| IntroductionParkinson' s disease ( PD) is a common f severe and progressive neurode-generative disease of the central nervous system, attacking the middle-aged and older people and characterized by resting tremor,cogwheel rigidity, bradykinesia , and loss of postural reflexes. The etiology of PD is still enigmatic. It is commonly regarded that many factors such as free redicals, toxicity of excitatory a-mino acids, dysfunction of mitochondria etc. cause selective loss of domaminer-gic neurons of the substantia nigra pars compacta (SNc) in the midbrain resulting in dificiency of the neurotransmitter dopamine at the nerve terminals of the nigrostriatal dopaminergic neurons in the striatum. And apoptosis is thought to be the main death form of these neurons.Flunarizine (Fz) a difluorinated piperazine derivative, is regarded as a selective calcium channel blocker preventing cell damage caused by calcium accumulation in neurons after ischemia and hypoxia. This compound improves blood flow by vasodilation or inhibition of vasoconstriction. For this reason, Fz has widely been used for therapeutic approach in cerebral blood flow disturbances and diseases of the central nervous system. It has also been reported that it caused Parkinsonian symptoms and transient loss of tyrosine hydroxylase (TH) immunoreactivity in dopaminergic neurons in SNc. What on earth is the effect of Fz on the neurons of SNc? To what degree does Fz affect the neurons? Does Fz induce cell apoptosis in these neurons? We tried to answer these questions by morphological methods in our experiment.The SN damage characterized by neuron loss occurs in pathlogical change of all PD patients.- Neuronal loss in the SN mainly occurs at SNc where the ventral lateral part is most seriously involved, ventral medial part less, and the dorsal part least. The number change in SNc was considered to study the effect of Fz on neurons in the SN of SD rats.TH is a nonheme ferritin distributing in the catecholaminergic neurons in the central nervous system. In the brain TH catalyses the reaction of hydroxyl-ation of tyrosine into L-Dopa which is the first step in biosynthesis of cate-cholamine (CA) in the body. Comparing with other enzymes catalyzing the synthesis of CA TH is least in content, slowest in synthetic rate, weakest in catalyzing activity and its substrates are most specific. TH is hence thought to be the rate-limiting enzyme in synthesis of CAs including DA. At the same time TH is also the marker protein of dopaminergic cells in the brain. TH immunocytochem-istry was utilized in our experiment to detect changes of TH immunoreactivity in neurons in SNc of SD rats after Fz administration.MATERIALS1. Experimental animals48 healthy Sprague-Dawley (SD) rats weighting about 200g,the gender not considered were supported by the Laboratory Animal Center of the China Medical University.2. Main AgentsTH immunohistohemical kitFlunarizine hydrochlorideTartaric acidThioninIn situ cell apoptosis detection kit (POD)3. Main instrumentsParaffin microtome, LEITZ, CemanyVertex Mixer ,XW-80-C , ShanghaiDigital Microcammera,OLYMPUS/C-5050 JapanImage Analyzing Instrument, Meta Morph/BX41 Japan - Microscope, OLYMPUS JapanMETHOD1. Animal grouping48 healthy adult SD rats, weighting about 200g and without considering the gender were randomly divided into A, B, C experimental , and D control groups, 12 in each. Rats in experimental groups were injected with Fz intraperi-neally at the doses of 10mg/kg b. wt. , 100 mg/kg b. wt. and 200 mg/kg b. wt. respectively. The control animals were given 1.5% tartaric acid of same volume as in the experimental groups.2. Specimen praparationAt various survival periods (1 day, 1 week, or 1 month) after Fz injections, the animals (subdivided into Al ~ A3, Bl ~ B3, Cl ~ C3 and Dl ~ D3 subgroups) were transcardially perfused with 4% paraformaldehyde in 0. 1M phosphate buffer (pH 7.4). The rats'brains were stripped and postfixed for 18 hours. Routine dehydration, clearing, paraffin embedding were practiced and 7jxm-thick sections were cut for Nissl staining , TH immuncytochemistry and cell apoptosis detection.3. Nissl stainingSections for Nissl staining were drawn from the serial sections at 2-section intervals with 1 section for TH immunocytochemistry the other for cell apoptosis detection. The sections were routinely dewaxed to hydrated , stained in the the Nissl staining solution at 37°C for 1 hour, differentiated in 80% alcohol, dehydrated in 100% alcohol I, II , cleared in xylol I, II respectively, mounted with neutral gum, observed under light microscope.4. TH immunocytochemistrySections of the SN and striatum were routinely dewaxed to hydrated, treated with 3%H2O2, treated with complex digestion fluid, 5% BSA fluid,first antibody, biotinated goat-anti-mouse IgG, SABC, colorated with DAB, slightly counter stained in hematoxylin, dehydrated, cleared, and mounted for observa-tion under microscope.5. Cell apoptosis detectionSections of the SN and striatum were routinely dewaxed to hydrated, treated with 3% H2O2, digested with 0. 01M TBS freshly diluted proteinase K ( 1: 200) , added labeled buffer, blocking fluid, anti-DIG-biotin(1: 100 diluted) , SABC also 1' 100 diluted by the antibody dilution, colorated with DAB, slightly counter stained with hematoxylin , dehydrated, cleared, mounted and observed under microscope.6. Quantitative analysis of product of immunoreactionImages of the sections were collected with Meta Morph/BX41 (Olympus, Japan). 10 sections were randomly chosen. In each section cells of 5 view fields of SNc were detected at the magnification of 200 times under microscope. Totally 50 view fields were detected in each group. Relative content of product of TH immunoreaction of the neurons in SNc in both experimental and control groups were analyzed with Meta Morph/BX41 image analyzing software ( Olympus, Japan) and shown as average optic density and integrated optic density.7. Statistical treatmentAll data were analyzed with SAS statistical analysis software and shown in the form of mean ± standard deviations (x ± s). T test was used for comparation between two means.RESULTS1. Effect of Fz on the behavior of SD ratsRats in the control group showed normal activities, with nimble movements , and quick reactions to the environment.Rats in the experimental groups injected with Fz showed Parkinsoninian symptoms obviously including significantly-decreased activities, rigidity, slowness, postural instability on the first day. Ingestion and drinking also decreased drastically. The rats became insensitive to the outside environment. From day 2 to day 7 the rats in experimental groups showed more movements. Those in group A showed more movements than in group B and C with rats of the leastmovement in group C showing more extrapyramidal symptoms. After the 8th day, rats in group A3 had more and more movements and rats in group B3 and C3 also showed recovery of different degrees.2. Nissl stainingIt was shown at the lower magnification that SNc was triangular in shape. The ventral part is broader and and the dorsal part is narrower. Abundant neurons were densely distributed in it. Under high magnification many large pyramidal , spindal, polygonal , bipolar neurons could be observed in SNc. Their cytoplasm was purple-red in color with a vacuolated nucleus with a clear nucleolus in the center. No significant difference was found between the numbers of neurons in this area betwwen experimental and control groups (p >0.05).3. TH immunocytochemistryMany large pyramidal, polygonal, spindal, TH positive cells were distributed in SNc in the control group. TH positive cells scattered in the ventral teg-mental area. Yellow or yellow-brown granules were in the cytoplasm of these TH positive cells. Their nuclei were spherical and vacuolar in TH staining and blue in color with a clear nucleolus in after counter stain with hematoxylin.After Fz administration, regular changes of TH immunoreactivity of SNc neurons in the experimental groups were observed; better TH immunoreactivity of SNc neurons in rats with less Fz injected of same survival period or in rats with longer survival period of same dose of Fz administrated. Significant differences were found between the goups.TH immunoreactivity of neurons in the ventral tegmental area were not affected.4. Cell apoptosis detection of SNcTUNEL was used to detect cell apoptosis of the neurons in SNc in our experiment. No apoptotic neuron was found in SNc in all sections. But some apop-totic cells were found at other parts of the brain sections. These cells had a yellow nucleus or had yellow brown granules in their nucleus.5. Quantitative analysis of product of TH immunoreaction of SNc neurons After administration of Fz, the values of average optic density and integrated optic density of the neurons in SNc of the rats with a longer survival periodwere larger than those with a shorter survival period in the experimental group of the same Fz dose. With the same survival period, larger average optic density, and integrated optic density were detected in the sections of rats injected with low-dosed Fz, and vice versa.DISCUSSION1. Effect of Fz on dopaminergic neurons in SNc of SD ratsFz is a selective calcium channel blocker preventing cell damage caused by calcium overload in neurons. It was reported that Fz caused Parkinsonian signs. In our experiment, extrapyramidal symptoms occurred in all rats injected with Fz. This was consistent with the clinical reports. Regular changes of TH immunoreactivity of the neurons in SNc of the rats at different survival periods were observed: In group A, the rats received the least dose of Fz and the quick recovery of the Parkinsonian symptoms and TH immunoreactivity of the neurons in SNc were observed. Contrary phenomena were found group C. Nissl staining showed no decrease in cell number in SNc in the experimental group than those in the control. Fz induced Parkinsonian symptoms but did not cause cell death or cell loss in these neurons. After drug withdrawl TH immunoreactivity of these neurons could recover with the time passing and the recovery was dependent on time and dose of the drug used i. e. the less the dose is the better the recovery is and the longer the time is the better the recovery, and vice versa. This is also consistent with the early reports in which patients used this drug showed Parkinsonian symptoms improved after drug withdrawl.2. Fz and cell apoptosisEvidences support the effect of cell apoptosis in PD, including evidence from cell culture model, from animal model, from PD patient study, etc. All these studies depended upon morphological change of apoptic cells were detected by existence of morphological markers. In our experiment, TUNEL method was utilized to detect the apoptotic cells in SNc after Fz administration. But no typical apoptotic cells were observed in this area. Nissl staining showed that no significant difference in cell number between the experimental and control groups. |
PDF Full Text Request