| ObjectiveParkinson’s disease is a neurodegenerative disorder that is closely related to exposure to environmental toxicants.Studies in China and other countries demonstrated that long-term exposure to pesticides and other environmental toxicants promotes the development of PD.Exposed to paraquat(PQ)and maneb(Mb),two commonly used pesticides,has been proved to be closely associated with PD.Epidemiological investigation showed that the combined exposure of PQ and Mb binereases the risk of PD.Experimental animals exposed to PQ and Mb also display dopaminergic neurodegeneration and gait abnormalities that are are similar to be observed in patients with PD.At present,PQ and Mb co-exposure has become a widely accepted method to generate experimental PD model.Recently,it has been showed that PQ and Mb-induced rodent PD model displays not only motor symptom,but also non-motor symptom(NMS),such as anxiety,depression,learning and memory impairment.However the mechanisms behind PQ and Mb-induced NMS remain unclear.The locus ceruleus(LC)is a dense group of norepinephrine(NE)producing neurons in the upper part of the rhoMic head in the lateral edge of the fourth ventricle that regulates a variety of systemic activities,including sensory,emotional regulation,maintenance of awakening and learning and memory and so on.Patients with PD showed degeneration of LC/NE neuron,which is more serious and even earlier than that of dopaminergic neuron in the substantia nigra.We recently found that comined exposure to PQ and Mb results in loss of LC/NE neuron and decrease of NE content in the brain of mice.However,are impaired LC/NE neuron and associated with reduction of NE level contribute to NMS induced by PQ and Mb co-exposure?If so,what is the underlying mechanism?None of these questions is answered previously.Therefore,in this study,we used N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine(DSP4),a specific toxicant for LC/NE neuron,to induce LC/NE neurodegeneration and NE deletion in mice and followed by PQ and Mb co-exposure.The alterations of NMS,including constipation,depression,learning and memory as well as neuronal damage and neuroinflammatory responses in different brain regions of mice were investigated to elucidate the role of LC/NE in PQ and Mb-induced NMS.MethodsEighty adult SPF male C57BL/6J mice were fed adaptively for 7 days and then were randomly divided into four groups(n=20 per group):control group(Con),model group(PQ+Mb),DSP4 treatment group(DSP-4+PQ+Mb)and DSP-4 alone group(DSP4).Mice in PQ+Mb group were administrated(i.p.,5 μl/g body weight)with comined paraquat(10 mg/kg)and maneb(30 mg/kg)for consecutive 4 weeks(twice per week)according to our previous report.Mice in DSP-4+PQ+Mb group received a single system injection of DSP-4(50 mg/kg)one week prior to PQ+Mb exposure.Control and DSP-4 alone mice received an equal volume of 0.9%saline or DSP-4,respectively.After 4 weeks of initial PQ+Mb treatment,fecal collection,forced swimming and morris water maze experiments were used to measure constipation,depression and learning and memory ability of mice,respectively.After finishing behavioral measurement,mice(n=5)were anesthetized and were perfused with normal saline for 5 minutes,and then 4%paraformaldehyde for 10 minutes.The brains were collected and frozen sections were prepared.Immunohistochemical staining was performed to detect the damage of dopamine neuron(TH),hippocampal and cortical neuron(NeuN),neuronal synapse(PSD95)and microglial activation(Iba-1).The other mice(n=15 per group)were perfused with normal saline only.The brain was rapidly dissected and brain regions including midbrain,striatum,brainstem,hippocampus and cortex were separated on mice,followed by frozen in liquid nitrogen and transferred to-80 C.Western blot was used to detect the expressions of NeuN,PSD95,Iba-land qRT-PCR was used to detect the mRNA level of microglial M1/M2 polarizing markers.Prism5.0 statistical analysis software was used to analyze the experimental results.Results1.The effects of DSP-4 on NE content in different brain regions Compared with Con,NE contents in midbrain,hippocampus and cortical regions were significantly decreased 1 day after DSP-4 injection,and 48.8%,72.2%and 45.5%of reduction(p<0.05)at day 28,respectively,were observed.2.The effects of NE deficiency on body weight of mice exposed to PQ+Mb There was no significant change in body weight of mice in PQ+Mb group compared with controls,but the body weight of DSP4+PQ+Mb group was significantly lower than that of both Con and PQ+Mb mice(p<0.01).3.The effects of NE deletion on constipation-like behavior in mice exposed to PQ+Mb The defecation times of mice in PQ+Mb and DSP-4+PQ+Mb group were significantly lower than those of Con group(p<0 05),but there was no significant difference between PQ+Mb and DSP-4+PQ+Mb groups(p>0.05).Analysis of fecal dry/wet ratio showed no significant difference among groups(p>0.05).4.The effects of NE deletion on depressive behavior in mice exposed to PQ+Mb In forced swimming experiments,there was no significant difference in the time of struggling in water among Con,PQ+Mb and DSP-4+PQ+Mb groups(p>0.05).Consistently,no significant difference of struggle times among groups was observed(p>0.05).5.The effects of NE deficiency on learning and memory in mice exposed to PQ+Mb There was no significant difference in escape latency and swimming distance between PQ+Mb and Con groups(p>0.05).However,compared with Con and PQ+Mb group,mice in DSP-4+PQ+Mb group showed increased escape latency and swimming distance(p<0.05).The results of space exploration showed that compared with Con,the latency of first-time crossing in mice of both PQ+Mb and DSP-4+PQ+Mb groups was longer(p<0.05),while the time percentage of target quadrant was lower(p<0.05).However,no significant difference of above-mentioned parameters between DSP-4+PQ+Mb and PQ+Mb group was observed(p<0.05).6.The effects of NE deficiency on dopaminergic neuron in mice exposed to PQ+Mb Immunohistochemical staining showed that the number of dopaminergic neuron(TH cell)in the substantia nigra in PQ+Mb-treated mice was significantly lower than that in Con group(P<0.05).Compared with PQ+Mb group,the number of dopaminergic neuron in DSP-4+PQ+Mb group decreased further,and the difference was statistically significant(p<0.01).The light density of TH staining in the striatum was significantly decreased in PQ+Mb group after DSP-4 injection,and the difference was statistically significant(p<0.01).The results showed that DSP-4 exacerbated axonal injury of dopaminergic neuron in PQ+Mb mice.TH staining in the hippocampus and cortex showed that compared with Con group,the density of TH staining in PQ+Mb group did not change significantly,but TH staining optical density in DSP-4+PQ+Mb-treated mice was decreased significantly(p<0.01).DSP-4 alone did not affect the survival of dopaminergic neuron in mice.Consistently,Western blot showed that compared with Con and PQ+Mb groups,the expressions of TH in the hippocampus and cortex of DSP-4+PQ+Mb-treated mice were significantly decreased(p<0.01),although PQ+Mb alone failed to affect TH expression.7.The effects of NE deletion on hippocampal neuron in mice exposed to PQ+Mb The immunohistochemistry staining showed that the number of Neu-N neurons and the expression of synaptophysin PSD-95 in hippocampus in PQ+Mb-treated mice group were not significantly different from those of Con group.The number of Neu-N neurons and the expression of synaptophysin PSD-95 in hippocampus of DSP-4+PQ+Mb group were significantly lower than those of Con and PQ+Mb groups.The difference was statistically significant(p<0.01).Consistently,the expression of Neu-N and PSD-95 in hippocampus of PQ Mb group was not significantly different from that of Con group(p>0.05).Compared with Con and PQ+Mb-treated group,the expression of Neu-N and PSD-95 protein in hippocampus of DSP-4+PQ+Mb group decreased significantly(P<0.05).The results showed that DSP-4 aggravated the neuronal and synaptic injury in the hippocampus of PQ+Mb treated mice.8.Effect of NE deletion on cortical nerve injury in mice exposed to PQ+Mb The immunohistochemistry staining showed that the number of Neu-N neurons and the expression of synaptophysin PSD-95 in cortical in PQ+Mb-treated mice group were not significantly different from those of Con group.The number of Neu-N neurons and the expression of synaptophysin PSD-95 in cortical of DSP-4+PQ+Mb group were significantly lower than those of Con and PQ+Mb groups.The difference was statistically significant(p<0.01).Consistently,the expression of Neu-N and PSD-95 in cortical of PQ Mb group was not significantly different from that of Con group(p>0.05).Compared with Con and PQ+Mb-treated group,the expression of Neu-N and PSD-95 protein in hippocampus of DSP-4+PQ+Mb group decreased significantly(p<0.05).The results showed that DSP-4 aggravated the neuronal and synaptic injury in the cortical of PQ+Mb treated mice.9.Effect of NE deletion on the level of neuroinflammation in mice exposed to PQ+Mb The immunohistochemistry staining showed that the number of microglia activation and the expression of Iba-1 in hippocampus and cortex of PQ+Mb-treated mice group were higher than those in Con group.The number of microglia and the expression of Iba-1 protein in hippocampus and cortex of DSP-4+PQ+Mb group were significantly higher than those in Con and PQ Mb group.The difference was statistically significant(P<0.01).Consistently,The expression of Iba-1 protein in hippocampus and cortex of PQ+Mb group was higher than that in Con group.Compared with Con and PQ+Mb groups,the expression of Iba-1 protein in hippocampus and cortex of DSP-4+PQ+Mb group was significantly higher than that of Con and PQ+Mb group(p<0.05).RT-PCR results showed that iNOS,in hippocampus of PQ+Mb group was significantly higher than that of DSP-4+PQ+Mb group(p<0.05).The levels of TNF-α and IL-1β were higher than those of Con group.Compared with Con and PQ+Mb groups,the levels of iNOS,TNF-α and IL-1β in DSP-4+PQ+Mb group were higher than those in DSP-4+PQ+Mb group(P<0.05).On the contrary,the expression of Ym-1 in DSP-4+PQ+Mb group was significantly lower than that in PQ+Mb group(p<0.05),but there was no significant change in Arg-1 expression(P>0.05).Conclusion1.NE deletion had no significant effect on constipation and depressive behaviors in PQ+Mb-treated mice.2.NE deficiency aggravated learning and memory dysfunction in PQ+Mb-treated mice.3.NE deficiency significantly exacerbated the damage of dopaminergic neuron as well as hippocampal and cortical neuron in PQ+Mb-treated mice.4.NE deficiency enhances microglial activation and Ml polarization in PQ+Mb-treated mice. |
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