| Objective The aims were triple. First, we investigated whether the early loss of axon and dendrites of substantia nigra dopaminergic neurons was specific for MPTP-induced Parkinson's diseas in C57BL mice model (including acute model, subacute model and chronic model), to explore the role of axonal degeneration in the degeneration of substantia nigra dopaminergic neurons as well as the association with the level of dopamine in striatum and conclude the mutual relationship of axonal degeneration and dopaminergic neuron degeneration. As a secondary aim, we investigated whether the phenomenon of spontaneous recovery of MPTP-induced PD mice model (including acute model, subacute model and chronic model) may be relevant to the regenerative sprouting of remained axon. To explore the role of axonal regenerative sprouting in nigrostriatal dopaminergic system of spontaneous recovery of MPTP-induced PD mice model. As well as axonal regenerative sprouting influence for the level of dopamine in striatum and conclude the major changes of morphology in the striatum and substantia nigra of midbrain of spontaneous recovery of MPTP-induced PD mice model. Third, we first raise reinjury of spontaneous recovery of MPTP-induced PD mice model (including acute model, subacute model and chronic model) and probe the role and mechanism of axonal degeneration in reinjury of spontaneous recovery of MPTP-induced PD mice model (including acute model, subacute model and chronic model). We hope provide a new evidence of theory for PD in the pathogenesy, recovery mechanism, prevention, therapeutic avenues and rehabilitation. Methods and results First experiment ( The degeneration of dopaminergic neuron and axon on MPTP-induced Parkinson's disease in C57BL mice) : The PD models were formed with intraperitoneal injections of MPTP in old C57BL mice, according to dosage of MPTP, intermission time and persistence time, fifty-five old mice random were divided into control group (saline group), acute group, subacute group and chronic group. Then according to severe degree of PD symptoms and results of behavioral test (for example, Pole test, Swim-test, Traction test and Elevated body swing test) to determine clinical stage of animal model (for example, preclinical phase, clinical early phase, clinical intermediate stage and clinical advanced stage. Three mice models were executed and perfused in every group at preclinical phase, clinical early phase, clinical intermediate stage and clinical advanced stage, respectively. One mouse model's substantia nigra of midbrain and striatum was treated by Tyrosine hydroxylase (TH)-immunocytochemistry and FJC dyeing, to survey the dopaminergic neuron, axon, striatal TH-ir fibers and axon terminal. Another one mouse model's substantia nigra of midbrain and striatum was treated by electron microscope, to survey the ultramicrostructure of the dopaminergic neuron, axon, striatal TH-ir fibers and axon terminal. Third model's DA, DOPAC and HVA contents in the striatum were measured by High performance liquid chromato-graphy and electrochemical detection. We observed the symptoms of PD mice models and recorded the time that symptoms occurrenced sooner and later, such as tremor, rigidity and bradykinesia, then comparison with the morphological changes of DAG neuron body, axon, dendrite, axon terminal and synapse, at last conclude which one first take place degeneration or necrosis. First experiment results: All group models showed a series of motor functional impairment resembling symptoms of PD and the symptoms become more and more serious. The number of TH-ir neurons gradual decreased (29.6% at preclinical phase, 44.5% at clinical early phase, 66.7% at clinical intermediate stage and 78.4% at clinical advanced stage, respectively) compared to age-matched saline-treated animals. As well as the level of dopamine in striatal also gradual fall-off (56.3% at preclinical phae, 71.5% at clinical early phase, 84.1% at clinical intermediate stage and 91.3% at clinical advanced stage, respectively) in comparison with age-matched saline-treated animals. there are significant difference (P< 0.01). The striatal TH-ir fiber optical density value gradually decreased significantly in comparison with age-matched saline-treated animals. Not only the number and striatal dopamine concentration of TH-ir neurons in SNc obviously decreased by a slow, progressive procedure, but also the number of axons and dendrites declines, and the descending degree of the latter are more severe than the former. The axon of remained DAG neurons was thin and short; crudeness or fineness was inequable and showed a string-of-beads or courser circuity, has an appearance of axoplasmic transport disturbance, even the axon has disappeared completely in some remained DAG neurons. Although left the cell bodies intact but caused a striking fragmentation and apparent degeneration of the dendrites. FJC-ir cell ( namely dopaminergic neurodegenerative neuron) only were discovered in SNc of MPTP-induced PD mice models, the number of FJC-ir cell in the acute model exceed the subacute model and the subacute model exceed chronic model. Although FJC-ir cell remained intact cell bodies, but the axon and dendrites were not immunolabeled or very severe damage, only fewer neurons exhibited relic axonal root. These results indicate that early loss of anox and dendrites is a characteristic morphological feature in the suite of events leading to dopaminergic neurons death. Results of transmission electron microscope: under the circumstances of shape of ultramicrostructure and cellular organ in substantia nigra DAG neurons were normal, such as the cellular nucleus was large and orbicular-ovate, the nuclear membrane was clear and integrity, the lipofuscin scattered in the cytoplasm, there were a lot of rough endoplasmic reticulum, free ribosome, Golgi's body and mitochondria, the axon of DAG neurons in striatum already have appeared an early appearances of degeneration, for example local intumescentia, the mitochondria was swell in intumescentia, the mitochondrial cristae was blurry and even disappearance completely, the vacuolization was found in mitochondria, the structure of neurofilament and neurotubule were disorder and discontinuation in local intumescentia, the microtubule disaggregation was observed in axon. It indicated that the axonal degeneration was earlier than cell body degeneration. Second experiment (The regeneration of dopaminergic neuron and axon on MPTP-induced Parkinson's disease in C57BL mice): Fifty-five young mice random were divided into control group (saline group), acute group, subacute group and chronic group.we observed the MPTP-induced PD mice model if have a phenomenon spontaneous recovery and again determined the clinical stage during the recovery period by behavioral test. Three mice models were executed and perfused at preclinical phase, clinical early phase, clinical intermediate stage and clinical advanced stage, respectively. Among three PD mice model's substantia nigra of midbrain and striatum was treated by Tyrosine hydroxylase (TH)-immunocytochemistry and FJC dyeing, to survey the degeneration, necrosis and regeneration of dopaminergic neuron, axon, striatal TH-ir fibers and axon terminal. DA, DOPAC and HVA contents in the stiatum were measured in other three PD mice models. the last three PD mice model's substantia nigra of midbrain and striatum was treated by electron microscope, to survey the ultramicrostructure of the dopaminergic neuron, axon, striatal TH-ir fibers and axon terminal, and in comparison with first part experiment, at last conclude a conclusion if MPTP-induced PD mice model have a phenomenon of spontaneous recovery, whether or not the striatal TH-ir fibers optical density value and DA contents have a gradually increased tendency with the improvement of PD animal model'symptoms. As well as the recovery of PD animal model with relation to regeneration of axon and the sprouting of dopaminergic axonal terminals. Second experiment results: All group MPTP-induced C57BL young mice have a substantial reduction of striatal dopamine level, the TH-ir fibers optical density value and number of TH-ir cell bodies were also markedly reduced after the toxin treatment. The symptoms of PD were observed following MPTP treatment, especially, acute model and chronic model showed more serious symptoms than subacute model. Thereafter, the symptoms of acute and subacute models showed a spontaneous recovery phenomenon within a few weeks after the toxin treatment. Especially acute PD model, the recovery rate almost gets 100%. However, the chronic PD mice were somewhat different from the acute and subacute PD mice, which showed a typical PD symptoms and become more and more serious, only a fewer models showed a slightly recovery. In the the recovery period, the dopamine content in striatum progressively increased significantly (by 10.7% at clinical advanced stage, by 35.1% at clinical intermediate stage, by 44.8% at clinical early phase and by 67.5% at preclinical phase, respectively) in comparison with age-matched saline-treated animals, from clinical advanced stage to preclinical phase onwards, although the number of TH-ir neurons remained fall off , from clinical advanced stage to preclinical phase, the number of TH-ir neurons declined by 76.3%, 78.4%, 82.4% and 85.2% in comparison with age-matched saline-treated animals, respectively. Tyrosine hydroxylase (TH)-immunocytochemistry was employed for the manifestation of the striatal TH-ir fibers and confocal microscopy was used to determine the optical density value of TH-ir fibers in the striatum. During the recovery period, the striatal TH-ir fiber optical density value of MPTP-induced PD model progressively increased from clinical advanced stage to preclinical phase. TH- immunohistochemical analysis showed that although the number of remained TH-ir neurons in stage of recovery was less than clinical advanced stage, but the cell body of TH-ir neurons is large and full, kytoplasm abundant and nucleolus in centre, the morphous of neurons have recovered normal fusiform shape or polygon. The anox and dendritic processes were frequently apparent and intensive and fasciculation in the remained dopaminergic neurons in substantia nigra. These results indicate that MPTP-treated mice have a potential for spontaneous regenerative sprouting in nigrostriatal dopaminergic system. There are a few FJC-ir cell, the FJC-ir cell bodies appeared to shrink, lose cytoplasmic material, and become detached from neighboring cells, the morphous became irregular and disorder, the volume of degenerating neurons became smaller and concentrationer. It is different from normal morphous of cell。Results of transmission electron microscope: The shape of ultramicrostructure and cellular organ in substantia nigra DAG neurons were normal, such as the cellular nucleus was large and orbicular-ovate, the nuclear membrane was clear and integrity, the reductus was disappearance, the lipofuscin was few in the cytoplasm, there were a lot of rough endoplasmic reticulum, free ribosome, Golgi's body and mitochondria.The number of synapsis increased obviously in the striatum, and the recovery of synaptic quantity preceded the recovery of cell body, The number of mitochondria increased obviously in synapse, it's shape and structure were integrity. The axonal swell and local intumescentia have already disappeared completely, the neural sheath, neurotubule and neurofilament lined up in order and intensive, the continuity was fine and natural, the neural sheath fragmentation and solution wasn't observed in the striatum. During the recovery period, the DAG neurons, ultramicrostructure of axon and the synaptic quantity in striatum were more and more advancement with the improvement of symptoms of PD models. Third experiment (The reinjury of the regeneration of dopaminergic neuron and axon on MPTP-induced Parkinson's disease in C57BL mice): Thirty-five young mice random were divided into control group (saline group), acute group, subacute group and chronic group. We careful observed the MPTP-induced PD mice model if have a phenomenon spontaneous recovery and again determined the clinical stage during the recovery period by behavioral test. Then chose MPTP-induced PD mice model which recovery from clinical advanced stage to preclinical phase and again raised from ten day to fifteen day after stabilization of symptoms. The reinjury PD model was raised by injecting MPTP with a dose 30 mg/kg again. The reinjury PD model were killed on second day. Some reinjury PD mice model's substantia nigra of midbrain and striatum was treated by Tyrosine hydroxylase (TH)-immunocytochemistry and FJC dyeing, to survey the degeneration, necrosis and regeneration of dopaminergic neuron, axon, striatal TH-ir fibers and axon terminal. DA, DOPAC and HVA contents in the stiatum were measured in other reinjury PD mice models, one reinjury PD mouse model's substantia nigra of midbrain and striatum was treated by electron microscope, to survey the ultramicrostructure and restorative regeneration of the dopaminergic neurons, axon, distal dendrites, striatal TH-ir fibers, axon terminals, cell organ and synapse and in comparison with first part and second experiment results. Third experiment results: fourteen PD mice models advanced to clinical advanced stage, among the total eight PD mice models showed a spontaneous recovery tendency, the degree of recovery from clinical intermediate stage to preclinical phase. When we again injected MPTP (30mg/kg), the acute toxic reaction was light than first injection, but the reinjury PD mice models fast appearanced a symptoms of clinical advanced stage after injecting MPTP ten hours. Some PD models gradually died. We have observed a robust depletion of striatal DA and its metabolite, DOPAC and HVA. the dopamine content in striatum depleted by 94.2%, DOPAC decreased by 85.1% and HVA declined by 76.8% in comparison with age-matched saline-treated animals, the striatal TH-ir fibers optical density value was extreme low in comparison with age-matched saline-treated animals, the number of TH-ir neurons was scanty in SNc, only about 6.5±0.6, The number of dopaminergic neurons in the substantia nigra were declined about 91.9%in comparison with age-matched saline-treated animals. The axons and dendrites of remained DAG neurons were not apparent, occasionally can find a short axonal root in residual processes. The TH-ir fibers were sparseness and courser disorder. FJC-dyeing found only a few FJC-ir cells in SNc, but the cell body of FJC-ir neurons is approximately normocellular figure, such as kytoplasm abundant, fusiform shape or polygon. It indicated that these cells died in a short time. Results of transmission electron microscope: the axon of DAG neurons in striatum appeared neural sheath disaggregation, the structure of neurofilament and neurotubule were severe disorder and discontinuation, the microtubule disaggregation was observed in axon, the quantity of neurofilament and neurotubule decreased obviously and majority were fragmentation, only a fewer neurofilament and neurotubule were continuous. The mitochondrion was swelling and disaggregation, the mitochondrial cristae was blurry and even disappearance completely. Not only the number of synapse decreased obviously but also the configuration of remained synapse was shrinkage. Conclusions First: the findings suggest that disruption and degeneration of the dendritic arbor and axon is an early indicator of neurodegeneration of dopaminergic neurons in substantia nigra on MPTP-induced PD in C57BL mice. Dying-back neuropathies are characterized by a sequence that progresses from loss of synaptic function to distal axonopathy and eventually to dopaminergic neuron death, dopaminergic neuron death commonly occurs in a sequence of morphological changes that begins with loss of TH-immunolabeled dendritic processes and axon. Second: There is a spontaneous recovery phenomenon in MPTP-induced PD mice model, especially acute PD model, the recovery of morphous and function in axon and axon terminal may precede the recovery of DAG neuron cell body and function. In other words, MPTP-treated mice have a potential for spontaneous regenerative sprouting in nigrostriatal dopaminergic system, the regenerative sprouting in axon and axon terminal are the premise and fundament for spontaneous recovery in MPTP-induced PD mice model. Third, it is very easy that the spontaneous recovery in MPTP-induced PD mice model to be injury by MPTP. The fast degeneration and necrosis of the regenerative sprouting in axon and axon terminal are first and major pathologic changes in reinjury of spontaneous recovery of MPTP-induced PD mice model. Third: The results that we have developed may prove valuable in investigating the mechanisms of degeneration and recovery of substantia nigra dopaminergic neurons; it is important enlightenment significance for PD in the strategy of prevention, therapeutic avenues and rehabilitation. |
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