| ObjectivesThe present study was designed to investigate whether the mitochondrial dysfunction account for the development of OPIDN. We examined the morphological changes of mitochondria and mitochondrial dysfunction in a dose-and time-dependent manner following treatment with TOCP at three different doses.Methods96 adult Roman hens were randomly divided into four groups, i.e. three experimental groups (low-dosage, medium-dosage, and high-dosage, n=24) and the corresponding time-matched control group (n=24). The hens in experimental groups were treated with TOCP by gavage at single dosages of 185,375,750 mg/kg-bw, respectively. TOCP was dissolved in corn oil and administered at 0.65 ml/kg·bw, while hens in control group received an equivalent volume of corn oil by gavage. Neurological testing was examined by delayed neurotoxicitic symptoms. OPIDN was assessed by an 8-point graded scale. On day 1,5,15,21 post-dosing, hens respectively selected from experimental groups and control group were sacrificed and nerve tissues were dissected for the determination of mitochondrial functions. Another 16 hens were used for electron microscopic study. Hens were randomly divided into 2 groups: TOCP-treated group (n=12) received TOCP by gavage at dosage of 750 mg/kg-bw and control group (n=4) received an equivalent volume of corn oil by gavage. The hens were respectively sacrificed on day 1,5,15,21 post-dosing, and one control hen was sacrificed simultaneously at each time point. The selected hens were anesthetized, perfused, and then the cerebral cortex cinerea and the ventral horn of lumbar spinal cord were dissected for electron microscopy. The preparation of mitochondria was proceeded as method of density gradient centrifugation. Mitochondrial swelling was assayed by decrease of A540 nm. The isolated mitochondria were modulated to 0.5 mg/mL and incubated in the assay buffer. MPT was initiated by adding 50 uM calcium chloride, and monitored by measuring the decrease of A540 nm (△A540nm) at 37℃using an ultraviolet spectrophotometer in five min.△ψm was detected by monitoring the fluorescence quenching of Rh123 dynamically. Fluorescence with excitation at 503 nm and emission at 527 nm was detected in a reaction buffer using F-4500FL Spectrophotometer. The mitochondria were diluted to 0.5mg/mL in the buffer and incubated for 3 min. The fluorescence was recorded again, and the alteration of the△ψm was detected by the decrease of the fluorescence. ATP concentration in hen's cerebrum, cerebellum, spinal cord and sciatic nerve was determined by Firefly Luciferase Bioluminescence (FLB) method. MTT reduction was used to assess the activity of the mitochondrial respiratory chain. The dye is converted to water-insoluble purple formazan on the reductive cleavage of its tetrazolium ring by the succinate dehydrogenase system of the active mitochondria. The mitochondrial ATPase 6 mRNA level in hens'nerve tissues was determined by Reverse Transcription Polymerase Chain Reaction (RT-PCR). The expression of ATPase beta in cerebrum, cerebellum, spinal cord and tibial nerve of hens treated with TOCP were analyzed by western blotting.ResultsHens exhibited obvious reductions of body weights after administration of TOCP. On day 16 post-dosing, the body weight of hens in 375 mg/kg and 750 mg/kg TOCP-treated group began to decrease (79.4% and 74.2% of that of control group hens, P<0.05). The mean body weight loss progressively increased as time went on. On day 21 post-dosing, the mean body weight of the three experimental groups respectively decreased to 71.2%,69.3% and 66.0% of that of control group hens (P<0.05).Hens in 750 mg/kg-bw TOCP-treated group began to show slightly abnormal gait on day 6 post-dosing. Clinical signs progressed to disturbances in gait and unsteadiness, and signs were severe on day 10 post-dosing. Most of the hens reached total hindlimb paralysis on day 16 post-dosing. Hens in 375 mg/kg-bw TOCP-treated group began to take on slightly abnormal gait on day 7 post-dosing with daily progression, reached total hindlimb paralysis on day 17 post-dosing. Hens in 185 mg/kg-bw TOCP-treated group had slightly abnormal gait on day 8 post-dosing and were exacerbated on day 11 post-dosing. Not all hens in this group reached total hindlimb paralysis on day 21 post-dosing. In contrast, no clinical signs of delayed neurotoxicity were observed in control hens.In neurons of cerebrum, the progressive morphological changes of mitochondria were observed. On day 5 post-dosing there were slight abnormalities, exhibited as mild swelling and the slight decrease of mitochondrial cristae. On day 15 post-dosing, most of the mitochondria were swollen, and the mitochondrial cristae ruptured, decreased and even disappeared. On day 21 post-dosing, vacuolation of mitochondria, and swollen endoplasmic reticulum were much more obvious. The changes of mitochondrial structure in oligodendrocytes of cerebrum were similar with those in neurons. Changes began to take on from day 5 post-dosing and developed in a time-dependent fashion. Swelling and mitochondrial cristae rupture and dissolution were observed at day 21 post-dosing.The ultrastructural alterations of mitochondria in spinal cord were much more obvious than those in cerebrum. In neurons, the structure of mitochondria represented changes on day 5 post-dosing. On day 15 post-dosing, the swelling and vacuolation of mitochondria were more obvious and subsequently almost all mitochondria in neurons were vacuolated on day 21 post-dosing. In oligodendrocytes, the vacuolation of mitochondria was much more obvious than that in neurons. Swelling and vacuolation of mitochondria began to represent on day 5 post-dosing, and nearly all of the mitochondria were swollen and vacuolated on day 21 post-dosing.In tibial nerve, the vacuolization of mitochondria could be observed under the electron microscopy on day 1 post-dosing. The axons represented release of myelin lamellar partly and the edge of microfilaments and microtubules in axoplasm was blurred, showing like floc. On day 5 post-dosing, the changes were more obvious. Axonal myelin sheath was invaginate forming a circuitous sac-like structure. The microfilaments and microtubules were proliferative abnormally and the density of axoplasm were increased. On day 15 post-dosing, the number of mitochondria were decreased. On day 21 post-dosing, swell and vacuolization of mitochondria were much more obvious. Universal release of myelin lamellar was observed and some microfilaments and microtubules cross-linkinged and fused, forming irregular electron dense-sparse area.Ca2+-induced MPT in cerebrum had no significant changes after administration of TOCP. However, in spinal cord,△A540nm of the three treated groups was all significantly decreased on day 1 post-dosing compared to the control group (P<0.05,△A540nm of the three treated groups respectively decreased by 36.8%,43.8%and 42.1%). On day 5 and 15 post-dosing, the decrease trend of△A540nm was still kept in the 375 and 750 mg/kg-bw TOCP-treated groups hens (P<0.05). On day 21 post-posing,△A540nm of the three experimental groups slightly increased, while there was no significant change compared to the control group.Although TOCP could result in decrease of△ψm in hens'nervous tissue, there was no significant difference between experimental groups and control group. Also there was no significant difference of△ψm in the three treated groups as time went on.In cerebrum, compared with control group, the ATP concentration in three TOCP groups was decreased, but there was no significant difference. On day 21 post-dosing, a significant decrease in ATP concentration was observed on day 15 post-dosing in low-dosage group (7.55%), medium-dosage (12.6%), and high-dosage (19.3%) of TOCP treated animals. In cerebellum, the decrease of ATP concentration was not obvious on day 1 and 5 post-dosing. On day 15 post-dosing, the ATP concentration in medium-dosage group decreased by 13.3%(P< 0.05). On day 21 post-dosing, compared with control group, the ATP concentration in medium-dosage and high-dosage groups were respectively decreased by 12.9%(P< 0.05) and 18.3%. In spinal cord, there was no significant difference in ATP concentration on day 1 post-dosing. The decrease of ATP concentration in three different dosage groups was kept during the whole experiment. On day 5 post-dosing, the ATP concentration in three TOCP groups respectively decreased by 5.20%,15.7% and 30.0%(P< 0.05). On day 21 post-dosing, a significant decrease in ATP concentration was observed on in low-dosage group (20.5%, P< 0.05), medium-dosage (30.6%, P< 0.05), and high-dosage (36.1%, P< 0.01) of TOCP treated animals. In sciatic nerve, the ATP concentration in three dosage groups was all slightly decreased on day 1 post-dosing. The decrease trend has been keeping on day 5 and 15 post-dosing. Compared with control group, the ATP concentration decreased by 16.5%,42.9% and 46.8%(P< 0.05) on day 15 post-dosing. On day 21 post-dosing, the ATP concentration represented a little increase, but was still significantly decreased compared with control group.In cerebrum, there was no significant decrease in the activity of mitochondrial succinate dehydrogenase on day 1 and 5 post-dosing. On day 15 and 21 post-dosing, MTT reduction in high-dosage group was significantly decreased (16.8% on day 15 and 30.7% on day 21). In cerebellum, the alteration of the activity of mitochondrial succinate dehydrogenase was not obvious on day 1 post-dosing. On day 5 post-dosing, it declined slightly. A significant decrease in the activity of mitochondrial succinate dehydrogenase was observed on day 21 post-dosing in low-dosage group (9.63%), medium-dosage (29.9%), and high-dosage (31.9%) of TOCP treated animals. In spinal cord, compared with the control group, the activity of mitochondrial succinate dehydrogenase in high-dose group decreased by 28.0% (P< 0.05) on day 5 post-dosing. The decrease trend of it was still kept in three TOCP-treated groups hens on day 15 post-dosing (P<0.05). At the end of the experiment, the activity of mitochondrial succinate dehydrogenase was slightly increased, but still decreased compared with control group. In sciatic nerve, the alteration of the activity of mitochondrial succinate dehydrogenase was not obvious on day 1 post-dosing. On day 5, the activity of mitochondrial succinate dehydrogenase in medium-dosage was decreased by 18.5% (P< 0.01). A significant decrease in the activity of mitochondrial succinate dehydrogenase was observed on day 15 in low-dosage group (13.6%), medium-dosage (22.3%), and high-dosage (42.0%) of TOCP treated animals (P< 0. 05). On day 21, the activity of mitochondrial succinate dehydrogenase was increased slightly, but still significantly decreased compared with control group.In cerebrum, compared with control group, the ATPase 6 mRNA level in three TOCP groups all dereased significantly (P< 0.05). On day 21 post-dosing, a significant decrease in ATPase 6 mRNA level was observed on in low-dosage group (34%), medium-dosage (32%), and high-dosage (23%) of TOCP treated animals (P<0. 01). In spinal cord, the decreased of ATPase 6 mRNA level began to represent on day 1 post-dosing. The ATPase 6 mRNA level in high-dosage group decreased by 33%(P< 0. 01), which was the most obvious. On day 21 post-dosing, the ATPase 6 mRNA level in three different TOCP groups respectively decreased by 23%,44%and 35%(P< 0.01). In sciatic nerve, the change of ATPase 6 mRNA level in high-dosage group was more obvious than those in low-and medium-dosage groups. On day 21 post-dosing, the ATPase 6 mRNA level in TOCP groups decreased by 12%,20% and 35%(P< 0.01) respectively.Western blotting results showed, in cerebrum there was no significant change ATPase beta protein level in low-dosage group on day 1 post-dosing. ATPase beta protein level increased by 6.96% and 8.75% (P< 0.05) respectively. On day 5 post-dosing, the ATPase beta protein level began to increase in low-dosage group and decrease in medium-and high-dosage groups. On day 15 post-dosing, the ATPase beta protein level in low-dosage group also decreased. On day 21 post-dosing, the ATPase beta protein level in three dosage groups respectively decreased by 15.3% (P< 0.05), 27.9%(P< 0.01) and 35.0% (P< 0.05). In cerebellum, the decrease of ATPase beta protein level in TOCP groups began from day 1 post-dosing, respectively decreased by 11.3% (P< 0.05),15.0% (P< 0.01) and 16.5% (P< 0.05). The decrease trend has been keeping on day 5 and 15 post-dosing. On day 21 post-dosing, the level of ATPase beta increased slightly, but still significantly decreased compared with control group. In spinal cord, there was no significant change of ATPase beta protein level in low-dosage group on day 1 post-dosing, whereas in medium-and high-dosage groups ATPase beta protein level increased slightly. On day 5 post-dosing, the ATPase beta protein level began to increase in low-dosage group and decrease in medium-and high-dosage groups. On day 21 post-dosing, the ATPase beta protein level in three dosage groups respectively decreased by 22.7% (P< 0.01),31.8% (P< 0.05) and 43.9% (P< 0.01). In sciatic nerve, the decreased of ATPase beta protein level in three dosage groups all began to represent on day 1 post-dosing, decreased by 15.3%,25.1% (P< 0.01) and 33.3% (P< 0.01). This decrease trend was kept on day 5 and 15 post-dosing (P< 0.05). On day 21 post-dosing, the ATPase beta protein level in low-dosage group kept on dereasing while in medium-and high-dosage groups the level of ATPase beta protein began to increase slightly. But the levels were still much more lower than control group. The ATPase beta protein level respectively decreased by 30.6%,39.4% and 50.4%(P< 0.01) in three TOCP groups.Conclusions1. TOCP could result in structural changes of hens'nerve tissues, including cerebrum, cerebellum, spinal cord and tibial nerve from day 5 post-dosing when the clinical signs of OPIDN began to take on. Swelling mitochondria, vacuolation, decrease of mitochondrial cristae could be observed under the electron microscopy.2. The mitochondrial membrane permeability of hens'nerve tissues was increased and the mitochondrial membrane potential was decreased after the administration of TOCP, which indicated the injury of mitochondrial function. TOCP could result in the decrease of mitochondrial ATP concentration and the activity of mitochondrial respiratory chain enzymes.3. In hens'nerve tissues, ATPase 6 mRNA level and ATPase beta protein level were decreased because of the administration of TOCP.4. The changes of mitochondrial dysfunction might be related to the development of OPIDN induced by OPs.
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