Study On Apoptosis Of Oligodendrocyte And Etiology In Heroin Spongiform Leucoencephalopathy

Background and PurposeHeroin Spongiform Leucoencephalopathy (HSLE) is a kind of leucodystrophy related with snorting of heroin. This disease was first reported by Wolters E in 1982 after he studied 47 patents in Amsterdam, Netherlands, which had following features:1, falling ill after snorting of heroin by nose; 2, The clinical symptoms began with apathy, bradyphrenia, cerebellar dysarthria and ataxia; 3, Medical image showed that cerebral and cerebellar white matter had extensive and symmetric changes; 4, The characteristic change of pathology was spongiform vacuoles degeneration of white matte in the cerebrum and cerebellum. Within the following two decades, many individual cases were reported in Germany, Italy, Spain, Switzerland, Norway, Belgium, American and Taiwan. 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 the majority of cases had a history of inhaling heroin vapor with noses, whose clinical manifestations were characterized with signs of cerebellar and pyramidal tract lesions. Their CT, MRI and pathological findings were quite similar with those patients reported in Netherlands, Dr Lu Bing-Xun considered it was the second outbreak throughout the world. The onset of this disease was related with snorting of heroin and the pathology of alba showed spongiform change(spongiform vacuole degeneration), consequently, it was designated as Heroin Spongiform Leucoencephalopathy (HSLE). HSLE was different from the known cerebropathy related with heroin and other metabolic cerebropathy with spongiform changes as well. Currently, the etiology and pathogenesis of HSLE were still unclear, the neuropathological documents were quite limit and various reported findings weren’t coincidence with each other completely. Therefore, we try to further find the pathology of the disease based on the investigation we had studied before and to reveal the pathogenesy of HSLE by surveying the correlative animal experiments and clinical researches.For the purpose of understanding the pathogenesy of HSLE, we conducted the following experiments respectively: fumigate Sprague Dawley rats with heroin and heroin dopant extractive and induce the spongiform vacuolar degeneration in Sprague Dawley rats with dicyclohexanoneoxalyldihydrazone(cuprizone). And we had discovered the apoptosis in lesions as an original study clue. Base on these researches, we explored the apoptosis of oligodendrocyte and its mechanisms, and analyzed the conscious disturbance of HSLE patients and clinical features of acute heroin-toxic encephalopathy to illuminate the pathogenesy of HSLE.Materials and Methods60 Sprague-Dawley (SD) rats(provided by experimental animal center of Southern medical university) were divided into two groups. Group 1: 30 rats (weighted 200-220g, male and female were equal) were divided into experiment group, heroin group and control group randomly. They were fumigated with INH, diazepam, barbitone and talcum powder which were detected from heroin dopant and heroin once a day, 20 minutes every time for two weeks, respectively. The rats’ brains were dissected and stained with H. E staining, then the alba’s changes were observed. Group 2: 30 SD rats(age 4-6 weeks, weight 180-200g, equal male and female) were divided into three groups randomly, the study group were infused by intragastric administration with 2% cuprizone (10mg/100g weight/24h) once a day for 4 and 6 weeks, while the control group were infused with normal sodium for 6 weeks. All samples of the brains were stained with H. E staining, caspase-3 RT-PCR and Hoechst 33258 cells were observed under electron microscope.4 cases of brain biopsies samples of HSLE aged 26-38 years were enrolled from March in 2000 to April in 2004 in Nangfang hospital, 2 controllers aged 54 and 66 years. Microtubule protein of Tau1, Tau3 and MAP2 were studied by immunohistochemical method.4 cases of brain biopsies samples of HSLE aged 26-38 years were enrolled from March in 2000 to April in 2004 in Nangfang hospital, 5 controllers aged 54-80years. The frontal lobes, cerebella and corpus callosums were drawn and stained with H. E and K. B staining. GFAP, MBP, COX-2, CNPase and caspase-3 were stained with immunohistochemical method and immunofluoresce. Apoptosis of TUNEL cells were detected under the electron microscope, and quantitative analysis was conducted by micro-image analysis instrument.39 cases of HSLE aged 23-45 years hospitalized from Mar 2000 to Oct 2006 in Nangfang hospital were carried out clinical analysis, the patients with conscious disturbance were compared with the patients without conscious disturbance and 6 patients with Acute Heroin Toxicity encephalopathy.ResultsHistological examination showed that the structures of brain grey and white matter in the experiment of fumigateing SD rats and heroin rats had no obvious changes. The whiter matter had yet no vaculous change in H. E staining.The results of experiment in SD rats infused with cuprizone were the following: there was slightly conglutination between partial meniges and skull. The color of the brains’ surfaces was gray and the boundary between grey and white matter was clear. The structure of grey and white matter in cerebellar hemisphere was clear. There was no expansion in the alba.The results under electron microscope showed there were vacuolization in myeline sheath layer, and medullary sheathes of multiple sites were split by the turbine vacuolus.Under H. E staining, 7 cases out of 10 samples showed vacuole changes in the brain stem and 4 cases in the deep layers of cerebral white matter. There were no vacuole changes in cerebellar white matter.Hoechst33258 apoptosis staining found that 7 and 5 cases of experiment 4 weeks and 7 and 6 cases of experiment 6 weeks animals showed positive apoptosis cells under caryon-fluorescence staining in corpus callosum and deep layers of cerebral white matter respectively. There were no DNA positive changes in control group.IHC of microtubule-associated protein showed there were no positive express of Tau1, Tau3 and MAP2 in HSLE.The examination results of 4 cases HSLE brain autopsies showed as the following:Under H. E staining, the cerebellar whiter matter of HSLE presented obvious vacuole changes of unequal size. Compared with control group, the structures in every layer of cerebellar whiter matter of HSLE had been destructed, and the vacuole changed more from superficial layer to deep layer. The boundary between grey and white matter in frontal lobe of HSLE took on obvious vacuolization and heterocentric structures, but U-fibers were relative integrity. While the boundary between grey and white matter in frontal lobe of controllers was clear.MBP immunohistochemical study indicated that there existed significant difference in gray scale value of deep layer in white matter between HSLE and controllers (t=3.93, P=0.011). There were statistically significant differences of gray scale value among deep layer and superficial layer of white matter and gray matter(t=4.587, P=0.006; t=5.412, P=0.003).GFAP immunnhistochemical study showed there were two significant features of astrocytes in HSLE focal zone, they were: 1, astrocytes cumulated clustering near the margin of focal zone, while they distributed well in control group. 2, astrocytes correlatively decreased in the center of focal zone compared with the controllers, but their cell body increased obviously and there were thick-dyed granulations surrounding them. There were statistically significant differences between deep layer and superficial layer of white matter of HSLE (t=23.43, P=0.000). Deep and superficial layers in white matter of HSLE all had statistically significant differences from those of control group (t=22.88, P=0.000; t=10.16, P=0.000 respectively). The results manifested that Astrocytes in focal zone changed hypertrophy but no hyperplasy.Caspase-3 immunohistochemical staining showed that there were leonine Caspase-3 protein-positive oligodendrocyes in the focal zone of HSLE, which had statistically significant differences with controllers(t=10.38, P=0.000). The distributions of caspase-3 protein-positive oligodendrocyes were more predominance in superficial layer of white matter at the site of minimal-changed myelin sheath than in deep layer.TUNEL apoptosis detection showed there were oligodendrocyte apoptosis in white matter HSLE and apoptotic bodies in the deep layer, which appeared coloration and partial condense of caryon of caryon. There were statistically significant different apoptosis express of oligodendrocytes in cerebellum, corpus callosum and white matter of frontal lobe between HSLE and control group (Mean±SD were 8.08± 4.42; 12.83±5.20; 12.92±4.80, respectively, P=0.000).Immunofluorescence staining showed there appeared yellow double staining positive markers using the COX-2＋Caspase and CNPase+Caspase. The results indicated that apoptosis and changes of COX-2 were generated in oligodendrocytes,COX-2 immunnhistochemical staining found there were yellowish-brown or sorrel COX-2 positive express in the kytoplasm. The numbers of COX-2 positive cells in white matter of cerebellum, frontal lobe and corpus callosum in HSLE (Mean±SD were 12.83±3.81, 15.33±4.81 and 3.08±5.28 respectively) were significant higher than those in control group(Mean±SD were 1.42±1.16, 0.83±0.94, 0.58±0.67 respectively).CNPase immunohistochemical staining manifested that kytoplasm were yellowish-brown or dark brown. The numbers of CNPase positive cells in white matter of cerebellum, frontal lobe and corpus callosum in HSLE (Mean±SD were 12.50±5.39; 13.08±4.14; 12.75±4.03, respectively) were lower than those in control group(Mean±SD were 16.75±5.88; 17.42±4.08 and 17.67±3.77, respectively. There were difference between them (t=2.219, P=0.048; t=2.229, P=0.048 and t=3.335, P=0.007).The correlation analysis between the express of COX-2/CNPase and caspase-3 showed as following: CNPase and COX-2 had opposite manifestation, COX-2 and CNPase showed up-regulation and down-regulation respectively in focal zone of HSLE. Caspase-3 was positive and negative correlation with COX-2(r=0.848, P=0.004) and CNPase(r=-0.849, P=0.004) respectively. COX-2 was negative correlation with CNPase(r=-0.688, P=0.040).Clinical analysis between conscious disturbance group (CDG) and non-conscious disturbance group (NCDG) showed that routine examination and C. S. F test were no difference between CDG and NCDG, except plasma protein decreased in CDG. All patients with CDG appeared moderate or severe abnormal electroencephalogram, especially, 3 patients who had clear consciousness on admission but turned light coma appeared very severe abnormal electroencephalogram. Only 10% patients with NCDG appeared mild electroencephalogram abnormality. The changes of imageology showed the lesion extent in NCDG was obviously smaller than that in CDG (Mean=70.09, Std Deviation=39.65 and Mean=41.79, Std Deviation=11.40, respectively), there was statistically significant differences between them(t=3.427, P=0.005). Cerebral perfusion imaging of SPECT showed radioactivity rarefaction in white matter mainly. NCDG mainly displayed symmetric hypoperfusion imaging in occipital lobe of cerebral posterior longitudinal division. While CDG revealed expansion and radioactivity rarefaction or defect in white matter of cerebrum and cerebellum, and low blood supplement focuses in gray matter.Clinical analysis between conscious disturbance group (CDG) of HSLE and acute heroin poisoning encephalopathy (AHPE) disclosed that AHPE had a higher probability of complicating infection such as hepatitis B, C and HIV was more in AHPE than that in CDG. There were no difference in biochemistry examination between CDG and AHPE. AHPE had more abnormal appearance than CDG of HSLE in the examination of blood gas analysis. Electroencephalogram showed both groups had moderate or severe diffuse slow wave changes. In MRI imaging, HSLE patients had extensive and symmetric increased T1 weighted (T1W) signal and increased T2 weighted (T2W) signal in sub-cortical white matter of bilateral cerebral hemisphere and cerebellum, as well as corpus callosum, brain stem, posterior limb of internal capsule, centrum ovale and corona radiata. MRI imaging of AHPE showed diffuse cerebral swelling or nucleus damage and focal albocinereous damage. AHPE had a lower fatality than CDG, 6 out of 9 HSLE patients died while none of AHPE died. Conclusions1, Perfusion method using bis(cyclohexanone)oxalydihydrazone in SD rats can be made animal models with vacuolou change in white matter and this change was closely related with the cell apoptosis in white matter. The heroin impurity-isolated substances including INH, diazapam, barbital and talcum powder fumigating SD rats for two weeks can not induced animal model with vacuolou change in white matter.2, HSLE was not degenerative disease of nervous system.3, HSLE had extensive spongiform degeneration in white matter, and there were a large number of apoptotic oligodendrocytes in lesions, especially, in the rim of the lesion. CNPase down -regulation and COX-2 up-regulation caused oligodendrocyte apoptosis. The inhibitor of COX-2 such as indomethacin can treat HSLE patients.4, Abnormality of electroencephalogram was the important character of HSLE patients with conscious disturbance. The range of lesion was closely relative with the occurrence of conscious disturbance in HSLE patients. The focal low perfusion especially in gray matter might cause convulsion, and therefore induce conscious disturbance. MRI imaging in HSLE patients with conscious disturbance displayed extensive and symmetric increased T1 weighted (T1W) signal and increased T2 weighted (T2W) signal in sub-cortical white matter of cerebral hemisphere and cerebellum, as well as corpus callosum, brain stem, posterior limb of internal capsule, centrum ovale and corona radiata. AHPE patients showed diffuse cerebral swelling or nucleus damage and focal albocinereous damage in MRI. HSLE with conscious disturbance had higher fatality than AHPE.