Research progress in atrial fibrillation with cerebral small vessel disease / 中南大学学报(医学版)

Non-valvular atrial fibrillation is a common arrhythmia and a major risk factor for cardioembolic stroke. Small cerebral vascular disease is a syndrome of clinical, cognitive, imaging, and pathological manifestations caused by intracranial small vascular lesions. The imaging findings on cranial magnetic resonance usually shows recent subcortical small infarction, vascularised lacunae, white matter hypersignal, perivascular space enlargement, cerebral microhemorrhage, and brain atrophy. It is a major cause of neurological loss and cognitive function decline in the elderly. Current studies suggest that atrial fibrillation may increase the imaging load of cerebral small vessel disease through a series of mechanisms such as microembolization, hypoperfusion, inflammation, endothelial dysfunction, and lymphoid system dysfunction. The imaging of cerebral small vessel disease with atrial fibrillation has a potential relationship with cognitive function decline and is related to the occurrence and prognosis of stroke, even more has a potential role in suggesting the etiology and secondary prevention strategies of ischemic stroke.

Effect of total knee arthroplasty on osteoarthritis of knee joint / 中国基层医药

Objective:To observe the effect of total knee arthroplasty in the treatment of knee osteoarthritis.Methods:From April 2017 to April 2019, 80 patients with knee osteoarthritis admitted to Zhejiang Rongjun Hospital were selected in the research.The patients were divided into control group(40 cases) and observation group(40 cases) according to the random digital table method.The control group was operated by arthroscopy, and the observation group was operated by total knee replacement.The bleeding volume, incision healing time, hospitalization time, incidence of postoperative complication, knee joint activity, VAS score and HSS score were compared between the two groups.Results:The amount of bleeding in the observation group was (137.26±30.85)mL, which was less than that in the control group[(300.31±58.40)mL]( t=15.613, P<0.001). The healing time of incision and hospitalization time in the observation group were (4.20±1.37)d and (7.58±1.20)d, respectively, which were shorter than those in the control group[(6.89±2.43)d and (10.50±3.20)d], the differences was statistically significant( t=6.099, 5.404, all P<0.001). The incidence of postoperative complications in the observation group was 7.50%(3/40), which was lower than 25.00%(10/40) in the control group(χ 2=4.501, P<0.05). The knee joint mobility of the observation group was (106.71±15.38)° after operation, which was higher than that of the control group[(95.51±11.62)°]( t=3.675, P<0.001). The VAS score and HSS score of the observation group were (2.50±0.68)points and (89.27±5.65)points, which were better than those of the control group[(4.53±0.71)points and (69.39±5.41)points], the differences were statistically significant( t=13.059, 16.073, all P<0.001). Conclusion:Total knee arthroplasty is effective in the treatment of knee osteoarthritis, which is helpful for the recovery of knee function.

Efficacy of the traditional Chinese medicine NAOAN capsule for primary prevention of stroke in high-risk population / 中华健康管理学杂志

Objective To evaluate the efficacy of the traditional Chinese medicine NAOAN capsule in primary prevention of stroke in high?risk populations. Methods A multicenter, randomized controlled study was performed in community setting, involving 1 088 individuals at high risk of stroke, with cerebrovascular function scores<75 and 10?year Framingham stroke risk ≥6%. Subjects were recruited in communities at Beijing, Shanghai, Changsha, and Chengdu communities, through resident committees or the village unit. A total of 559 subjects were randomized into a group treated with the NAOAN capsule, and 529 subjects in an aspirin treatment group. Follow?up was performed every 2 months for 2 years. At the mid?point and end of the intervention, we compared cerebrovascular function differences between the 2 groups. Results During the 2?year community trial, 531 subjects in the NAOAN capsule group and 465 in the aspirin group followed the protocol. Cerebrovascular function scores increased from 45.2±19.7 at baseline to 61.7±26.5 after the 2?year trial (t=－12.931, P<0.001) in the NAOAN capsule group, and from 47.2±18.9 at baseline to 53.7 ± 25.1 (t=－5.058, P<0.001) in the aspirin group; greater increases in cerebrovascular function scores were found in the NAOAN capsule group than that in the aspirin group (t=4.906, P<0.001). Conclusions Cerebrovascular function in individuals at high risk of stroke was improved by taking NAOAN capsule. Cerebrovascular function scores improved more with NAOAN capsules than with aspirin.

Effects of estrogen or/and progesterone on the differential expression of αvβ3 in bovine endometrial epithelial cells in vitro / 中国实验动物学报

Objective The aim of this study is to explore the changes of expression of integrin αvβ3 in bovine u-terine epithelial cells in vitro induced by estrogen or/and progesterone alone or in combination,and to provide a new refer-ence marker for determining bovine uterine receptivity state. Methods RT-PCR was used to analyze the transcriptional changes of αvβ3 expression in bovine endometrium treated by different concentrations of estrogen,progesterone alone or in combination. Results The expression of integrin αvβ3 reached the highest level when the culture medium was added with progesterone at the concentration of 10 -7mg/mL,and the expression of αv and β3 in the 10 -7mg/mL concentration group was significantly higher than that of the control one(P<0.05). Moreover,the expression of αv was highest in the 10 -10 mg/mL E2group,but the expression of β3 was the lowest in that one. In addition,adding with both estrogen and progester-one,the transcriptional level of integrin αvβ3 was significantly higher than that in the control one. The transcriptional level of αv in the treatment group was significantly higher than that in the control group(P<0.05),but the transcriptional level of β3 in this group was not(P>0.05). Conclusions It can be concluded that integrin αvβ3 can be used as a new poten-tial reference marker gene for detecting the bovine uterine receptive status.

Investigation on the detection of transient ischemic attack in cerebrovascular disease surveillance in Hunan Province / 中华健康管理学杂志

Objective To determine the incidence and prevalence of transient ischemic attack (TIA) and to evaluate its epidemiological situation in Hunan province.Methods Seven monitoring points were randomly selected from the province,a total of 8 311 subjects aged≥50 years were then chosen by stratified sampling.The cases counted in prevalence was defined as patients diagnosed before 24:00 o'clock August 31st,2013,and the new diagnosis for incident counting was defined as those diagnosed between 00:00 September 1st,2012 and 24:00 August 31st,2013.Results Among all 8 311 screened subjects,the number of TIA patients was 24 (288.8 per 100 000 people),the incidence of TIA was 7 (85.2 per 100 000 people).Standardized prevalence and incidence were 283.2 and 82.4 per 100 000 respectively using 2010 China census population.Among them,the standardized incidence rate of female was higher than that of male (114.8 per 100 000 person-years vs.48.8 per 100 000 person-years),and the prevalence rate of males was higher than that of female (288.2 per 100 000 people vs.273.2 per 100 000 people).Hypertension is the most important risk factor for TIA (55.2％).Conclusion The incidence and prevalence of TIA in Hunan province are higher than the national average.Hypertension is the main risk factor.

Advance in research on the genetic etiology of spontaneous intracerebral hemorrhage / 中华医学遗传学杂志

Spontaneous intracerebral hemorrhage (SICH) is a form of brain parenchymal hemorrhage caused by a variety of non-traumatic reasons, resulting in cerebral artery, veins or capillaries rupture. The etiology of SICH is variable, with hypertensive intracerebral hemorrhage being the most common, accounting for 60% ~ 81% of all cases. Cerebral amyloid angiopathy, drug use related hemorrhage, Moyamoya disease are also important causes of SICH. Previous studies showed that genetic factors play an important role in the pathogenesis of SICH. Here the genetic mechanisms of SICH and classification of its etiology are reviewed.

Association between tumor necrosis factor superfamily member 4 gene polymorphism and risk of asymptomatic carotid vulnerable plaque in a Chinese population / 中华流行病学杂志

Objective Vulnerable plaque of carotid artery is one of the risk factors of atherosclerotic cerebral infarction.Detection and treatment of vulnerable atherosclerotic plaque of carotid artery before symptoms of cerebral infarction is an effective way to prevent atherosclerotic cerebral infarction.Tumor necrosis factor superfamily member 4 (TNFSF4) plays a key role in the process of atherosclerosis,a common risk factor for both myocardial and cerebral infarctions.Studies have indicated that the single nucleotide polymorphism (SNP) rs3850641 in TNFSF4 is associated with higher risk of myocardial infarction and SNP rs3861950 in TNFSF4 is associated with higher risk of atherosclerosis cerebral infarction (ACI),but little is known about the association between TNFSF4 variations and vulnerable plaque of carotid artery.Methods A case-control study involving 510 patients with asymptomatic vulnerable plaque of carotid artery and 485 age and sex matched healthy subjects without vulnerable plaque of carotid artery was conducted in Hunan province.Asymptomatic vulnerable plaque of carotid artery means vulnerable plaque of carotid artery without cerebral infarction.Two SNPs of TNFSF4,rs3850641 and rs3861950,were genotyped by the TaqMan SNP genotyping method,and verified partly by Genomic DNA Sequencing.Results The results revealed a significant allelic association between rs3861950 and asymptomatic vulnerable plaque of carotid artery in case group (x2=9.13,P=0.003;OR=1.41,95％ CI:1.12-1.76).Compared with control subjects,the difference in genotype was significant in case group (x2=25.28,P＜ 0.000 1).However,there was no significant association between rs3850641 and asymptomatic vulnerable plaque of carotid artery(OR=1.16,95％CI:0.92-1.46;x2＝ 1.47,P=0.225).Conclusion TNFSF4 gene polymorphism rs3861950 was associated with the risk of vulnerable plaques of carotid artery in a Chinese population,which might be middle phenotype indicating higher risk of cerebral infarction.

Role of mitochondrial dysfunction in hypoxic-ischemic brain damage of mice / 中国医师杂志

Objective The hypoxic-ischemic(HI) cardio-cerebral damage caused by cardiac arrest in perioperative period is the main cause of acute and chronic disability in children patients.To investigate role of mitochondrial dysfunction in hypoxic-ischemic brain damage of mice.Methods The hypoxic-ischemic mice model was established by the bilateral carotid artery occlusion and hypoxia treatment.The neurobehavior of mice in HI model group,sham-operated group,and comparative group were evaluated within 48 hours after operation.After 48 hours,the mice were killed to evaluate the brain water content,mitochondria content,swelling,antioxidant capacity,and respiratory function.Results Within 0,24 hours after operation,the abnormal rate of the neurobehavior of HI model mice was 83.33％,which was significantly higher than comparative and sham-operated groups.The water content of right brain was significantly increased evidently compared to the other two groups (P ＜ 0.05).The content and swelling of mitochondria in brain were increased.The activity of superoxide dismutase (SOD),the glutathione (GSH) content,respiratory state 3 (ST3),and respiration control of rate (RCR) were significantly decreased; while the content of malondialdehyde (MDA) and ST4 were significantly increased (P ＜ 0.05).Conclusions The brain tissue showed different swelling,the mitochondrial function occurred disorder,which might play an important role in hypoxic-ischemic brain damage of mice.

Ultrastructural observation of dormant mouse embryos cultured in vitro after freezing-thawing / 中国实验动物学报

Objective The aim of this study was to investigate the differences of the cell ultrastucture of normal mouse hatched blastocysts and their dormant ones cultured in vitro after freezing-thawing, and to explore whether the dor-mant embryos have a better anti-freezing shock property than the normal hatched mouse embryos .Methods By transmis-sion electron microscopy , the ultrastructure of these two types of mouse embryos was observed and analyzed .Results By comparative analysis of their ultrastructure , the results showed that the dormant embryos before freezing are being austerity and with lower energy metabolism at a ‘ground state ’ .After freezing-thawing and culture , their cellular structure seemed to be similar to that of the normal embryos cultured in vitro before freezing.However, after freezing-thawing and culture, the number of mitochondria decreased , the nuclei were loose , and their heterochromatin also increased .Conclusions From the ultrastructural observation , compared with the normal mouse hatched embryos , the cellular state of dormant mouse em-bryos after freezing-thawing is more favorable for material storage and energy metabolism , thus, indicating that they have a better anti-freezing property than normal hatched embryos .

Influence of apurinic/apyrimidinic endonuclease on repair of rat brain regions distant from the focal cerebral ischemia site / 中华老年医学杂志

Objective To investigate changes in the expression of apurinic/apyrimidinic endonuclease (APE) and the oxidative DNA damage marker 8 OHdG in distant hippocampus regions of the rat brain after focal cerebral ischemia of the middle cerebral artery.Methods SD rats were divided into the sham surgery group and the pMCAO group (induced by middle cerebral artery occlusion).Pathological changes in brain tissues were examined at 2 h,6 h,12 h,24 h,48 h and 72 h.The expression of APE and 8-OHdG was measured by immunohistochemical staining methods.TUNEL staining was performed to detect apoptosis.Results Reduction of APE expression in the CA1 region of the hippocampus on the ischemia side appeared at 2 h in the pMCAO group and continued as ischemia persisted (F=11.91,P＜0.05).The expression of 8OHdG and TUNEL immunoreactivity in the CA1 region of the hippocampus on the ischemia side were first observed at 6h in the pMCAO group and intensified during the remainder of induced ischemia (F=9.23 and 10.46 respectively,P＜0.05 for both).Compared with the sham group,8-OHdG expression and TUNEL immunoreactivity in the pMCAO group were at nearly the same levels from 24 h to 72h.Conclusions Oxidative DNA damage occurs in hippocampus regions of the rat brain after experimentally induced focal cerebral ischemia of the middle cerebral artery.APE expression declines in regions distant from focal cerebral ischemia.Development and accumulation of oxidative DNA damage can induce apoptosis in certain brain regions.

Gene polymorphisms of intracerebral hemorrhage in Chinese population: a systematic review / 中华神经科杂志

Objective To assess the genes polymorphisms associated with intracerebral hemorrhage (ICH) in Chinese quantitatively or qualitatively by searching all case control studies related comprehensively.Methods Odds ratio (OR) and 95％ confidence intervals (95％ CI) were determined for each polymorphism using fixed or random model with Revman 5.1.Results Statistically significant associations with ICH were detected with both ε4-carrier genotypes and ε2-carrier genotypes of apolipoprotein E (OR =1.59,95％ CI 1.23-2.04,P ＜ 0.01 and OR =1.92,95％ CI 1.50-2.47,P ＜0.01),methylenetetrahydrofolate reductase C677T (OR =2.08,95％ CI 1.52-2.83,P ＜ 0.01) and angiotensin converting enzyme insertion/deficiency (OR =2.34,95％ CI 1.82-3.02,P ＜ 0.01),but no statistical association was detected for plasminogen activator inhibitor type-1 (OR =1.27,95％ C10.84-1.93,P =0.26).Furthermore,two out of ten polymorphisms,each of which was investigated in a multicenter study with sample size larger than 400,also suggested a positive association with ICH.They were apolipoprotein A (OR =1.64,95％ CI 1.21-2.21,P ＜0.01) and vessel endothelial growth factor 2 (OR =3.22 ;95％ CI 1.63-6.37,P ＜ 0.01) respectively.Conclusions ε2 and ε4 carriers of apolipoprotein E,angiotensin converting enzyme I/D,methylene tetrahydrofolate reductase C677T,lower repeats of TTTTA of apolipoprotein A and vascular endothelial cell growth factor 2 rs2305948 polymorphisms are the possible genetic risk factors in Chinese.In conclusion,several genes are identified as to be susceptible to ICH in Chinese by our well defined criteria,however,the evidence for majority of studies is limited.

Expression of OX40/OX40L mRNA in experimental allergic neuritis under influence of Rho-kinase inhibitor / 中华神经科杂志

Objective To study the expression of mRNA of OX40 and OX40L in the sciatic nerve,spleen,peripheral blood mononuclear cells and lymph nodes of EAN under the influence of Rho-kinase inhibitor.Methods All 54 female Lewis rats were divided into 3 groups:the EAN group,the EAN+ Rho-kinase inhibitor group and the complete Freund's adjuvant(CFA)group.The rats were sacrificed at 9,17 and 26 days after immunized.Ox40 and OX40L mRNA were detected by RT-PCR which came from spleens,sciatic nerves,peripheral blood mononuclear cells and lymphonodes.Results In EAN+ Rho-kinase inhibitor group,the mRNA expression of OX40 were 0.266±0.031,0.298±0.024 and 0.113±0.018 at 9.17 and 26 days in the sciatic nerve,the expression were 0.453±0.030,0.496±0.100 and 0.220±0.016 in the lymph nodes.The mRNA expression of OX40L were 0.247±0.018.0.298±0.026 and 0.165±0.013 in the sciatic nerve,the expression were 0.283±0.027,0.306±0.011 and 0.161±0.012 in the lymph nodes.The mRNA expression of OX40 and OX40L in EAN+Rho-kinase inhibitor group was lower than EAN group at the three time points(t=2.24-4.89,P＜0.05),and the demyelination and inflammation cells infiltrating were ameliorated in spinal nerve.CFA group didn't show any clinical manifestation.Conclusion Rho-kinase inhibitor may ameliorate tlIe development of EAN through inhabiting the OX40 and OX40L activation.

Effect of Rho-kinase inhibitor on experimental allergic neuritis / 中国医师杂志

Objective To study the effect of Rho-kinase inhibitor on experimental allergic neuritis. Methods 54 female Lewis rats were divided into three groups; EAN group, EAN + Rho-kinase inhibitor group, and CFA group. The rats were sacrificed on the 9th day, 17th day, and 26th day after immunized. The changes of weight, EAN incidence, and mean day of onset, mean maximum clinical score, and histopa-thology were observed. Results The clinical course in EAN group reached peak on the 17th day. Compared with EAN group, the weights of Rho - kinase inhibitor group were increased, while EAN incidence, mean day of onset delay, and the clinical scores in Rho-kinase inhibitor group were significantly decreased, ( P < 0.01) , and the demyelization and inflammation cells infiltrating was ameliorated in spinal nerve. CFA group didnt show any clinical manifestation. Conclusions Rho - kinase inhibitor may ameliorate the development of EAN through inhabiting the Rho/ROK signal pathway.

Study on differentiation of adipose-derived stem cells into neural cells induced by supernatant liquid from brain tissue in rats / 中华老年医学杂志

Objective To observe the effect of supernatant liquid from brain tissue on the differentiation of adipose-derived stem cells (ADSCs) into neural cells in normal brain tissues, the homogenate of infarcted cerebral hemisphere and the opposite side in the rats. Methods The ADSCs were obtained from rat retroperitoneal adipose tissue. The normal brain tissues, the homogenate of the infarcted cerebral hemisphere and thc opposite side got from middle cerebral artery occlusion in rats were used to induce ADSCs. Immunocytochemistry or immunofluorescence were used to identify the cell types at the 3rd day. Positive expression rate was counted by fluorescence microscope. Results (1)The neuron-specific enolase (NSE) positive cells, microtubule-associated protein 2 (MAP-2) positive cells and glial fibrillary acidic protein (GFAP) positive cells were much more in the homogenate of the infracted cerebral hemisphere than in others (P＜0.05). (2)The NSE positive cells, MAP-2 positive cells and GFAP positive cells were much more in the homogenate of the normal brain tissues and the opposite side than normal level ( P ＜ 0. 05 ) . Conclusions The homogenate of the infracted cerebral hemisphere and the opposite side can induce adipose-derived stem cells into neural-like cells and express neural cells markers in rats.

Relationship between T704C polymorphism of angiotensinogen gene and cerebral hemorrhage in Han people in Changsha / 中南大学学报(医学版)

OBJECTIVE@#To investigate the relationship between T704C polymorphism of angiotensinogen (AGT) gene and cerebral hemorrhage and its impact on the levels of blood pressure in Han people in Changsha.@*METHODS@#A total of 273 cerebral hemorrhage patients (the cerebral hemorrhage group) and 140 normal controls (the control group) were collected from Jan. 2005 to Jan. 2009. DNA was extracted from their peripheral blood samples. The polymorphism of AGT-T704C was analyzed by SNaPshot and direct DNA sequencing. The possible risk factors of cerebral hemorrhage were investigated at the same time. Each group was divided into 2 subgroups (a high blood pressure subgroup and a normal blood pressure subgroup) according to whether they had essential hypertension. Logistic regression analysis was used to detect the relationship between cerebral hemorrhage and all its possible risk factors and AGT-T704C polymorphism.@*RESULTS@#The drinking history, coronary heart disease history, essential hypertension history, and blood levels of lipids were shown significant difference between the cerebral hemorrhage group and the control group (P0.05).@*CONCLUSION@#The polymorphism of AGT-T704C may not be associated with cerebral hemorrhage and not related to the levels of lipids and blood pressure in Han people in Changsha. Hypertension history, systolic blood pressure level, and high density lipoprotein cholesterol level are the main risk factors of cerebral hemorrhage in Han people in Changsha.

Dynamic change OX40/OX40L mRNA in experimental allergic neuritis / 中南大学学报(医学版)

OBJECTIVE@#To examine the expression of mRNA of Oxford 40(OX40) and Oxford 40 ligand(OX40L) in the sciatic nerve, spleen, peripheral blood mononuclear cells and lymph nodes of experimental allegic neuritis (EAN).@*METHODS@#Thirty-six Lewis rats were randomly assigned into an EAN group and a CFA group. The rats were sacrificed on 9th, 17th, and 26th day after immunization. OX40 and OX40L mRNA was detected by reverse transcription polymerase chain reaction in the spleen, sciatic nerves, peripheral blood mononuclear cells and lymphonodes.@*RESULTS@#The peak of clinical course came on 17th day after the immunization in EAN. The mRNA expression of OX40/OX40L was higher on 8th day and 17th day than that on 26th day after the immunization (P<0.05). There was significant difference between the EAN group and the CFA group at the 3 time points (P<0.05); rats in the CFA group didn't have any clinical manifestations. The mRNA expression of OX40 and OX40L in the EAN group raised in the sciatic nerves and lymph nodes at the above 3 time points (P<0.05). Weak expression was seen in the peripheral blood mononuclear cells.@*CONCLUSION@#OX40 and OX40L may play a role in the pathogenesis of experimental allegic neuritis.

Association between polymorphism of rs3212855 and rs5515 of KLK1 gene with cerebral hemorrhage in Changsha Han population / 中南大学学报(医学版)

OBJECTIVE@#To explore the association between single nucleotide polymorphism (SNPs) of KLK1 gene and cerebral hemorrhage in Changsha Han population.@*METHODS@#We enrolled 273 patients with cerebral hemorrhage and 140 normal people. The SNPs (including rs3212855 and rs5515) of KLK1 gene were analyzed by Snapshot method and direct sequencing.@*RESULTS@#We found rs5515 was not a polymorphic site in Changsha Han population. Genotype and allele frequency in rs3212855 were not different between patients with cerebral hemorrhage and the controls (P>0.05). The blood pressure level was not different between the genotype subgroups.@*CONCLUSION@#Neither rs5515 nor rs3212855 is associated with cerebral hemorrhage.

The study on the correlation of C1773T polymorphism of LDL receptor gene (LDLR) with cerebral hemorrhage and its impact on the levels of serum lipids in Chinese / 中国神经精神疾病杂志

Objective To investigate the relationship between C1773T polymorphism of LDL receptor gene (LDLR) and cerebral hemorrhage and the impact of C1773T polymorphism of LDLR on the levels of serum lipids in Chinese Han in Changsha, Hunan province. Methods Two hundred seventy-three cerebral hemorrhage patients and 140 normal controls were recruited in the present study. The C1773T polymorphism of LDLR was analyzed by SNaPshot and direct DNA sequencing. The triglyceride (TG), total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C) levels were examined using oxidase method. Results The CC, CT and TT genotype frequencies of LDLR polymorphism were 0.703/0.278/0.019、0.707/0.250/0.043 and the allele C and T frequencies of LDLR polymorphism in the cerebral hemorrhage group and the control group were 0.842/0.158,0.832/0.168 respectively. The differences in genotype and allele frequencies of LDLR polymorphism were no significant between cerebral hemorrhage group and the control group (P>0.05). There were no significant differences in the levels of lipids among the CC, CT and TT genotype in either cerebral hemorrhage group or the control group (P＞0.05). Conclusions The LDLR-C1773T polymorphism may not be associated with cerebral hemorrhage nor be related to hyperlipemia in Chinese Han in Changsha.

Radix isatidis and infectious diseases caused by viruses / 中国中药杂志

Radix Isatidis (Banlangen in Chinese), used to clearing away heat and toxic material, is a traditional Chinese medicinal (TCM) herb. It is frequently used for preventing and treating infectious diseases caused by viruses. To provide scientific basis for the effect of Radix Isatidis on infectious diseases, the traditional effect and new research development on pharmacological activities are summarized in the review. According to the existed problems in the clinical application, the weak links and shortages of quality research and industrialized production of Radix Isatidis are discussed. It could present the new ideas for improving the technology of Radix Isatidis preparation, and promoting the rational use of the preparation in the clinical treatment.