The Preliminary Study Of The Dynamic Change Of The Cerebrospinal Fluid Proteins Of Patients With Acute Traumatic Brain Injury Using 2-dimensional Electrophoresis

Completion of the"Human Genome Project"has stimulated scientists to begin looking for the next step in unraveling normal and abnormal functions within biological systems. Consequently, there is new focus on the role of proteins in these processes. Proteomics is a burgeoning field that may provide a valuable approach to evaluate the post-traumatic central nervous system. 2-dimensional polyacrylamide electrophoresis is a dominant method of separating proteins in the study of proteomics. Like other central nervous system pathologies, TBI changes the composition of cerebrospinal fluid (CSF), which may represent a unique clinical window to study the pathophysiology of brain injury.Materials and methods:Six cerebrospinal fluid samples were collected from 3 patients with acute traumatic brain injury. The samples were obtained by lumbar puncture on the first and the 10th post-traumatic day respectively from each patient. In sample preparation, overdrive centrifugation and precipitation with acetone were applied. The method of Bradford was used to determine the final protein concentration. Immobilized gradient strips of 13cm in length and 3-10 in pH range were chosen in the first phase of isoelectric focusing(IEF). The program was set as follows: phase 1: 300V, 1min 0.001 kVh;phase 2: 3500V, 1h and 30min, 2.9 kVh; phase 3: 3500V,3h and 10min-4h, 11.1-14.1 kVh. Total: 4h and 40min-5h and 30min, 14-17 kVh. During the sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE), 12% concentration polyacrylamide was used and the program was set according to the standard below: step1: 15 mA/gel, 15min; step 2: 20 mA/gel, 5h. Silver nitrate was employed to stain the proteins. The map was scanned and then analyzed using the Image Master 2D-Elite software.Results:6 different 2D-PAGE gel maps were harvested. Approximately 258 spots were detected in gel map A1, and 87,271,256,263,242 in gel map A2, B1, B2, C1, C2 respectively. 85 spots in map A2 was found matched with map A1, 80 spots down-regulated in volume by >50%, and no new spots showed up. 240 spots was detected in map B2 matched with map B1, 20 spots down-regulated, 6 spots up-regulated in volume by >50% and 6 new spots appeared. 221 spots in map C2 matched with map C1, 18 spots down-regulated, 4 spots up-regulated in abundance by >50% and 5 new spots were identification. Spot a and b were both found in the gel maps of B2 and C2 .Conclusion:1. Map B1, B2 andC1, C2 show relatively good reproducibility when 2-D PAGE was applied to study the dynamic change of the cerebrospinal fluid proteins of patients with traumatic brain injury.2. At different post-traumatic time, the composition and contentof CSF proteins showed dynamic characterizations. Some decreased while some increased significantly in abundance, and some new proteins just appeared.3. After literature review, it is hypothesized that the proteins down-regulated significantly might be plasma proteins, contaminating the CSF following subarachnoid hemorrhage, or acute phase reactants which degraded and were eliminated as time progressed. The up-regulated and new proteins are likely to be involved in the pathological process of brain tissue inflammatory damage or cell apoptosis,and should be treated as spots of special interest, especially for spots a and b. Further analysis, using mass spectrometry, is needed to characterize their structure and determine their possible functions which would be potentially helpful in investigating the underlying molecular events that exacerbate TBI.4. Due to experimental error and lack of laboratory experience, high resolution of the 2D-PAGE gels was not achieved, especially for map A2. The reproducibility between samples from different patients was neither good, probably because the different degree of contamination by blood and different severity of brain injury.5. Changes of spots in number and intensity on the gels don't necessarily correspond to that for proteins. Consequently, the following mass spectrometry of the spots of interest is indispensable for characterization of the proteins. Furthermore, the way in which proteins change is more complicated than what the gel can tell.There is a long way ahead.