BACKGROUNDAlthough traumatic brain injury can lead to opening the blood-brain barrier and leaking of blood substances (including water) into brain tissue, few studies of brain antigens leaking into the blood and the pathways have been reported. Brain antigens result in damage to brain tissues by stimulating the immune system to produce anti-brain antibodies, but no treatment has been reported to reduce the production of anti-brain antibodies and protect the brain tissue. The aim of the study is to confirm the relationship between immune injury and arachnoid granulations following traumatic brain injury, and provide some new methods to inhibit the immune injury.

METHODSIn part one, methylene blue was injected into the rabbits' cisterna magna after traumatic brain injury, and concentrations of methylene blue and tumor necrosis factor (TNF)-α in blood were detected to determine the permeability of arachnoid granulations. In part two, umbilical cord mesenchymal stem cells and immature dendritic cells were injected into veins, and concentrations of interleukin 1 (IL-1), IL-10, interferon (IFN)-γ, transforming growth factor (TGF)-β, anti-brain antibodies (ABAb), and IL-12 were measured by ELISA on days 1, 3, 7, 14 and 21 after injury, and the numbers of leukocytes in the blood were counted. Twenty-one days after injury, expression of glutamate in brain tissue was determined by immunohistochemical staining, and neuronal degeneration was detected by H&E staining.

RESULTSIn part one, blood concentrations of methylene blue and TNF-α in the traumatic brain injury group were higher than in the control group (P < 0.05). Concentrations of methylene blue and TNF-α in the trauma cerebrospinal fluid (CSF) injected group were higher than in the control cerebrospinal fluid injected group (P < 0.05). In part two, concentrations of IL-1, IFN-γ, ABAb, IL-12, expression of glutamate (Glu), neuronal degeneration and number of peripheral blood leukocytes were lower in the group with cell treatment compared to the control group. IL-10 and TGF-β were elevated compared to the control group.

CONCLUSIONSTraumatic brain injury can lead to stronger arachnoid granulations (AGs) permeability; umbilical cord mesenchymal stem cells and immature dendritic cells can induce immune tolerance and reduce inflammation and anti-brain antibodies to protect the brain tissue.

Adipocytes ; cytology ; Animals ; Antigens ; blood ; metabolism ; Brain Injuries ; blood ; cerebrospinal fluid ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Dendritic Cells ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Interleukin-1 ; blood ; cerebrospinal fluid ; Interleukin-10 ; blood ; cerebrospinal fluid ; Interleukin-12 ; blood ; cerebrospinal fluid ; Mesenchymal Stromal Cells ; cytology ; Methylene Blue ; metabolism ; Osteoblasts ; cytology ; Rabbits ; Transforming Growth Factor beta ; blood ; cerebrospinal fluid ; Tumor Necrosis Factor-alpha ; blood ; cerebrospinal fluid

OBJECTIVETo evaluate the application of XT-4000i automatic blood cell analyzer in white cell count and classification of cerebrospinal fluid (CSF).

METHODSThe fluid model of XT-4000i automatic blood cell analyzer was directly used to detect the white cell count and classification in 200 samples of CSF, and compared with the results obtained by manually microscopic counting method. White blood cell classification was performed by manual method under microscope with Wright's staining,and instrumental method with fluorescence staining and flow cytometry.

RESULTSThere is no statistical difference between instrumental and manual method in detecting the absolute counting of WBC, RBC, mononuclear cell and multinucleate cells (P>0.05), and there is a good correlation between the two methods (r=0.987, 0.999, 0.981 and 0.983 for WBC, RBC, mononuclear cell and multinucleate cell counts). Tumor cells in the samples with high fluorescent staining by instrumental method can be identified with Wright's staining by microscope method, which were consistent with the clinical diagnosis.

CONCLUSIONThe fluid model of XT-4000i automatic blood cell analyzer was rapid and accurate in CSF white cell count and classification,and it also can provide preliminary information for tumor cells screening. The fluid model of XT-4000i automatic blood cell analyzer in white cell count and classification of CSF has the value of popularization in clinical application.

Autoanalysis ; instrumentation ; methods ; Cerebrospinal Fluid ; cytology ; Humans ; Leukocyte Count ; instrumentation ; methods ; Leukocytes ; classification

AIMTo observe the distribution and expression of p-p38MAPK in the distal cerebrospinal fluid contacting neurons in brain of rat by noise stress.

METHODSBy a double-labelled method combing the tracing of CB-HRP and the immunohistochemical technique p-p38MAPK, the distribution and expression of p-p38MAPK in the distal cerebrospinal fluid contacting neurons(csf cn) were observed following noise stress. Expression of p-p38MAPK and double-labelled of CB-HRP/p-p38MAPK were also observed in rat brain after noise stress.

RESULTSTwo groups of CB-HRP labeled neuron clusters consistently appeared in certain regions of the brainstem but none in other regions of the brain. Without noise stress exposure, only a few neurons were found double-labeled by CB-HRP/p-p38MAPK. After 1 day noise stress exposure, only few neurons double-labeled by CB-HRP/p-p38MAPK were observed in the above-mentioned regions. After 5 days, the number of neurons double-labeled by CB-HRP/p-p38MAPK increased significantly compared with the control group (P < 0.05). After 10 days, the number of neurons double-labeled by CB-HRP/p-p38MAPK increased significantly compared with the control group (P < 0.05). After 20 days, both of the numbers of neurons double-labeled by CB-HRP/p-p38MAPK increased significantly compared with that of the control group (P < 0.01).

CONCLUSIONTwo groups of distal cerebrospinal fluid contacting neuron clusters consistently existed in certain regions of the brain parenchyma, and in these clusters only a few neurons con rained p-p38MAPK. After noise stress exposure of different durations (days 1, 5, 10, 20), the number of distal cerebrospinal fluid contacting neurons with p-p38MAPK increased significantly with increasing days. The results indicate that distal cerebrospinal fluid contacting neurons are special neurons existing consistently in brain, including distal cerebrospinal fluid contacting neurons with p-p38MAPK which may participate in the whole procedure of signal transduction or central modulation in noise stress response and play greater roles with increasing days.

Animals ; Brain ; cytology ; Male ; Neurons ; metabolism ; Noise ; Rats ; Rats, Sprague-Dawley ; Stress, Psychological ; cerebrospinal fluid ; p38 Mitogen-Activated Protein Kinases ; cerebrospinal fluid

The purpose of this study was to develop a sensitive method for the detection of malignant B lymphocytes in cerebrospinal fluid (CSF) from patients with diffuse large B-cell lymphoma (DLBCL) who were considered as risk of central nervous system (CNS) involvement. Nine CSF samples were collected and then centrifuged. The cell precipitate was lysed directly. The supernatant was used to detect immunoglobulin heavy chain (IgH) gene rearrangement (characteristic changes of malignant B lymphocytes ) by BIOMED-2 PCR. The sensitivity of this method was compared with that of cytology defection and flow cytometry. In addition, through a series of quantity/concentration of tumor cells, the sensitivity differences caused by two sample handling methods (direct cell lysis vs traditional DNA extraction) were analyzed, and the sensitivity of direct cell lysis combined with BIOMED-2 PCR was evaluated. The results showed that the positive clonality of IgH gene rearrangement were detected by BIOMED-2 PCR in 5 cases, but the positive were detected by cytology defection/flow cytometry only in 2 cases, which indicated that the BIOMED-2 PCR assay gives a better yield. In addition, when combined with BIOMED-2 PCR, direct cell lysis produced sensitivity much higher than DNA extraction. The former can enable clonality detection from a minimum of 0.1%/20 tumor cells. It is concluded the method of direct cell lysis combined with BIOMED-2 PCR is sensitive and suitable for paucicellular CSF detection. It may aid the diagnosis of CNS involvement in patients with DLBCL.
Aged ; B-Lymphocytes ; pathology ; Case-Control Studies ; Cerebrospinal Fluid ; cytology ; Humans ; Lymphoma, Large B-Cell, Diffuse ; cerebrospinal fluid ; diagnosis ; Middle Aged ; Polymerase Chain Reaction ; methods

In order to set up a base for stem cells to be widely used in clinical medicine, we tried to optimize, in this study, the technique that induces human mesenchymal stem cells (hMSCs) to differentiate into neural stem cells by using cerebrospinal fluid (CSF) from the different groups. After the induction, presence of neural stem cells was confirmed with microscope observation, flow cytometry analysis, immunohistochemistry and fluorescent immunohistochemistry. At the same time, we also compared and analysed the data of the number of stem cells when it totally met the requirements for clinical treatment and the days required. At last, we confirmed that hMSCs could be induced to differentiate into neural stem cells, and that the number of cells totally met the requirements for clinical treatment. But there were some differences both in the number of cells and the days required. Among the groups, the group that marrow mesenchymal stem cells from patients own induced by CSF from healthy volunteers used the shortest time and the quantity of the cells was significantly higher than those of the others.
Cell Differentiation ; Cerebrospinal Fluid ; chemistry ; Culture Media ; chemistry ; Flow Cytometry ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells ; cytology ; Neural Stem Cells ; cytology

BACKGROUND: To enumerate leukocyte count in cerebrospinal fluid (CSF) is important for diagnosing bacterial meningitis. Using automated hematology analyzer for enumeration of leukocyte in CSF is below the sensitivity, so microscopic hemocytometric method is standard method. But this requires sufficient practical experience and has limitation of accuracy and stability. So we developed new manual method and evaluated it. METHODS: We designed new method using transparent ruler tape. We performed correlation, accuracy and precision test by counting leukocyte in diluted EDTA blood with three methods: new method, Neubauer and Nageotte hemocytometry. Twenty two CSF were used for stability test, which determines leukocyte count according to time (within one hour and after 2, 4 and 12 hr), by new method and Neubauer hemocytometry at room temperature. RESULTS: There was no clinical significant difference between three methods in correlation test, whereas Neubauer and Nageotte hemocytometry showed a bias to underestimation relative to the results obtained with new method in case with low leukocyte count. The new method showed the lowest CV and most accurate result. In stability test, leukocyte counts decreased being 44.4%, 72.1% of initial values after 2 hr, 14.8%, 31.1%, after 4 hr and 4.2%, 8.7%, after 12 hr, by Nageotte hemocytometry and new method, respectively. CONCLUSIONS: The new method we devised is simple, easy and applicable to use in a laboratory and offers advantages of improved precision and stability. It may be sufficient for replacing standard methods for leukocyte counting in CSF.
Cerebrospinal Fluid/*cytology ; Female ; Humans ; Leukocyte Count/instrumentation/*methods ; Male ; Regression Analysis ; Reproducibility of Results ; Sensitivity and Specificity ; Time Factors

It is very important to probe into the axonal regeneration and functional recovery of central nervous system (CNS) after implantation of cells into cerebrospinal fluid (CSF) for spinal cord injury (SCI). Transplantation of cells via CSF poses great potentials for SCI in clinic. Studies on administration of cells via CSF indicate that the method is safe and convenient. The method is more suitable to treating multiple lesions of the CNS since it does not produce open lesions. However, there are disputes over its promotion effects on axonal regeneration and functional recovery of spinal cord after injury; and some questions, such as the mechanisms of functional recovery of spinal cord, the proper time window of cell transplantation, and cell types of transplantation, still need to be handled. This review summarized the method of cell transplantation via CSF for treatment of SCI.
Animals ; Cell Transplantation ; methods ; Cerebrospinal Fluid ; cytology ; physiology ; Humans ; Nerve Regeneration ; physiology ; Spinal Cord Injuries ; pathology ; surgery

The present study aimed to explore the effects of 5-HT(1A) receptors in the distal cerebral spinal fluid-contacting neurons (CSF-CNs) in rat brain parenchyma in neuropathic pain. The model of neuropathic pain with chronic constriction injury (CCI) of the sciatic nerve was made in Sprague-Dawley rats. The behavioral studies of animal were scored and the paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were measured. The distribution and expression of 5-HT(1A) receptors were observed in the distal CSF-CNs in brain parenchyma with double labeling of cholera toxin subunit B with horseradish peroxidase (CB-HRP) and 5-HT(1A) receptors with immunhistochemistry. The relationship between 5-HT(1A) receptors in distal CSF-CNs and neuropathic pain was analyzed. The results were as follows. On days 1, 3, 7, 14 of neuropathic pain, the PWL was 19.37±2.74, 12.04±1.77, 8.74±1.15 and 12.31±1.94, respectively; the PWT was 18.58±3.62, 13.05±1.81, 6.66±1.43 and 11.55±2.01, respectively. CB-HRP-labeled neurons of two clusters were always found in definite region but not in other area in brain parenchyma. The number of neurons double-labeled with CB-HRP/5-HT(1A) receptors in each group was 276.14±36.00, 161.72±28.41, 108.64±6.81, and 139.76±44.64, which was about 95%, 60%, 40% and 55% of all CB-HRP-labeled neurons in the four courses of neuropathic pain, respectively. It is suggested that the distal CSF-CNs are always located in a special region in rat brain parenchyma and most of them have 5-HT(1A) receptors. A negative correlation is found between the expression of 5-HT(1A) receptors and neuropathic pain.
Animals ; Brain ; cytology ; Cerebrospinal Fluid ; Neuralgia ; physiopathology ; Neurons ; physiology ; Rats ; Rats, Sprague-Dawley ; Receptor, Serotonin, 5-HT1A ; physiology

BACKGROUND: The clinical usefulness of flow cytometry (FCM) for the diagnosis of leptomeningeal diseases (LMD) in non-Hodgkin lymphomas has been suggested in previous studies but needs to be further validated. With this regards, we evaluated the use of FCM for LMD in a series of Korean patients with non-Hodgkin lymphoma. METHODS: FCM and cytomorphology were conducted using samples obtained from clinically suspected LMD patients, follow-up LMD patients, and those with high risk of developing tumorigenic diseases. We then compared results of FCM and cytomorphology. In total, 55 and 47 CSF samples were analyzed by FCM and cytomorphology, respectively. RESULTS: Of the samples analyzed, 25.5% (14/55) and 12.8% (6/47) were positive by FCM and cytomorphology, respectively. No samples were determined as negative by FCM but positive by cytomorphology. Seven patients were positive only by FCM and negative by cytomorphology, and six among them were clinically confirmed to have LMD either by follow-up cytomorphology or imaging study. CONCLUSIONS: We observed a high detection rate of tumor cells by FCM compared with cytomorphology. FCM study can be useful in early sensitive detection of LMD.
Adult ; Aged ; Female ; Flow Cytometry ; Glucose/cerebrospinal fluid ; Humans ; Leukocytes/cytology ; Lymphoma, Large B-Cell, Diffuse/diagnosis/mortality ; Lymphoma, Non-Hodgkin/*complications ; Male ; Meningeal Neoplasms/cerebrospinal fluid/complications/*diagnosis ; Middle Aged ; Prognosis ; Retrospective Studies ; Survival Rate

Adolescent ; C-Reactive Protein ; metabolism ; Cell Aggregation ; Child ; Child, Preschool ; Cytodiagnosis ; methods ; Female ; Humans ; Infant ; Leukocytes ; cytology ; Male ; Meningitis, Bacterial ; blood ; cerebrospinal fluid ; diagnosis ; Meningitis, Viral ; blood ; cerebrospinal fluid ; diagnosis ; Sensitivity and Specificity