Hen Spinal Cord Research Articles

Activation of mitochondria-mediated apoptotic pathway in tri-ortho-cresyl phosphate-induced delayed neuropathy

Previous studies suggest that abnormal neurons death has been implicated in organophosphate-induced delayed neuropathy (OPIDN). However, the precise mechanism of neuronal death in OPIDN remains largely unknown. In this study, adult hens were treated with a dosage of 750mg/kg tri-ortho-cresyl phosphate (TOCP) by gavage, and then sacrificed on the time-points of 1, 5, 10, and 21days after dosing TOCP, respectively. The apoptotic change of spinal cord neurons induced by TOCP was examined, and the role of mitochondria-mediated apoptosis of neurons during OPIDN was investigated. TUNEL assays showed that apoptotic neurons in hen spinal cords began to appear on day 5 following TOCP exposure. Immunohistochemistry and western blot analysis revealed a translocation of cytochrome C from mitochondria to cytoplasm after dosing TOCP. Moreover, the level of Bcl-2, Bcl-xl, Pro-caspase3 and Pro-caspase9 in hen spinal cord was significantly decreased, whereas that of Bax and cleaved-PARP was significantly elevated. Taken together, these findings indicate that the administration of TOCP can induce neuron apoptosis in hen spinal cords, which might be mediated by the activation of mitochondrial apoptotic pathway.

Aberrant activation of CDK5 is involved in the pathogenesis of OPIDN

Exposure to triorthocresyl phosphate (TOCP) may result in a late neurological complication, i.e. organophosphate-induced delayed neuropathy (OPIDN). The aim of this study was to examine changes in levels of cyclin-dependent kinase 5 (CDK5) and of its activator, p35/p25, in the spinal cord of hens treated by TOCP. After exposure to a single dose of TOCP, groups of adult hens were examined in 3, 5, 7, 9, 14, and 18 days after exposure. CDK5, p35/p25 expression and distribution in the lumbar spinal cord were evaluated by immunohistochemistry and Western blotting. The hens showed signs of OPIDN around day 9 after exposure. The number of p (phosphorylated) -CDK5 and p35 positive cells increased significantly. Co-localization and mislocalization of p-CDK5 and p35/p25 was identified and became evident in neurons around the 9th day. Meanwhile, CDK5, p-CDK5, p35, p25 protein levels and p25/p35 ratio were increased, and peaked around the 9th day, then decreased. Some hens' unilateral common peroneal was treated by roscovitine 3 days after TOCP exposure. Axonal transport of these nerves was faster than of their opposite side and of those simply treated by TOCP. These findings indicate aberrant activation of CDK5 may be involved in the pathogenesis of OPIDN.

Spinal cord neuronal apoptosis induced by triorthocresyl phosphate poisoning in hens

To investigate the role of neuronal apoptosis in organophosphorus poisoning-induced delayed neuropathy (OPIDN) and its dynamic pathological changes. To establish OPIDN animal model, triorthocresyl phosphate (TOCP)was given to hens with a single dose (1 000 mg/kg, im). Changes of neuropathology, number of neurons and apoptotic cells in the third lumbar spinal cord were observed by HE, Nissl and TUNEL methods 3, 5, 7, 10, 14, 18 days after injection. The hens showed OPIDN typical signs (progressive ataxia and hypotonia) about 9 days after TOCP exposure. HE staining revealed dark red nucleus in neurons of anterior horn of lumbar spinal cord 5 days after exposure, but this phenomenon disappeared 18 days later. Nissl method showed that the number of neurons in anterior horn of spinal cord decreased [from (82 +/- 4) cell/mm(2) to (66 +/- 6) cell/mm(2)]. TUNEL positive cells began to appear [(22 +/- 2) cell/mm(2)] 5 days after TOCP exposure, and reached the peak [(27 +/- 3) cell/mm(2)] 7 days later, and disappeared 18 days later. Neuronal apoptosis in anterior horn of spinal cord of hens appeared in OPIDN, suggesting that cellular apoptosis may play an important role in the pathogenesis of OPIDN.

Characterization of binding sites for diisopropyl phosphorofluoridate in spinal cord cytosol

Gel filtration chromatography was performed on cytosol preparation of hen spinal cord to find molecular target(s) for organophosphorus-induced delayed neurotoxicity (OPIDN). Three binding peaks of [ 3H]diisopropyl phosphorofluoridate (DFP), an organophosphate that induces OPIDN, were separated from the cytosol preparation. The activities of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) that has been proposed as a screening method for OPIDN eluted in the fractions within these two DFP binding peaks. However, the other peak had none of the activities of AChE and NTE. Therefore, this DFP binding proteins in cytosol may be peculiar to the pathogenesis of OPIDN.

A Comparative Study of Binding Sites for Diisopropyl Phosphorofluoridate in Membrane and Cytosol Preparations from Spinal Cord and Brain of Hens

Biochemical events in the initiation of organophosphorus induced delayed neurotoxicity (OPIDN) are not well understood. To find new putative target(s) for OPIDN, we investigated the biochemical and pharmacological characteristics of [ 3H ]diisopropyl phosphorofluoridate (DFP) binding to membrane and cytosol preparations from the brain and spinal cord of hens in vitro. [ 3H ]DFP binding to both preparations was determined by the specific binding obtained by subtracting non-specific binding from total binding. The specific binding sites of [ 3H ]DFP were found not only on membrane but also in cytosol. K d values were higher and B max values were lower in cytosol than in membrane. Moreover, the K d values in both membrane and cytosol preparations from spinal cord were lower than those of brain. The B max values in membrane and cytosol were similar between brain and spinal cord. The specific binding to both preparations was markedly displaced by unlabeled DFP. The specific binding of DFP to the membrane was highly or partly displaced by organophosphorus compounds (OPs) or a carbamate, respectively. However, both the OPs and the carbamate had considerably weaker blocking effects on the specific binding of DFP to cytosol. None of the compounds known to interact with neuropathy target esterase (NTE) had a strong blocking effect on the specific binding of DFP to either membrane or cytosol. These results show that the specific binding of DFP to the membrane may be binding with cholinesterase (ChE). However, cytosol, especially in spinal cord, may have DFP binding sites other than ChE and NTE.

Correlation of Binding Sites for Diisopropyl Phosphorofluoridate with Cholinesterase and Neuropathy Target Esterase in Membrane and Cytosol Preparations from Hen

To find new putative target(s) for organophosphorus induced delayed neurotoxicity (OPIDN), we investigated the biochemical and pharmacological characteristics of [ 3H]diisopropyl phosphorofluoridate (DFP) binding to membrane and cytosol preparations from the brain and spinal cord of hens. Specific [ 3H]DFP binding was determined by subtracting non-specific binding from total binding. The binding sites of [ 3H]DFP, an organophosphate that induces OPIDN, were found not only on membrane but also in cytosol. Reduction of subsequent ex vivo specific [ 3H]DFP binding by in vivo pretreatment with unlabeled DFP was found in cytosol, not membrane. The reduced binding lasted to the onset of OPIDN, especially in spinal cord. These results suggest that the specific DFP binding sites in cytosol, rather than on membrane, are the most important with regard to the initiation of OPIDN. Inhibitors of cholinesterase (ChE) and neuropathy target esterase (NTE) other than DFP did not affect specific [ 3H]DFP binding to either membranes or cytosol in vivo. Additionally, inhibition of the activities of these esterases by these compounds was not consistent with either the degree of inhibition of the [ 3H]DFP binding or a time-dependent manner of OPIDN. These results suggest that DFP binding site(s) involved in the initiation of OPIDN may be different from the active sites of ChE and NTE.

Altered expression of neurofilament subunits in diisopropyl phosphorofluoridate-treated hen spinal cord and their presence in axonal aggregations

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester, which produces organophosphorus ester-induced delayed neuropathy (OPIDN) in hen and other sensitive species. A single dose of DFP (1.7 mg/kg, sc.) produces mild ataxia in 7–14 days in hens, which develops into severe ataxia or paralysis with the progression of disease. OPIDN is associated with axonal swellings and degeneration of axons. This study was carried out to investigate the expression of neurofilament (NF) subunits in the spinal cord of DFP-treated hens. Hens were treated with a single dose of DFP and sacrificed 1, 5, 10, and 20 days post-treatment. Western blot analysis showed increased expression of middle molecular weight neurofilament protein (NF-M), and decreased expression of high molecular weight (NF-H) and low molecular weight (NF-L) neurofilament proteins in the 2 M urea extracts of spinal cord particulate fraction. These changes were observed within 24 h of DFP administration and persisted for 10–20 days. Thus, there was increase in the stoichiometry of NF-M:NF-L in the spinal cord of DFP-treated hens. Immunoprecipitation, cross-linking, and two-dimensional polyacrylamide gel electrophoresis showed the presence of heterodimers, but not heterotetramers, in the hen spinal cord extract. Immunohistochemical staining revealed the presence of all three NF subunits in the cytoskeletal inclusions in DFP-treated hen spinal cord cross-sections. The results suggested that each NF subunit might be accumulated by a different mechanism in the axonal aggregations of DFP-treated hen.

Comparison of the Relative Inhibition of Acetylcholinesterase and Neuropathy Target Esterase in Rats and Hens Given Cholinesterase Inhibitors

Comparison of the Relative Inhibition of Acetylcholinesterase and Neuropathy Target Esterase in Rats and Hens Given Cholinesterase Inhibitors. Ehrich, M., Jortner, B. S., and Padilla, S. (1995). Fundam. Appl. Toxicol.24, 94-101.Inhibition of neuropathy target esterase (NTE, neurotoxic esterase) and acetylcholinesterase (AChE) activities was compared in brain and spinal cords of adult White Leghorn hens and adult male Long Evan rats 4-48 hr after administration of tri-ortho-tolyl phosphate (TOTP po. 50-500 mg/kg to hens: 300-1000 mg/kg to rats), phenyl saligenin phosphate (PSP im 0.1-2.5 mg/kg to hens; 5-24 mg/kg to rats), mipafox (3-30 mg/kg ip to hens and rats), diisopropyl phosphorofluoridate (DFP sc, 0.25-1.0 mg/kg to hens; 1-3 mg/kg to rats), dichlorvos (5-60 mg/kg ip to hens; 5-30 mg/kg to rats), malathion (75-300 mg/kg po to hens; 600-2000 mg/kg to rats), and carbaryl (300-560 mg/kg ip to hens; 30-170 mg/kg to rats). Inhibitions of NTE and AChE were dose-related after administration of all compounds to both species. Hens and rats given TOTP, PSP, mipafox, and DFP demonstrated delayed neuropathy 3 weeks later, with spinal cord lesions and clinical signs more notable in hens. Ratios of NTE/AChE inhibition in hen spinal cord, averaged over the doses used, were 2.6 after TOTP. 5.2 after PSP, 1.3 after mipafox, and 0.9 after DFP, which contrast with 0.53 after dichlorvos, 1.0 after malathion, and 0.46 after carbaryl. Rat NTE/AChE inhibition ratios were 0.9 after TOTP, 2.6 after PSP, 1.0 after mipafox, 0.62 after DFP, 1.3 after dichlorvos, 2.2 after malathion, and 1.1 after carbaryl. The lower NTE, AChE ratios in rats given dosages of the four organophosphorus compounds that caused delayed neuropathy interferred with survival, an effect that was not a problem in hens. This observation, along with the absence of overt and specific clinical signs and the restricted presence of neuropathological lesions in rats, suggests that the hen remains the animal of choice for testing for organophosphorus-induced delayed neuropathy.

Cell mixing in the spinal cords of mouse chimeras

With the aim of determining whether there is significant cell mixing during development of the spinal cord, experimental chimeric mice containing two genetically distinct cell populations were produced by aggregating BALB c or BALB c × LPT hybrid embryos with C3H HeN embryos. The BALB c and LPT hybrid spinal cord cells were distinguished histochemically from the C3H HeN spinal cord cells by using β-glucuronidase as an independent cell genotype marker. BALB c and LPT hybrid cells have high levels of β-glucuronidase activity, while the C3H HeN cells have low levels. The spinal cords of the chimeras were dissected out regionally (i.e., cervical, thoracic, and lumbar areas) and were sectioned serially. Each region was then analyzed by scoring large- and medium-sized neuronal cell bodies (⩾ 10 μm) whose genotypes were distinguished by their β-glucuronidase levels. Observations of seven chimeric mice, with coat colors that varied from one extreme (5% albino) to the other (90% albino), suggest that each chimeric spinal cord is a relatively homogeneous population throughout its length. On average only 4 to 5 percentage point differences were observed when comparing left-right, cranial-caudal, and dorsal-ventral regions within a given chimera. The cell mixing, however, is not total, and regional variations were noted. Maximum left-right differences between different spinal cord levels never exceeded 18 percentage points, while in the entire cord the maximum left-right difference was 11 percentage points. When considering dorsal-ventral differences, 18 and 15 percentage points were observed within the spinal cord levels and the entire cord, respectively. However, when comparisons were made between smaller subregions (e.g., right-dorsal-cervical vs left-ventral-lumbar), larger differences of up to 30 percentage points were observed. In addition, the genotype proportions in the spinal cord were closely correlated with the visually estimated proportions of coat color genotypes.

Comparison of endogenous phosphorylation of hen and rat spinal cord proteins and partial characterization of optimal phosphorylation conditions for hen spinal cord

The optimal conditions for the endogenous phosphorylation of hen spinal cord cytosolic and membrane proteins with 5 μM [ γ- 32P]ATP, 10 mM MgCl 2, were determined by 10% SDS-polyacrylamide gel electrophoresis, autoradiography, and microdensitometry. Phosphate incorporation increased linearly with concentrations ranging from 35–75 μg/100 μl for cytosolic proteins and 21–125 μg/200 μl for membrane proteins. Optimal incubation times, temperatures, and pH values were 60 s, 30°C, and 6.0, respectively, for spinal cord cytosolic proteins and 15 s, 45°C, and 8.0, respectively, for spinal cord membranes. Prominent species differences in protein phosphorylation between these fractions in hens and similarly prepared fractions in rats, co-electrophoresed, include 80K and 30K protein phosphate acceptors unique to rat spinal cord cytosol, 60K and 16K protein phosphate acceptors characteristic of rat spinal cord membranes, a 50K protein phosphate acceptor present only in hen spinal cord membranes, and greater phosphorylation of a more abundant 20K protein in both hen spinal cord fractions. The functional significance of these differences is presently unclear. However, their characterization provides a basis from which to launch future investigations of the biochemistry, pharmacology, and toxicology of spinal cord protein phosphorylation and indicates that caution should be exercised in the choice of an animal model with characteristics appropriate to those of the system it is representing.

Synaptic morphology in the spinal cord of normal and tricresylphosphate (TCP) poisoned hen

A study has been made of synaptic degeneration at an ultrastructural level using 6% glutaraldehyde perfusion, fixation method in the anterior horn of hen's spinal cord, after application of a single dose of 0.5 cm3 TCP on the comb. The degenerative changes are seen in the synaptic vesicles and mitochondria of pre-synaptic bag. A comparison of these findings is made with degenerative changes caused by mechanical interference and x-irradiation. It is concluded that the toxic effects of TCP are certainly manifested in the synaptic areas which show changes similar to those caused by the mechanical interference and by x-irradiation.