Study On Neural Pathology And Oligodendrocyte Apoptosis In Heroin Spongiform Leucoencephalopathy Pathogenesis

Heroin spongiform leukoencephalopathy(HSLE)is a kind of central nervous system organic diseases with white matter spongiform vacuolar degeneration for the pathological features after snorting heroin.Cerebellar ataxia is the main symptoms. This disease was first reported by Wolters E in 1982 after he studied 47 patients in Amsterdam Netherlands.Within the following two decades,many individual cases were reported in some country.In 2000,Dr Lu Bing-Xun firstly diagnosed and reported 28 cases HSLE patients in Guangdong province of China.This was the first outbreak in Chinese mainland and patient had a history of inhaling heroin vapor with noses.Medical image showed that cerebral and cerebellar white matter had extensive and symmetric changes,which was spongiform vacuoles degeneration of white matte, myelin bases protein(MBP)was increase in the cerebrospinal fluid.Its incidence may be related to the central nervous system demyelination.But specific mechanisms unclear.Single intravenous injection of heroin were not found HSLE incidence. Substantial adulterant has been identified from the retail heroin powder.Such as diazepam,isoniazid,talc powder,etc.HSLE are probably caused by heroin adulterant, rather than the heroin itself.Research has shown that secondary oligodendrocyte apoptosis can be caused by radiation,hypoxia-ischemia.Bcl-2/bax change can direct cause neuronal apoptosis pathway.Cuprizone can cause demyelination of white matter,and significantly distribute the splenium of corpus callosum and post-fornix, but the genu of corpus callosum and rostral cerebellar pebuncle is no obvious demyelination.This study adopt imaging and neuropathological to analyze features of cerebral edema and demyelination of white matter in HSLE,used heroin adulterant and cuprizone to prepare rat model of white matter changes,and compared to HSLE, detect oligodendrocytes apoptosis and bcl-2/bax to study the HSLE possible originate from blood vessel and oligodendrocyte apoptosis pathogenesis.1.Study of characteristics of Neural Pathology in Heroin spongiform leucoencephalopathy imaging and neuropathological1.1 PurposesThrough the MRI examination,HE staining and MBP immunohistochemical method,to study and explore HSLE features of vacuolar degeneration and the demyelination.1.2 MethodsSix cases of patients HSLE(Nanfang Hospital,Southern Medical University). The incidences were after withdrawal.MRI was used to Collect T1WI,T2WI, enhancement,FLAIR,DWI and MRA image,comprehensive analysis localizion, qualitation and cerebral vessels change.4 cases of brain autopsies samples of HSLE and 5 controllers.The frontal lobes, cerebella and corpus callosum were drawn.Demyelination and vacuolation were detected by HE stained and MBP immunohistochemisty under the light microscope. Ultra-structrue were detected by electron microscope method.1.3 Results1.3.1 MRI of HSLE patients Cerebral cortex of HSLE patients become thinning,white matter expansion,no significant edema surrounding lesions,no significant mass effect.Lesions appear long T1WI,long T2WI.There are cerebellar white matter,splenium of corpus callosum, internal capsule posterior branch and occipital white matter lesions in 6 patients. FLAIR showed high signal lesions,like than T2WI slightly reduced,suggest myelination decrease.Enhanced no enhancement,refer to the blood-brain barrier without damage.MRA showed internal carotid artery,anterior cerebral artery,middle cerebral artery,posterior cerebral artery running normal,the arterial trunk slender, some rigid,branch red,and in direct proportion to the severity of clinical symptoms.1.3.2 Electron microscopHSLE Oligodendrocytes show multiple vacuolar degenerate.Swollen mitochondria and endoplasmic reticulum can be seen in their reliquus cytoplasm.1.3.3 HE stainingThe encephalon white matter of HSLE presented obvious rarefaction and unequal size vacuole changes.Vacuoles run along the axon.The vacuole changed more from superficial layer to deep layer.The structures were heterocentric.Lower white matter of cerebellar granule show larger vacuoles,to the deeper white matter vacuoles gradually small and intensive.Perivascular Vacuolating were more obvious. There is a small amount of vacuolated changes surrounding the small blood vessels and the capillary.1.3.4 MBP immunohistochemistyCompare with the control group,cerebral White matter,cerebellar white matter and corpus callosum showing stained slight and uneven,vacuolated areas no staining, There were growing heavier myelin fiber collapse and fracture from superficial layer of white matter to deep layer.HSLE cerebral cortex can be seen a small amount of filament-like fibers,dyeing uniformity,compared with the control group no significant difference.1.4 Conclusions(1)Myelination were obvious decreased in HSLE white matter vacuolar area;(2)HSLE white matter exist Extensive demyelination and vacuolar degeneration;(3)Secondary vascular changes increase the vacuolar degeneration.We conjecture cerebral microvascular changes related to vacuole formation.2 Research of bcl-2/bax gene expression and oligodendrocyte apoptosis in the heroin spongiform leukoencephalopathy pathogenesis2.1 PurposesDetect cells apoptosis and its regulatory factors in HSLE brain tissue,research the role of oligodendrocyte apoptosis in HSLE pathogenesis.2.2 Methods4 cases of brain autopsies samples of HSLE and 5 controllers.The frontal lobes, cerebella and corpus callosum were drawn.Caspase-3 and bcl-2/bax were stained with immunohistochemical method.TUNEL cells apoptosis were detected under the light microscope.Quantitative analysis was conducted by micro-image analysis instrument.Statistical analysis use SPSS 13.0 software package.Data of the positive cells write as(x~-±s).Comparison of different brain regions in HSLE and control group used repeated measure analysis of variance,multiple comparisons using LSD method,the same brain area comparison between the two groups using independent sample t test,TUNEL apoptotic cell counts and expression of caspase-3 used the Pearson correlation test.2.3 Results2.3.1 TUNEL positive countsThere were oligodendrocyte apoptosis in white matter HSLE and apoptotic bodies obviously in vacuolar area,which appeared coloration and partial condense of caryon.Count was significantly higher than control group,compared with the control group the difference was statistical significance(F=327.913,P=0.000);site to compare statistically significant differences(F=68.100,P=0.000),multiple comparisons between sites,cerebellar white matter and corpus callosum was no significant difference(P=0.310),cerebral gray matter and cerebellar white matter,the corpus callosum have statistically significant difference(P=0.018,P=0.017). Different brain regions between two groups there were significant interaction effect (F=83.715,P=0.000).2.3.2 Caspase-3 immunohistochemisty positive expressionThere were leonine Caspase-3 protein positive oligodendrocyes in the focal zone of HSLE,Counts was significantly higher than control group,compared with the control group the difference was statistical significance(F=387.955,P=0.000); Comparison at sites statistically significant differences(F=215.727,P=0.000), multiple comparisons between sites,cerebellar white matter and corpus callosum was no significant difference(P=0.096),cerebral gray matter and cerebellar white matter, the corpus callosum have statistically significant difference(P=0.020,P=0.022). There were direct correlation between TUNEL-positive count and caspase-3 positive counts of HSLE(pearson correlation coefficient r=0.924,P=0.000).Myelins were mainly formed by oligodendrocyte.This indicate HSLE exist extensive white matter oligodendrocyte apoptosis.2.3.3 Bcl-2 gene expressionThe difference was not significant between HSLE and the control group (F=0.001,P=0.978),brain gray matter,white matter and corpus callosum to compare the difference was not significant(F=0.003,P=0.999).2.3.4 Bax gene expression The differences of HSLE comparison with the control group were statistical significant(F=11.098,P=0.013).Inter-site difference was significant statistical significance(F=5.632,P=0.016).Multiple sites comparison,cerebellar white matter and corpus callosum was no significant difference(P=0.952),cerebral gray matter and cerebellar white matter(P=0.032)and corpus callosum(P=0.035)difference was significant statistical significance.Bcl-2 is promote the survival family.Bax is the pro-apoptotic family.Bax high expression increase the mitochondrial cytochrome C release,resulting in Caspases-9 and Caspases-3 activation and induced cells apoptosis. Bcl-2 and Bax ratio may play a key role in determining the cell survival or death.2.4 Conclusions(1)A large number of apoptotic cells in white matter vacuolation areas,speculated oligodendrocyte apoptosis are probably one of HSLE pathogenesis;(2)Bcl-2/bax expression change are one of oligodendrocyte apoptosis path;(3)These were related to Oligodendrocyte poisoning and mitochondrial dysfunction.3 Characteristics of vacuolar degeneration and oligodendrocyte apoptosis in alba of SD rats with cuprizone induced3.1 PurposesThrough explore and the establish alba vacuolar degeneration model,further study its etiology and pathogenesis,for etiology and pathogenesis of HSLE study provides new research content and ideas.3.2 Methods72 Sprague-Dawley(SD)rats.Rats were randomly divided into four groups: adulterant smoking group(AS),adulterant feeding group(AF),cuprizone feeding group(Cu),and control saline feeding group(con).Each group set 3 time point:2,4 and 6 weeks.We adopt heroin adulterant(INH,diazepam,barbitone and talcum powder)and cuprizone induce SD rats leukoencephalopathy model.Adulterant by ratio:diazepam:isoniazid:barbitone:talcum powder=2.5:30:100:200 mix grinding into powder.Adulterant mixed powder exposed to the volume of each rat by weight 25mg/100g respectively.1%cuprizone were fed rats(10mg/100g weight).While the control group was fed with equal saline.Once a day.The rats' brains were detected with HE staining and MBP immunohistochemisty staining.Cells apoptosis was counted by TUNEL and caspase-3 immunohistochemisty staining under light microscope,ultra-structure was observed by electron microcope.Statistical method:Apply SPSS 13.0 software package.The positive counts data of TUNEL and caspase-3 of SD rat model as(x~-±s).Comparision between the same brain regions in each group and of different time points used factorial variance analysis respectively.3.3 Results3.3.1 The whole brain samples of SD rats72 cases of SD rat brain were not seen congestive and swelling.Rats of Cuprizone 6 weeks has mild adhesions between the meninges and the skull.Cerebral, cerebellar gray whiter matter structure clear,no expansion of white matter.3.3.2 HE stainingBrain gray matter and white matter no vacuolation in AS group,AF groups,Cu 2w,4w group and Con group.Rats of Cu 6w group show the white matter vacuolization,vacuoles were bar shape and running along the fibers.The brain stem and occipital lobe white matter vacuolation were the most obvious,no significant changes in blood vessels,no obvious inflammatory cell infiltration,no necrosis,no obvious glial cell hyperplasia,cerebral gray matter nerve cells were not loss and degeneration. 3.3.3 Electron microscopCu 4w group of rats show more disordered arrangement of myelin,but no typical demyelination.Cu 6w group rats corpus callosum can be seen myelin swelling and myelin lamellar Vacuolating.3.3.4 MBP immunohistochemicalWhite matter was sparse in Cu 6w group rats.Myelin lamellar split, vacuolization without coloring.The brain stem and top-occipital white matter most obvious.3.3.5 TUNEL positive cellsMore positive apoptotic nuclei were seen in white matter of Cu 4w group,deeply stained unevenly.Large number of apoptotic cells were seen in Cu 6w group,which main located in vacuole area of the ventricle white matter,cerebellar white matter, corpus callosum and brainstem.TUNEL counts in each group the difference was significant statistical significance(F=309.934,P=0.000).The difference between different times was significant statistical significance(F=163.839,P=0.000),Cu 6w group and the 2w,4w group were significantly statistical significance(P=0.000),4w group compared with the 2w group had significant statistical significance(P=0.000), with Cu 6w group were the highest,followed by Cu 4w group.prompted Cu 4w groups have emerged apoptosis in white matter,extended over time,to 6w the apoptosis increase.3.3.6 Caspase-3 positive expressionThis result and TUNEL were accordant.Rats brain gray matter in each group there is a small amount of caspase-3 positive expression,the difference was not significant(F=0.485,P=0.694).Cu 4w group revealed higher apoptosis positive granules.large number of apoptotic cells were count in Cu 6w group.The difference of Caspase-3 expression in each group was significant statistical significance (F=84.018,P=0.000).The differences between different time have significant statistical significance(F=23.611,P=0.000),Cu 6w group and 2w group,4w group were significantly statistical significance(P=0.000),4w group compared with 2w group have significant statistical significance(P=0.003),while Cu 6w group were the highest,followed by Cu 4w group.Prompt Cu 4w group white matter appeared apoptotic signal,apoptosis increaseas by 6w.Cells apoptosis is consistent with myelin lesion morphology of HE staining and MBP staining,and it is earlier than morphology change.Description apoptosis cause white matter vacuolar degeneration. Cell apoptosis may be the originating causes of vacuolar degeneration in white matter.4 Conclusions(1)Smoking or feeding rats can not induce SD rats alba white matter changes by diazepam,isoniazid,barbitone and talcum powder adulterant;(2)Cuprizone oral perfusion may cause SD rats alba vacuolar degeneration;(3)There were a large number of apoptosis in alba lesions of SD rats by cuprizone induced.Suggesting that apoptosis was related to alba demyelination and vacuolar degeneration;(4)Damage target of cuprizone were in the central nervous system oligodendrocytes. The oligodendrocyte apoptosis was earlier than alba demyelination at the happened time.Guess apoptosis cause demyelination and vacuolar degeneration in white matter of SD rats by cuprizone induced;(5)Vacuolation was induced by cuprizone in SD rats.Vacuolating time variability were dose-related,with the time prolonged,the total dose increase,demyelination aggravate;(6)Cuprizone was used to induced white matter degeneration,no significant brain edema,no obvious vascular relevance; (7)The cuprizone-induced white matter degeneration were different with HSLE incidence reasons,Neuropathological changes were different,but there are cell apoptosis.Summary of The full text1,Myelination were obvious decreased in HSLE white matter vacuolar area;2,HSLE white matter exist Extensive demyelination and vacuolar degeneration;3,Secondary vascular changes increase the vacuolar degeneration.We conjecture cerebral microvascular changes related to vacuole formation.4,A large number of apoptotic cells in white matter vacuolation areas,speculated oligodendrocyte apoptosis are probably one of HSLE pathogenesis;5,Bcl-2/bax expression change are one of oligodendrocyte apoptosis path;6,These were related to Oligodendrocyte poisoning and mitochondrial dysfunction;7,Smoking or feeding rats can not induce SD rats alba white matter changes by diazepam,isoniazid,barbitone and talcum powder adulterant;8,Cuprizone oral perfusion may cause SD rats alba vacuolar degeneration;9,There were a large number of apoptosis in alba lesions of SD rats by cuprizone induced.Suggesting that apoptosis was related to alba demyelination and vacuolar degeneration;10,Damage target of cuprizone were in the central nervous system oligodendrocytes.The oligodendrocyte apoptosis was earlier than alba demyelination at the happened time.Guess apoptosis cause demyelination and vacuolar degeneration in white matter of SD rats with cuprizone induced;11,Vacuolation were induced by cuprizone in SD rats.Vacuolating time variability were dose-related,with the time prolonged,the total dose increase, demyelination aggravate;12,Cuprizone were used to induced white matter degeneration,slight cells swelling,no obvious vascular relevance;13,The cuprizone-induced white matter degeneration were different with HSLE incidence reasons,Neuropathological changes were different,but there are cell apoptosis.