| The serious EV71 encephalitis is a severe disease caused by entero virus 71 type that evoked a series of central nervous system impairment。EV71 possesses a strong neurotropism。The brainstem(pons and medulla oblongata) and spinal cord are both frequently involved, especially the brainstem, is the most infected region。EV71-infected disease is characteristic by fever, skin rashes in hand, foot and mouth, and neural abnormalities。About a third of infected children showed CNS involement and developed the fatal disease with rapid deterioration and high mortality and mutilation rate。According to a report by Ho M, the incidence rate of EV71 infection range from 0.055‰to 0.093‰(the mean is 0.078‰), the case fatality rate range from 7.7‰to 31.0%(the mean is17.8%)。The case fatality rate was significantly higher than any other age groups in infants and young children, who were more prone to develop the severe cases resulted from rapid clinical deterioration after EV71 infection。Children aged from 6 months to 12 months old accounted for 43.0% of all deaths, babies less than 6 months old accounted for 16.0%。The survivors who recovered from EV71 CNS infection often remained severe neurological sequelae。The report documented the number of total HFMD cases reached 1.5 million in the EV71 epidemic occurred in Taiwan in 1998, and the case fatality rate of neurogenic pulmonary edema resulted from impaired brainstem function due to EV71 infection was up to 64.3%。Unfortunately, there were 17.0 to 30.2 percent of children with CNS involvement who had no skin rashes or showed an atypical clinical manifestation with sparse rashes。Moreover, the sufferers often showed a wide variety of clinical presentations in EV71 outbreak period。To date, there is still a lack of sensitive methods for the early diagnosis and assessment of EV71 encephalitis and the early detection of brain injure。A small proportion of infected children resulted in severe brain injure or even death for the rapidly clinical deterioration due to impossible to administer appropriate treatment in a timely manner。Since EV71 was first identified in California, USA, in 1969, EV71 infection had been caused over 10 small and large outbreaks throughtout the world, and 4 of which were large-scale epidemic that ocurred in Bulgaria in 1975, in Hungary in 1978, in Malaysia in 1997, and the outbreaks in Taiwan in 1998 had a high fatality rate of 78 of the 405 inpatientS。In mainland of the People's Republic of China, HFMD was first reported in 1984 and EV71(C4 subtype) was first identified in Guangdong Province in 1998。To 2008, the involvements of EV71 epidemic occurred in the Republic of China mainland region were more than 20 provinces, autonomous regions and municipalities, and when the affected children amounted to nearly 500000, of whom 126 cases died。In recent years, the trend of EV71 induced epidemic is on rise, and the severity of CNS involement are becoming more and more serious。The serious EV71 encephalitis has been a severe threat to the safety of children that have drawn greater attention of international society and becomed a research focus。Up to now, the pathogenetic mechanism of the lesion in the CNS following EV71 infection remains obscure。There is still lack of objective diagnostic approach to early assess serious EV71 encephalitis。Many studies confirmed that metabolic changes prior to morphological changes in many diseases。1H-MRS is a non-invasive imaging technique in vivo that may be used to quantitative analysis of disturbed biochemical and metabolic function in local brain tissue。1H-MRS can detect the subtle changes on the type or quantity of metabolites such as NAA,Cho and Cr in region of interest (ROI) and metabolite ratios such as NAA/Cr,Cho/Cr and NAA/Cho ratio。NAA is exclusively located in mature neurons and axons,which is recognized as a neuronal and axonal marker,mainly reflect the quantity and function of the neurons。Cho is an indicator of gliocyte proliferation, myelination and membrane turnover, that can reflect the degree of neural celll damage。The value of NAA/Cr ratio also can reflect the severity of neural celll damage, the lower the ratio,the more severe the cell injure。Cho/Cr represent the activation and proliferation of glial cells and associate with the compensation for neural repair。The NAA/Cho+Cr ratio indicate the neuronal dysfunction or loss and the activities of gliocytes in the lesion。So,1H-MRS can promote the brain research not only on the morphology but into the molecular level and provide a new approach to reveal the essential nature of the disease mechanisms。Both clinical studies and animal experiments had confirmed that 1H-MRS could detect any metabolic abnormalities precede morphological changes and reflect the early ultrastructural changes in brain tissue。Therefore,1H-MRS can make an accurate estimation on the progression of brain injure。Cerebral metabolic changes can be detected by 1H-MRS significantly earlier than morphological changes observed with brain CT or conventional MRI。As a non-invasive imaging approach, 1H-MRS can detect differences in metabolic activity at onset of the disease as well as make the noninvasive investigation in the neuropathological mechanism of the serious EV71 infection。So,1H-MRS has an clinically important significance for early diagnosis and evaluation of EV71 encephalitis and EV71 related brain injure。NSE, MBP and S100βprotein are widely recognized as the biomarkers of neurons, myelination and glial cello NSE is a specific-marker for neuronal injury which mainly distributed in neurons and neuroendocrine cells o Many studies proved there have a significant correlation between NSE concentration and the number of neuronal death。The level of NSE can early predict the degree of neuronal damage。 S100P protein is synthesized and secreted primarily by CNS astrocytes, and recently, it has been suggested to have neurotoxicity at higher concentration。Research found that higher extracellular concentration of S100βprotein can stimulate the expression of inflammatory cytokines and induce cell apoptosiso The serum level of S100βprotein in patient with brain injury has a significant increase even before the detectable imaging abnormality。The experiment of mice model and clinical studies of S100βshowed there was a significant correlation between the serum level and the CSF level,S100βprotein is an early effective marker for the assessment of subclinical brain injure。MBP is synthesized and secreted by CNS oligodendrocytes and represent substantial damage to CNS。MBP is necessary for myelination and act as a myelin-specific marker。So, just mentioned above, by detecting MBP, NSE and S100βlevels in CSF or in serum of chindren with serious EV71 encephalitis, we can make a study on cellular injure mechanisms of neurons, glial cells and myelin formation, and these may help to clinical diagnosis and prognostic evaluation of serious EV71 encephalitis and brain injure。Now, the application of 1H-MRS and neural biomarkers to make a study on the serious EV71 encephalitis have not been reported in domestic and international documents。Therefore, The subject was designed to detect the changes of brainstem 1H-MRS metabolites and NSE, MBP and S100βprotein levels in serum and CSF in children with serious EV71 encephalitis, and get valuable information about the cellular injure mechanisms of severe EV71 encephalitis and the value of clinical application。The research may provide the theoretical basis for pathogenesis and offer a new method for clinical staging and prognostic judgement simultaneously。ObjectiveTo detect the characteristic changes of 1H-MRS biochemical metabolism and NSE,MBP and S100βlevels in serum and CFS in the acute phase in children with serious EV71 encephalitis, and investigate the cellular injure mechanisms and their significance in clinical staging and prognostic judgement。MethodsFrom June to December 2010, twenty-five children with neurological complications were transferred to pediatric intensive care unit in Guangdong General Hospital and then were diagnosed with severe EV71 infection by detection of EV71 specific nucleotides from anal swabs or feces samples were investigated。13 Male and 12 female, the mean age was 18.3 months with a range of 5-36 months。Referring to the《Guideline of diagnosis and treatment of enterovirus 71(EV71) infection(2008 edition)》published by Ministry of Health of the People s Republic China, the cases were classified into two groups according to the pathogenetic conditions within seven days of disease onset:16 cases in severe group,9 cases in critically ill group。By the suggestion on the clinical staging from Lin TY, the enrolled cases were subdivided into four stages:16 cases in stagesⅡ,5 in stagesⅢa,4 in stagesⅢb。Agreement with parents,10 children without any neurologic symptomes were assigned to normal contral group。6 Male,4 female, the mean age was 18.1 months with a range of 6-35 months。2. MRI and'H-MRS were performed for all study objects within 5 days of the onset of illness, by use a 3T MRI scanner (Achieva 3.0 T Philip Medical Systems)。MRI sequences:Transverse T2WI/FSE, T1 WI/FSE and T2-FLAIR and Axial, cornal and sagital SE enhanced T1WI examinations。Scaning protocol:Transverse T2WI/FSE (TR=4114ms, TE=110ms), T1WI/SE (TR=488ms, TE=15ms), T2-FLAIR (TR=6000ms, TE= 120ms, TI=2000ms), FOV= 24×24cm。Matrix 384×381.Slice thichness 5 mm, Space 1.0mm。1-MRS protocol:TR=1500ms, TE=144 ms。Auto-shimming and water suppression。The spectra were processed using spectroview software package。Single voxel 1H-MRS was acquired using point resolved spectroscopysequence (PRESS) over the region of interest (ROI) at the level of brainstem。The chief metabolites, including N-acetyl-as-partate (NAA), Creatine (Cr), choline contained compounds (Cho) and NAA/Cr, Cho/Cr, NAA/Cho and NAA/Cho+Cr ratios。Differences of 1-MRS metabolites in case group were compared to controll group。Two-group Independent-Sample T-Test was used to make a power analysis for the differences of H-MRS metabolites between case and control groups。If the homogeneity of variances assumption is met, LSD t-test is adjusted for multiple comparisons among groups (severe, critically ill and control groups,and stageⅡ, stageⅢand stageⅢb), whereas Welch test is recommended。3.The paired samples of cerebrospinal fluid (CSF,1.5ml) and peripheral blood(4ml, centrifuge at 1000 X g for 15 minutes, collect the supernatant) from 25 patients with EV71 encephalitis were collected within 5 days from the onset of the disease, and the CSF samples were placed directly into EP tubes and preserved in a-80℃freezer。0 sunjects of control group were health vonlenteer and only blood samples were collected from these subjects。Then, the serum and CSF levels of NSE, MBP and S-100βprotein were determined by enzyme-linked immunosorbent assay (ELISA)。The continuous vabible was described by x±s; Two-group Independent-Sample T-Test was used to make a power analysis for the differences of serum and CSF levels of NSE, MBP and S-100βprotein between case and control groups。If the homogeneity of variances assumption is met, LSD t-test is applied for multiple comparisons among the severe, critically ill and control grouops, and stageⅡ, stageⅢand stageⅢb groups, respectively, for the differences of serum and CSF levels of NSE, MBP and S-100βprotein, otherwise Welch test is recommended.The asssociation between the serum and the CSF concentrations of NSE, MBP and S-100βprotein was evaluated by Pearson correlation method(2-tailed)。P≤0.05 was considered statistically significant, P≤0.01 was considered as statistically significant and have a higher degree of assurance。Results1.There were no statistically significant differences in age and sex distribution between the case and control groups(Age:t=0.048, P=0.962; Sex:x2=0.185, P=0.723s)。No abnomalities on both 1H-MRS and MRI were detected in contro group。2. Neuroimaging detection on brain 1H-MRS in case group2.1 Routine MRI scans:of 25 cases,10 presented with detectable structural brain damage, including 5 with brainstem lesions and 3 with spinal cord injuries。The location of lesions:the involvement of brainstem(midbrain pontine tegmentum, medulla oblongata, C1-2 and bilateral occipital lobe) was seen in 1 patient; 1 patient with lesions of pontine tegmentum and medulla oblongata; 1 patient with lesions of C1-4 and medulla oblongata; 1 patient showed an extensive involvement of C6-C7, T1-12 and posterior corpus callosum; 1 patient with lesions of bilateral fronto-parietal lobes; 1 patient showed extensive damage to bilateral centrum ovale corona radiata, pons, medulla, cerebellum earthworm and medulla oblongata; 1 patient with lesions of bilateral parietooccipital lobes; 1 patient with lesions of bilateral frontal, parietal and temporal cortex; 1 patient with lesions of bilateral frontal and temporal cortex; 1 patient with the dorsal surface of medulla oblongata。The MRI signal intensity of the lesions:low or equal signal intensity on T1 WI and high signal intensity on T2WI and T2/FLAIR were detected。2.2 The findings of 1H-MRS:1H-MRS of the brainstem in 25 case patients demonstrated decreased NAA level and increased Cho level at different degrees。Compared with normal control group, the case patients showed decrease of NAA level(0.23±0.05) vs (0.29±0.07) (P<0.01) and increase of Cho level (0.33±0.04) vs (0.27±0.05) (P<0.01)。Metabolite ratio in case group demonstrated a statistically significant decrease of NAA/Cr ratio (1.36±0.32) vs (1.89±0.25) (P<0.01), NAA/Cho ratio (0.48±0.11) vs (0.71±0.17) (P<0.01) and NAA/Cho+Cr ratio (0.48±0.10) vs (1.02±0.28) (P<0.01), and a small increase of Cho/Cr ratio (1.90±0.4)vs (1.80±0.34) (P=0.477)。 2.3 The changes in'H-MRS metabolite ratios between the severe and critically ill groups:'H-MRS showed an significant elevation of Cho/Cr ratios in critically ill group (P<0.05), but only a mild increase of Cho/Cr ratios In severe group (P> 0.05)。1H-MRS in critically ill group demonstrated the marked reduction of NAA/Cr and NAA/Cho and NAA/Cho+Cr ratios (P<0.05, P<0.05, P<0.05 respectively).2.4 The changes in'H-MRS metabolite ratios at different stages:stageⅡand stageⅢ(Ⅲa,Ⅲb) compared with normal control group respectively:there was no marked changes in Cho/Cr ratios at stageⅡ(P>0.05), while a statistically significant increase of Cho/Cr ratios at stageⅢ(P<0.05)。StageⅢa compared with stageⅡ: there was a statistically significant increase of Cho/Cr ratios, dicrease of NAA/Cho+Cr and NAA/Cho ratios (P<0.01, P<0.01, P<0.05 respectively)。StageⅢb compared with stageⅡ:StageⅢb group exhibited a high statistical significance of reduction of NAA/Cr and NAA/Cho and NAA/Cho+Cr ratios and elevation of Cho/Cr ratio (All P<0.01)。StageⅢa compared with stageⅢb:StageⅢb group exhibited a high statistical significance of reduction of NAA/Cr and NAA/Cho+Cr ratios(P<0.01, P<0.05 respectively), and no statistical significance in Cho/Cr ratio (P>0.05).3.The findings of markers of brain injure3.1 Linear correlation analysis showed a positive correlation between NSE, MBP and S-100βin serum and CSF levels, r was 0.806,0.671,0.802 respectively (AllP< 0.01)。3.2 The results of neural markers in serum level between the case and normal control groups:The case group exhibited a statistically significance in the increase of serum NSE level (7.46±2.74) vs (4.13±0.68), and S-100βlevel (529.85±192.90) vs (325.61±63.14) (both P<0.01) and a samll increase of serum MBP level (1.45±0.79) vs (1.22±0.56) (P=0.409)。 3.3 Compared to control group, the critically ill group exhibited a statistically significant increase in serum NSE level(10.34±1.50)and MBP level(1.95±0.48) and S-100βlevel(744.31±101.57)(P<0.01, P<0.05, P<0.01 respectively), the severe group only showed a statistically significant increase in serum NSE level (5.80±1.70) and S-100βlevel (409.22±104.50) (both P<0.05)。There were a statistically significant differences in serum NSE, MBP and S-100βlevels in critically ill group compared with those in severe group (P<0.01)。3.4 The changes in NSE,MBP and S-100βlevels at different stages:With advancement of clinical stage, serum NSE, MBP and S-100βlevels increase gradually, the order of the marker level is as following:stageⅢb>stageⅢa>stageⅡ。stageⅡand stageⅢcompared with normal control group respectively, there were a statistically significant increase of NSE and S-100βlevels at stageⅡand NSE,MBP and S-100βlevels at stageⅢ(All P<0.01)。There were no statistical differences in serum NSE,MBP and S-100(3 levels at stageⅢb compared with those at stageⅢa (P>0.05)Conclusion1. The early characteristic findings of serious EV71 encephalitis were the reduction of NAA/Cr, NAA/Cho and NAA/Cho+Cr that suggested the neuronal injure occurred in the acute stage of CNS involvement and indicated the neuronal dysfunction or loss。The crinically ill cases may accompany by the increase of Cho/Cr, which demonstrated gliocyte proliferation or myelin breakdown。The more the'H-MRS metabolites changed, the more severe the disease was, and the worse the prognosis got。2. Elevated CSF and serum NSE and S-100(3 levels were presented in patients with serious EV71 encephalitis in the acute CNS involvement。These changes indicate the impairment of both neurons and astroglia cells may occurred in the early CNS involvement。Significantly elevated MBP level ocurred in crinically ill patients that suggested the oligodendrocyte damage or the breakdown of CNS myelin。The levels of NSE,MBP and S-100βin CSF or serum are associated with the severity of disease。3. The CSF and serum NSE,MBP and S-100βlevels showed a positive correlation。The serum level of neural markers can be used to assess the severity of serious EV71 encephalitis and brain injure。4. 1H-MRS is a non-invasive technology and can provide in vivo pathological diagnosis for serious EV71 encephalitis and brain injure, and offer objective quantitative evidence about the severity of disease and prognostic judgement。5. The 1H-MRS combined with MRI can provided objective imaging evidences for both the localization and functional diagnosis of serious EV71 encephalitis。... |
PDF Full Text Request