Research On Expression Of AQP4 In Early Human Traumatic Brain Edema

ObjectAfter acute craniocerebral injury, cerebral edema with different degrees will happen. The degree of edema and lasting time is closely related to the occurrence and development of delayed craniocerebral injury. Recently, two experiments directly testified that AQP4 participate in the formation of brain edema. Manley showed in the research of the model of acute overhydration cerebral edema that AQP4 expresses high in the brain tissue of edema model, and low in the normal rat brain; in the model of ischemia cerebral edema, compared with normal group, the application of gene knockout technology makes AQP4 lack expression in brain tissue. The result found out that big rats lacking AQP4 have strong viability. 24 hours after lasting cerebral arteries emphraxis ischemia, measuring cerebral hemisphere edema percentage reduced 35%, with clinical result obviously improved, indicating that AQP4 participates in forming cerebral edema and plays an important role in cerebral edema.Aquaporins (AQPs) are, found at the beginning of 1990s, a class of competence specific proteins with highly selectivity water channel existing in the membrane of biological cells, playing an important role in the adjustment of water metabolism. In 1994, the homology of aquaporins family was used to clone and separate a cerebral aquaporin -AQP4, whose basic structure is the same as that of other aquaporins, hydrophobic proteins with about 30kD molecular weight. Each monomer is made up of 6 transmembrane a-helices arranged in a right-handed bundle, with the amino and the carboxyl termini located on the cytoplasmic surface of the membrane. There are 5 interhelical loop regions (A - E) that form the extracellular and cytoplasmic vestibules. Loops B and E are hydrophobic loops which contain the highly, although not completely conserved Asn-Pro-Ala (NPA) motif.Researches show that the 6 transmembrane regions circle Loops B and E, forming a 3-D 'hourglass' structure where the water flows through. Immunohistochemistry found that the content of AQP4 protein is the highest in brain, distributing on the surface of cerebral pia mater, aqueduct, ependema and choroid plexus of cerebral ventricle system, consistent to the csf reabsorption position. Csf can reabsorb through AQP4 of other cells. AQP4 are highly expressed on astrocyte, especially those close to subarachnoid space, located around blood vessel and on the glial limiting membrane formed by closely wrapping capillary wall, as this membrane constitutes the second septum of blood-brain barrier, playing the role of adjusting water transportation; supraoptic nucleus, paraventricular nucleus and thirsty center of hypothalamus. The stretch sensitivity ion channel of the neurosecretory cell of supraoptic nucleus and the paraventricular nucleus is very sensitive to capacity changes such as cell expansion with reaction happening upon 1% change of osmotic pressure. Through the synthesis and release of AVP, adjust the renal excretion of water. It is inferred that AQP4 may be osmotic receptors or receptor, adjusting osmotic pressure at hypothalamus through intensifying the quick and sensitive cell capacity change in the subtle change of osmotic pressure. The hippocampal pyramidal cell layer of the dentate gyrus granule cell layer and multi-layer cell; the function of AQP4 distributing at the hippocampus is not clear at present.The distributing feature of AQP4 is important structure basis for the water adjustment and transportation between gliocyte, csf, and vessels, closely related to physiological and pathological process such as csf absorption, osmoregulation and cerebral edematization. But people are not clear about the specific physiological and pathological mechanism of cerebral edema caused after craniocerebral injury at present. About the role AQP4 plays in the traumatic cerebral edema, the conclusions are various with research methods and observation time different. What's more, the research targets of the most experiments are animals, rare research done on human beings in pathological state. Some researches think that AQP4 and its increase cause the formation of traumatic cerebral edema.Sun found that 24 hours after the craniocerebral injury of a rat caused by free falling body, the degree of the cerebral edema at the traumatic area is prominent. The expression of AQP4 obviously increases on the swollen astrocyte of the traumatic area, and decreases at neighboring areas, with no prominent change afar. It is therefore believed that the increase of AQP4 may be the main channel and reason causing cerebral edema at traumatic area. Some scholars, however, think that the increase or decrease of AQP4 expression is reactions made by astrocyte to adapt to the environmental change outside the cell, being an endogenous protection mechanism.Kiening etc. observed that the most serious edema happens 24 hours after the rat's brain trauma with gas percussion injury method. And the expression of AQP4mRNa reduces as the time prolongs, which is more prominent at the traumatic hemisphere. Therefore cellularity edema dominates at this stage. The reduction of AQP4 mRNA may reduce the further accumulation of water in the astrocyte.This experiment employs transmission electron microscopy to conduct ultrastructure observation on the cerebral tissue of human after edema caused by acute craniocerebral injury. With the immunohistochemistry method, the expression situation of AQP4 in the cerebral tissue of human is studied, with more direct understanding on the roles AQP4 and blood-brain barrier play in the occurrence of cerebral edema, providing theoretical basis for developing blocker for cerebral edematization and finding potential targeting therapy for cerebral edema, which has important clinical value for reducing death rate and disability rate.1. Source of Experimental Samples28 samples of traumatic cerebral tissue are drawn from Wuhan General Hospital of Guangzhou Military Area from March - September, 2006, when the brains of the patients with acute craniocerebral injury were opened for cleaning hematoma. Their injured cerebral tissues were obtained 2～24 hours before the attack. 18 males, 10 females, ages ranking from 15～60 years old, average age 35.3 years old, GSC scoring 3-10 scores, head CT indicates partial cerebral contusion hemorrhage of frontal lobe and emporal lobe, with prominent midline structure shifting. As normal cerebral tissue is hard to get, 8 relatively normal cerebral tissues obtained from idiopathic epilepsy patients are taken as comparison, with 5 males, 3 females, average age 23 years old.2. Apply routine HE staining and transmission electron microscopy to conduct ultrastructure observation on the 28 cerebral tissue of human after edema caused by acute craniocerebral injury. With the immunohistochemistry method, the expression sit ation of AQP4 in the cerebral tissue of human is studied3. Apply SPSS 10.0 software to analyze. Take average grey value representing AQP4 protein content on each immunohistochemistry staining section. The higher the grey value is, the lower content of this substance is, vice versa. The data are expressed with mean value +standard deviation (x±s), and two independent sample mean values t of SPSS are used for analysis. P＜0.01 indicates existence of significant difference.Result1. HE staining of cerebral tissue around traumatic area: seen through optical microscope, the cerebral tissue was loose, the gap around cells and vessels became bigger, the thickness of vessels was uneven, some vessels were cut or damaged, extensive bleeding in tissues.2. Transmission electron microscopy observation: swollen endothelial cells of capillary vessels of cerebral tissue around traumatic area, swollen mitochondrion of cells in the vessel, narrow canal, obvious blood-brain barrier damage, bubble-like changes around vessels, obvious swollen cells of cerebral tissue, bubble-like change of cytoplasm, enlargement of perinuclear endoplasmic reticulum, swollen mitochondrion, nucleus pyknic, chromatin in nucleus partially dissolved, electron density increased.3. Immunohistochemistry result shows: in normal cerebral tissue AQP4 is little expressed, while its expression rose obviously after the acute craniocerebral injury, (P＜0.001) mainly expressed in small vessels and glial cells.Conclusion:1. Damage of blood-brain barrier of cerebral tissue around traumatic area of the patient with acute craniocerebral injury is the morphology basis for the occurrence of cerebral edema.2. This experiment took human as research targets, with the restriction of factors such as ethnics, source of the experiment coming from traumatic cerebral tissues of frontal lobe and emporal lobe. Our research indicated that AQP4 proteins are mainly expressed in astrocyte and endothelial cells of microvessels, especially gathering in cytoplasm and plasmalemma of gliocyte directly touched by cerebral pia mater and capillary vessels. The result is fundamentally consistent to that of foreign scholars.3. Shortly after the occurrence of acute craniocerebral injury of human, the expression of AQP4 in cerebral tissues obviously increases.