 hours after injury. After PNI, SGC responded by increasing their intracellular potassium concentration and by increasing protein levels of Kir4.1, SK3 and Cx43. Nonetheless, this increase in protein levels was not accompanied by an increase in gene expression. Apoptosis results revealed that SGC decreased protein levels and gene expression of anti-apoptotic protein Bcl-2. Using in vitro methodologies, we developed primary trigeminal SGC cultures and evaluated how a decrease in the intracellular potassium concentration modulates apoptosis induced by the mitochondrial and death receptor pathways. SGC depleted of potassium after hypoosmotic shock showed a significant increase in early apoptosis after incubation with mitochondrial pathway apoptotic inducer staurosporine when compared to SGC with normal intracellular concentration. This research has revealed that SGC respond early to PNI by increasing their potassium buffering capacity. We also determined that the mitochondrial apoptotic pathway might be involved in the trigeminal SGC response to PNI. From our in vitro experiments we have revealed that potassium is an important modulator of apoptosis induced by the mitochondrial pathway in cultured trigeminal SGC.
  • Thumbnail Image
    Skin breaking strength in broiler chickens
    Kafri, Ilan (Virginia Polytechnic Institute and State University, 1985)
    A procedure was developed to examine factors contributing to skin strength of meat-type chickens. Skin breaking strength was initially measured in lines of chickens divergently selected for high (H) and low (L) juvenile body weight, their reciprocal crosses (HL and LH), and an F₂ generation derived from HL and LH matings. Skin of chicks from the LL line was weakest, that from the HH and LH matings strongest, and that from HL and F₂ matings was intermediate. Percentage heterosis for breaking strength was significantly positive while percentage recombination was not significant. Skin breaking strength and protein, fat, moisture, and total collagen concentrations of skin from the breast, thigh, and back of male and female commercial broilers were then examined in response to diets containing relatively wide or narrow ratios of calories to protein (C:P). Comparisons were made at 28, 42, and 56 days of age. Chickens fed diets containing wider C:P ratios had weaker skin than those fed diets with narrower C:P ratios, with the differences being greater at older than at younger ages. Males had stronger skin than females. Among body sites, breast skin was stronger than thigh skin with that from the back being intermediate in strength. Skin breaking strength did not appear to be consistently associated with either the protein, fat, or collagen concentrations in the skin. Skin from broiler chicks fed diets with differing C:P ratios was also examined histologically. Males had stronger and thinner skin than females, with the differences in thickness due primarily to differences in the thickness of the hypodermis. Regardless of sex, feeding diets containing wider C:P ratios resulted in weaker and thicker skin. The thicker skin was associated with an increase in the thickness of the hypodermis and a decrease in the thickness of the dermis and epidermis. With the exception of differences between back and thigh skin, increases in breaking strength occurring between skin from different sites were associated with a reduction in total skin thickness and in the thickness of the hypodermis. It was concluded that increased hypodermis thickness and/or decreased thickness of the dermis and epidermis reduced skin strength. Two experiments were conducted in which skin strength was examined in relation to heat stress and supplemental ascorbic acid. In the first experiment, chicks fed diets containing 0 or 100 mg of ascorbic acid per kg of diet were subjected to either a constant ambient temperature of 23C, a constant ambient temperature of 32C, or a treatment consisting of 32C from 1600 to 1200 hr and 400 from 1200 to 1600 hr (32/400). Both heat stress and supplemental ascorbic acid tended to increase collagen concentrations in the skin, but differences in breaking strength were inconsistent in response to these treatments. Supplemental ascorbic acid, however, partially alleviated reductions in body weight due to heat stress but did not decrease body temperatures of heat-stressed chicks. In the final experiment, diets containing either 0, 200, or 400 mg/kg of supplemental ascorbic acid were fed to chicks maintained at ambient temperatures of 23 and 34C. Contrary to previous observations, ascorbic acid failed to ameliorate the depressive effect of heat stress on body weight. Supplemental ascorbic acid tended to reduce adrenal, bursa of Fabricius, and spleen weights but had no effect on heterophil and lymphocyte numbers or ratios. Adrenal ascorbic acid concentrations were not significantly affected by supplemental ascorbic acid and plasma ascorbic acid levels were inconsistent among treatment groups. Neither supplemental ascorbic acid nor heat stress significantly influenced skin strength. It was concluded that supplemental ascorbic acid was not beneficial in improving skin strength in commercial broiler chicks.
  • Thumbnail Image
    Spinal cord gene expression changes in the chicken (Gallus gallus) model of phenyl saligenin phosphate induced delayed neurotoxicity
    Fox, Jonathan Howard (Virginia Tech, 2002-04-11)
    Some organophosphorus (OP) esters induce a central-peripheral distal axonopathy called organophosphorus ester-induced delayed neurotoxicity (OPIDN). In the chicken model neurological deficits and microscopic lesions develop 7-21 days after exposure. Neurotoxic esterase (NTE) is thought to be the initial target in OPIDN. Evidence indicates that neuropathic OP esters have to bind NTE and chemically ?age? for OPIDN induction. It was hypothesized that phenyl saligenin phosphate (PSP), a neuropathic OP ester that essentially irreversibly inhibits NTE as it undergoes the chemical aging process, results in changes in spinal cord gene expression that do not occur with phenylmethylsulfonyl fluoride (PMSF), a non-neuropathic compound that inhibits NTE without aging. This hypothesis was tested in Gallus gallus in experiments designed to detect differences in spinal cord gene expression between PSP, PMSF and vehicle-treated birds 24 hours after exposure. Two approaches were used. Targeted display was developed and used to screen approximately 15000 gel bands. Three candidate genes were identified by targeted display. One, designated P1 has 100% homology with expressed sequence tag pgp1n.pk010.m23, another, P2, is homologous to human KIAA1307, and a third, P3, is unidentified. Northern blotting was used to measure spinal cord expression of a-tubulin and other genes previously reported to be differentially expressed following exposure to di-isopropryl phosphorofluoridate, another agent causing OPIDN. Only expression of a-tubulin was altered in PSP-treated hens. Time course experiments were undertaken to determine spinal cord expression changes of P1, P2, P3 and a-tubulin transcripts at 12, 24, 36 and 48 hours post-exposure. Findings indicated decreases and increases, respectively, of P1 (22%, p=0.0011) and P2 (26%, p=0.0055) transcripts at 12 hours in PSP treated hen spinal cord compared to DMSO controls. An ~2.5 kb a-tubulin transcript was decreased across most time points with maximum change at 48 hours (33%, p=0.0479); an ~4.5 kb a-tubulin transcript was upregulated at 12 hours (38%, p=0.0125) and down regulated at 48 hours (28%, p=0.0576). Responses to PMSF were different than responses to PSP. Spinal cord in-situ hybridization experiments revealed, 1.) mainly neuronal expression of P1, P2 and a-tubulin transcripts, and, 2.) decreased expression of neuronal P1 and a-tubulin transcripts at 12 and 48 hours, respectively. Results indicate that PSP can induce changes in gene expression distinct from those induced with the non-neuropathic NTE inhibitor, PMSF. However, expression changes were low in frequency and magnitude, and their mechanistic importance remains to be fully established.
  • Thumbnail Image
    A study of the neurotoxicity of MPTP and analogs in human neuroblastoma SH-SY5Y cells
    Song, Xiaoou (Virginia Tech, 1996)
    Neuronal alterations resulting from exposure to Parkinsonian-inducing 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP) in an in vitro model SH-SYSY human neuroblastoma cells were explored using cytotoxic effects, neurochemical changes and pathological injury as endpoints. The results suggested that: MPTP entered the SH-SYS5Y human neuroblastoma cells through a non-dopamine transport mechanism, and was metabolized to 1-methyl-4-phenyl-2,3-dihydropyridium (MPDP⁺) and 1-methyl-4-phenylpyridium (MPP⁺) by monoamine oxidase (MAO). MPP⁺, the neurotoxic analog of MPTP, was taken up into cells through a dopamine (DA) uptake mechanism. MPTP, via its metabolite MPP⁺, inhibited NADH dehydrogenase activity. The MPTP-induced alterations of morphology included formation of blebs, attenuated neutrites, abnormal mitochondria with electron-density of matrix and disorganization of cristae, and abnormal aggregation of filamentous material of the cytoskeleton. MPTP was neurotoxic to the dopaminergic system, inhibiting monoamine oxidase (MAO) activity, and decreasing levels of dopamine (DA) and other catecholamines. In addition, MPTP enhanced ³H-DA release from cells, and its metabolite MPP⁺ inhibited ³H-DA uptake. MPTP was found to directly act on the cholinergic system in SH-SY5Y cells, causing dose-related decreases in the binding at muscarinic and nicotinic receptors. MPTP also inhibited acetylcholinesterase (AChE) activity and increased choline levels. The MPTP-induced increase in DA release and the decreases in catecholamines in SH-SYSY cells were blocked by pretreatment with acetylcholine receptor antagonists atropine and d-tubocurarine. MPTP caused increases in tau proteins, and also caused an increased expression of the reverse transcriptase polymerase chain reaction (RT-PCR) product after treatment for 2 to 5 days at 10⁻³ to 10⁻⁴ M. The results, for the first time, demonstrated that MPTP affected cytoskeletal associated tau protein and altered its mRNA. These results demonstrated that the human neuroblastoma cell line, SH-SYSY, can be used as an in vitro model for the study of the neurotoxicity of MPTP, including the mechanisms associated with exposure to this neurotoxicitant.