| Traumatic brain injury (TBI) is one of the most emergency issues in neurological surgery. Unrespectable pathological consequences are always found following traumatic contusion with a high morbidity and mortality. Survivors suffer mental problems and even permanent disabilities. As the fast development of neuroscience in recent 30 years, the mortality rate of traumatic brain injury decreases from 50% to 30% by now. Questions raise that how to promote the CNS functions of post traumatic patients from basic research and clinical prevention still remains a challenge.Studies have shown that the brain tissue damage resulted from both direct and indirect mechanical injury and secondary brain impairment. During the process of secondary brain impairment, a series of events of cells and molecules are related and cooperated and eventually leads to nerve cell death. Consequently, the brain structure and function also damaged. Therefore, researches on the mechanisms of secondary cell death become significance in uncovering the routine for the recovery of neuron cells function, and this will provide a potential therapeutic target for traumatic brain injury.Mechanisms of secondary brain injury are as follows:1) Mechanical damage caused by local tissue hypoxia2) Toxicity of excitatory amino acid3) Free radical damage4) Intracellular Ca2+ overloadAmong the TBI induced endogenous damaging factors, Ca2+ is the most important one which leads to the secondary injury through its occurrence and development. Furthermore, Ca2+ plays a key role in several signal pathways which leading to cell death. For example, secondary brain edema is considered as a final pathology phenomenon of variety of brain diseases, and the pathogenesis of which is the water transporting and balance disorders led by the blood barrier damage, cerebral anoxia, intracranial venous hypertension and other factors post brain injury. During the pathogenesis of brain edema, the distribution of water and electrolytes inside and outside the brain cells lost balance, resulting in increased brain volume and intracellular hypertension syndrome. Studies have demonstrated that the increased permeability of blood-brain barrier, cell membrane calcium channel, and calcium overload plays a key role in the development of traumatic brain edema. Under pathological condition of brain trauma, intracellular Ca2+ increases sharply to 200 times than that of normal cells. Increased Ca2+ can activate membrane phospholipid A2 and phospholipid C, and furthermore excite the polyunsaturated fatty acids. This eventually leads to the apoptosis and death of neurons.As an important intracellular messenger involved in neurotransmitter synthesis and release, neuronal calcium plays a key role in maintaining nerve excitability, synaptic plasticity and enzyme activates in the regulation of many cell functions.Studies have shown that neuronal calcium is also involved in ischemia, hypoxia and the toxic cell death and other pathological processes. This messenger function process of calcium is accomplished by changing the calcium ion concentration inside and outside the cytoplasm. The numbers of already known internal and external mechanisms that cell rely on to maintain the intracellular Ca2+ homeostasis is six.They are as follows:1) Cell membrane has a very low Ca2+ permeability, and this allows the extra-cellular Ca+ come into the cell following the ion gradient easily.2) The intracellular Ca2+ can be pumped out by the calcium pump on the membrane which activates by couples to ATP hydrolysis.3) The membrane sodium/calcium exchange system can exchange the intracellular ions by transmitting intracellular Na+ inside and releasing the Ca2+ out.4) The Ca2+ isolating function of mitochondria and endoplasmic reticulum.5) Some proteins and anions in the cytoplasm can bind Ca2+.6) The abilities of inner plasma membrane phospholipids polar end in binding of Ca2+.In summery, the calcium overloading process is consisted of a complex mechanism which various the intracellular Ca2+ ions.Clinical and experimental researches have shown that the calcium overload process also exists in traumatic brain injury. The variation of intracellular Ca2+ after brain injury can be detected by using a 45Ca immune autometallography technique, studies have demonstrated the time courses of calcium content in brain tissue suffered TBI and found that the calcium content of 2h injury animal brains is significantly increased, which peaked at 24h, lasting for 48h, then gradually declined. By using an electron microscope cytochemistry method, Huang et al. found a swelling phenomenon in capillaries and ultrastructure of nerve cells in a cerebral hemorrhage model induced by injection of collagenase. The swelling pathology changes can then be rescued by calcium antagonists. The other researches on DAI models found that there are a lot of harmful tiny calcium particles correlating with the degree of injury in injured myelin and these calcium particles become huge and tough in late axonal injury. He et al. considered that the existence of calcium overload in the injured axon of DAI is an important factor in the development of DAI. Reports comes from Zhou et al. indicate that Mg2+ can inhibit the excitatory role of amino acids in reducing the Ca2+ influx, presenting a significant protection to injured brain. Therefore, we can draw a preliminary conclusion that changes in intracellular Ca2+ is the final pathway of cell death, and in which the secondary cell damage happens. But no further studies showed the mechanisms of Ca2+ overload post traumatic brain injury in detail. As commonly known that Ca2+ overload correlates with the extracelluar Ca2+ flowing into cells through the damaged VDOCC and ROCC post TBI. In addition, ischemia and hypoxia, reduction of ATP, inactivate of calcium pump, mitochondria and endoplasmic reticulum secondarily to brain injury are factors leading to the calcium accumulation. In intracytoplasm, Ca2+ binds a special effector protein calmodulin (CaM) which then combines with the specific enzyme, resulting in microtubules and neuronal skeleton damage, hyperphosphorylation of proteins on presynaptic and postsynaptic membrane, disabilities of cellular energy metabolism.Researches on calcium metabolism have been stated numerously, among which some studies on Wnt5a/Frizzled-2 signal pathway in Xenopus and zebrafish embryos draws our attention. Related studies suggest that the Wnt gene family encodes a group of secreted glycoproteins implicated as ligands in cell signaling in both vertebrates and invertebrates. Wnts are involved in diverse developmental processes, including cell differentiation, cell migration, cell polarity, and cell proliferation. Wnt protein combines with the corresponding receptors of the Frizzled family to activate different signaling pathways within the cytoplasm. There are two primary kinds of Wnt related signal pathways:the canonical Wnt pathway (Wnt/p-catenin signal pathway) and the non-canonical Wnt pathway (Wnt/Ca2+ pathway, or Wnt5a/Frizzled-2 signal pathway). Wnts, including Wntl, Wnt3a, and Wnt8, can activate the canonical Wnt pathway. Comparatively, Wnt5a transduces binding of Frizzled-2 to induce the intracellular calcium release. Along with the continuous researches on Wnt/Ca2+pathway, the down stream signaling are found. Studies by Kuhl and Sheldahl proved that Frizzled-2 mediated Wnt5a/Ca2+pathway can activate two known calcium-regulated enzymes including calcium-calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ), which is activated in term of autophosphorylated on Thr286 (p-CaMK Ⅱ), and protein kinase C (PKC). Comparatively, the Frizzled-1 mediated Wnt8/β-catenin pathway can not activate CaMK Ⅱ and PKC. Therefore, researchers considered p-CaMKⅡ to be the marker protein of activated Wnt/Ca2+ signal pathway.Objective:Researches on Xenopus and zebrafish embryos have provided the first evidence thaf Wnt5a transduces by binding of Frizzled-2 receptor, leading to the intracellular Ca2+ release. Intracellular Ca2+ variation frequency was found two times higher when Wnt5a mRNA was injected into the embryos, this phenomenon indicates that the intracellular Ca5+ must be the secondary messenger of Wnt signal pathway. And it is clear that ectopic expression in zebrafish embryos of rat Frizzled-2 causes an increase in the release of intracellular calcium which is augmented by co-expression of Wnt5a. All these experiments about Wnt5a/Frizzled-2 signal pathway are carried out mainly on lower animals like Xenopus and Zebrafish, but how the existence of Wnt5a/Frizzled-2 pathway function in mammalian nerve cells, how it activates and whether it can influence the intracellular Ca2+ while activated are poorly known.Under normal physiological conditions, calcium is an important secondary messenger of nervous system, regulates diverse cellular functions and maintains the normal cell homeostasis. Calcium is an important media in exchanging information with outside environment. But under pathological conditions, like TBI, the cellular homeostasis is broken. Subsequently, intracellular Ca2+ accumulates, and then Ca2+ overload happens until cell death. In summery to the theories above, we hypothesis that there must be a relation with Wnt5a/Frizzled-2 pathway and Ca2+ overload post TBI, and this signal pathway plays a role in the process of Ca2+ overload after TBI.Methods and ResultsOur research was carried out through two parts:in vivo and in vitro. We demonstrated that the existence of Wnt5a/Frizzled-2 signal pathway in cultured rat hippocampal astrocytes, and when activated post TBI, it played an important role during calcium overload.In in vitro study, first we cultured neonatal rat hippocampal astrocytes, and detected the expression of Wnt5a and Frizzled-2 in normal cells by using QRT-PCR and Western blot. In our results, certain gene and protein levels of Wnt5a and Frizzled-2 were detected expressing in cultured neonatal hippocampal astrocytes. This indicates the possible existence of Wnt5a/Frizzled-2 pathway in nerve cells, at least in astrocytes. Fluorescent Ca2+ was also detected by confocal microscopy when the cells were post-incubated with fluo-3/AM, suggesting that Ca2+ inflow occurred in non-transfected cultured hippocampal astrocytes. Next, to examine the roles of Frizzled-2 in Wnt5a signaling and calcium inflow, we transfected rat neonatal hippocampal astrocytes with synthesized frizzled-2 plasmid and then analyzed the expression of Frizzle-2 and Wnt5a collected from Q-PCR and Western blot. When the cells were transfected, we observed that gene expressions of both Wnt5a and frizzled-2 were significantly increased approximately 4-fold in comparison to that in non-transfected control cells (P<0.01). Moreover, protein expressions of both Frizzled-2 and Wnt5a were significantly increased approximately 2-and 3-fold, respectively, when compared with that in non-transfected control cells (P< 0.01).In addition, it is known that phosphorylation of CaMK Ⅱ indicates the action of the Wnt5a/Frizzled-2 signaling pathway, and we found that p-CaMK Ⅱ was also significantly increased 3-fold after the transfection (P< 0.01), compared to non-transfected cells.To identify whether activation of the Wnt5a/Frizzled-2 pathway regulates Ca2+ release from intracellular stores, we examined whether changes in intracellular Ca+ inflow was affected by frizzled-2-transfection, indicated by the intensity of fluorescence of fluo-3/AM under confocal microscope after transfection. We found that the intensity was significantly increased 1.75-fold after the transfection (P< 0.05) when compared to the control group. This indicated that intracellular Ca2+inflow was increased in frizzled-2-transfected hippocampal astrocytes.As described above, we showed that the expression of Wnt5a was closely associated with the expression of the Frizzled-2 receptor, and that cells transfected with frizzled-2 plasmid dramatically increased intracellular Ca2+ inflow. Nevertheless, we could not draw a conclusion that Frizzled-2 transduces binding of Wnt5a and leads to the intracellular Ca2+ release. To resolve this problem, a special inhibitor called stealth RNAi was used to block the expression of the frizzled-2 gene. Compared to the treatment without Stealth RNAi, we found that the treatment with Stealth RNAi significantly inhibited both gene and protein expression of Frizzled-2 in transfected hippocampal astrocytes nearly to the control level, as demonstrated by Q-PCR and Western blot (P< 0.01). Moreover, the expressions of the Wnt5a gene and protein were also markedly down-regulated 2-and 4-fold, respectively, in transfected astrocytes following the suppression of the frizzled-2 gene by the treatment of Stealth RNAi (P<0.01), compared to that without this treatment.To examine whether the expression of p-CaMKII was blocked by the inhibition of frizzled-2 signaling, we detected its expression by Western blot after application of Stealth RNAi. We found that the treatment of stealth RNAi significantly inhibited the expression of p-caMK II by 1.5-fold in frizzled-2 transfected hippocampal astrocytes, indicating that the activation of the Wnt5a/Frizzled-2 pathway was suppressed by blocking frizzled-2.To examine whether intracellular Ca2+ inflow was affected by blocking frizzled-2 signaling, we measured the intensity of fluorescence of fluo-3/AM under confocal microscope after application of stealth RNAi. We found that stealth RNAi dramatically decreased the intensity of fluorescence 1.55-fold in frizzled-2 transfected hippocampal astrocytes, compared to that without the treatment. Taken together, we can draw a conclusion that the Wnt5a/Frizzled-2 pathway exists in rat astrocytes and activates Ca2+ release from intracellular stores. Furthermore, Ca2+ overload is known to be an important mechanism underlying TBI, to confirm the significance of inhibition of Wnt5a/Frizzled-2 pathway to attenuate intracellular Ca2+accumulation, we therefore made further tests in a rat model of traumatic brain injury (TBI). Firstly, we found that Wnt5a and Frizzled-2 were both stable expressed in normal hippocampi. However, after TBI, gene expressions of Wnt5a and frizzled-2 were significantly elevated in the ipsilateral hippocampus, when compared with that in normal hippocampus (P<0.01). Protein expressions of Frizzled-2 and Wnt5a were also elevated by 2-and 5-fold in the ipsilateral hippocampus, respectively, when compared to that in normal hippocampus (P< 0.01).While p-CaMKII was expressed at a very low level in normal hippocampus, however, its expression was markedly increased approximately 4-fold in the ipsilateral hippocampus after TBI (P<0.01;). This indicates that Wnt5a/Frizzled-2 signaling pathway was activated after TBI.Next, we detected the intensity of fluorescence of fluo-3/AM in the cells isolated from injured hippocampus. We found that the intensity of fluorescence was much stronger in the cells isolated from the injured hippocampus of rats with TBI than in normal hippocampal cells. The intensity was significantly increased 1.83-fold after TB.Since the Wnt5a/Frizzled-2 signaling pathway was activated after TBI and TBI can strongly increases intracellular Ca2+ inflow in injured hippocampus, these results may suggest that activation of Wnt5a/Frizzled-2 signaling pathway contributes to the increasing intracellular Ca2+ inflow post TBI.To determine the interaction between TBI-induced activation of Wnt5a/Frizzled-2 signaling and accumulation of intracellular Ca2+, we suppressed the expression of the frizzled-2 receptor gene by hippocampal injection of Stealth RNAi, and detected the effects of Stealth RNAi on gene and protein expression of Wnt5a/Frizzled-2 and intracellular Ca2+inflow after TBI. When the Stealth RNAi was applied to rat hippocampus 48 h prior to TBI through stereotactic injection together with invivofectamine, we observed a significant inhibitory effect on gene expression of frizzled-2 in the injured hippocampus, compared with that in the rats without the injection of stealth RNAi and invivofectamine, as demonstrated by the fact that gene expression of frizzled-2 was inhibited nearly 3-fold. Surprisingly, the expression of the Wnt5a gene was also down-regulated nearly 2.5-fold (P< 0.01). In addition, after injection of Stealth RNAi and invivofectamine, protein expressions of Frizzled-2 and Wnt5a in injured hippocampi was inhibited by nearly 3.5-fold and 50%, respectively, when compared to rats without the injection. Moreover, p-CaMKII in injured hippocampi was also inhibited nearly 50%, reaching up to the control level. Similarly, the intensity of Ca2+ fluorescence was significantly decreased 1.55-fold in injured hippocampal cells after the injection, when compared to those without the injection.ConclusionWe demonstrated the expression of Wnt5a/Frizzled-2 pathway in both neonatal astrocytes and adult hippocampal cells in vitro and in vivo, respectively. Moreover, TBI induced intracellular Ca2+ overload, the process of which was partly effected by the activation of Frizzled-2 mediated Wnt/Ca2+signaling pathway. These findings will provide an understanding of the mechanisms underlying the Wnt5a/Frizzled-2 signaling pathway via activation of calcium in the central nervous system under physiological and pathological conditions, and the specifically expressed component of this signal pathway like Wnt5a, Frizzled-2 and p-CaMKII post trauma would considered to be the potential research target for the further study of TBI. |
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