FK506 Prevents Cognitive Impairment And Brain Focal Edema Associated With Astrocyte Expression By Inhibiting Calcineurin

Epilepsy is one of the most common neurological disorders that affect people of all ages worldwide. There are about 9 million patients in china according epidemiologic survey. About 25% to 30% of patients with epilepsy were diagnosed as refractory seizure, which threats the patients health and life quality obviously, at the same time there is a higher disability and mortality. Status epilepticus (SE) is a medical emergency, generally refers to the occurrence of a single unremitting seizure with a duration longer than 5 minutes or frequent clinical seizures without an interictal return to the baseline clinical state. The pilocarpine-induced seizures have been one of the most frequently used models to research SE and temporal lobe epilepsy(TLE), because the alteration of behavior, electroencephalogram (EEG) and the hippocampal neuronal injury in pilocarpine-induced seizures in rats is similar to that in TLE patients.Status epilepticus causes a number of physiological and neurochemical changes such as brain focal edema, astrocyte activation and neuronal necrosis. Notably, calcium and calcium-regulated systems play an important role in the activity. Of these, one particular interest is calcineurin (CaN), a calcium-stimulated phosphatase.Seizures can result in increase in Ca2+ release and Calmodulin (CaN) activity, cause a number of physiological and neurochemical changes, leading to mitochondrial dysfunction,cognitive impairment, astrocyte activation and neuronal necrosis,which is ralated to hippocampus stiffen in temporal lobe. The changes may lead to permanent neurological impairments and susceptibility to spontaneous seizures.CaN is a member of the serine/threonine protein phosphatase family enriched in neural tissue. It has been found that CaN mediates many cellular processes CaN-mediated dephosphorylation is an important modulatory factor in many cellular processes, including development of learning and memory, regulation of long-term potentiation (LTP), neurotransmitter release and induction of apoptosis. It has been found that SE induced CaN activity and CaN concentration increase and CaN further participated in a series of pathological process after SE, including activation N-methyl D-aspartate (NMDA) and amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. CaN further participated in a series of pathological process after SE including the regulation of gene transcription, cytoskeleton protein synthesis, calcium protease, the bcI-2 death protein, many apoptosis related proteins and other molecules.CaN mediated dephosphorylation is an important process in the regulation of the synthesis of nitric oxide and oxidative stress development. It has been found that CaN is also rich in astrocytes in response to brain injury, aging, and^myloidosis, upregulation of CaN protein appears to initiate the activation of astrocyte and the induction of numerous immune/inflammatory signaling molecules.FK506 has been used in clinical settings for the prevention of allograft rejection as an immunosuppressive agent, which orchestrates its immunosuppressive action by binding to FK506 binding protein 12 (FKBP12). Recent studies have demonstrated that FK506 plays a role in neuroprotection and neurotrophy. In middle cerebral artery occlusion model, cortical damage was remarkably reduced by intravenous injection of FK506. FK506 was shown to protect neuron through preventing the sprouting of mossy fibres in the hippocampus. Despite the protective effect of FK506 was observed in experimental cerebral ischemia and seizure model, the mechanism remains unclear. The immunosuppressant rapamycin, which could also bind to FKBP12, has no effect of CaN inhibitor.Our current study was designed to establish lithium chloride-pilocarpin-induced SE (LPCSE) models to investigate the alteration of behavior, evaluate the ability of spatial learning and memory, brain focal edema, calcineurin and astrocyte expression. The findings have implications for potential clinical use of FK506 against SE.Part I Study of behavior and pathology in lithium-pilocarpine induced seizures in rats, and the effect of FK506 on improving ability of learning and memoryObjectiveTo investigate the alteration of behavior and pathology in lithium-pilocarpine-induced seizures in rats, and the effect of FK506 on improving ability of learning and memory, to explore the mechanisms of FK506.Method1. Pilocarpine was intraperitoneally injected to make modles of SE. The rats were randomly divided into 3 groups:control group, pilocarpine group (pilo group) and FK506 group.2. The seizure activity of rats induced by pilocarpine was scored using a modified Racine scale and the effect of FK506 on seizure behavior.3. To evaluate the ability of spatial learning and memory in a Morris water maze after SE and the effect of FK506 on cognitive impairment.4. To evaluate pathology features of hippocampal neurons of each group by H&E staining with hematoxylin and eosin at 12d after SE.Result1. Behavioral episodes induced by pilocarpine injections showed typical increases in their intensity and duration, gradually progressing towards status epilepticus. The rats treated with FK506 showed highly significant differences in their latency period reaching to stage IV-V and the profile of proportions of rats suffering from seizures of different intensities. The latency period of the group with FK506 was significant longer and the percentage of animals reaching stage V was considerably lower compared with pilo group. Control animals did not exhibit any behavioral seizure activity.2. Morris water maze showed the cognitive impairment of pilo group compared with control. The escape latency was significantly prolonged in pilo group compared with control. Compared with control group, the swimming distance in the target zone was significantly shorter in pilo group. In contrast to pilo group, the prolonged latency was significantly reduced by FK506 and longer swimming distance in the target zone in FK506 group was seen compared with pilo group. The time spent in the target zone and the crossings of the former platform position were more in FK506 group compared with pilo group. We demonstrated that FK506 improved the learning and memory of rats.3. Pilocarpine injection intraperitoneally resulted in a loss of neuron in pyramidal cells of hippocampus in CA3 region. It has been observed in some neurons that cytoplasm appears heavy staining and nucleolus disappears with pyknotic nucleus. The surviving neuron numbers after SE were significantly decreased compared with that of control. FK506 significantly attenuated the neuron loss induced by seizures. Conclusion1. Pilocarpine intraperitoneally injected can successfully make modle of of SE, which is safe, convenient and feasible. It has been one of the most frequently used models to research SE and TLE.2. Behavioral episodes induced by pilocarpine injections showed typical increases in their intensity and duration, gradually progressing towards status epilepticus. The latency period of the group with FK506 was significant longer and the percentage of animals reaching stage V was considerably lower compared with pilo group. It suggests that FK506 could inhibit status epilepticus induced by pilocarpine.3. Morris water maze showed the cognitive impairment of pilo group compared with control. FK506 improved the learning and memory of rats.4. Pilocarpine injection intraperitoneally resulted in a loss of neuron in pyramidal cells of hippocampus in CA3 region. FK506 significantly attenuated the neuron loss induced by seizures. It suggests that FK506 plays a role in protecting hippocampal neurons induced by pilocarpine.Part II FK506 prevents brain focal edema and astrocyte activation by inhibiting calcineurinObjectiveTo investigate the alteration of behavior and pathology in lithium-pilocarpine-induced seizures in mice, and to observe the brain focal edema, CaN and GFAP expression and the effect of FK506 after SE,to explore the mechanisms of FK506Method1.Pilocarpine was intraperitoneally injected to make modles of SE. The mice were randomly divided into four groups:the control group, the pilocarpine group (pilo group), the FK506 group and rapamycin group.2.The seizure activity of rats induced by pilocarpine was scored using a modified Racine scale and the effect of FK506 on seizure behavior.3.MRI scans at the level of the hippocampus were performed to evaluate the focal brain edema.4. To measure the brain water content by the wet-dry weight method.5. To detect GFAP immunoreactive positive cells in hippocampus by immunohistochemistry.6. By Westernblot analysis, we measured the CaN expression of hippocampus after SE and effect of FK506 on it.Results1. Behavioral episodes induced by pilocarpine followed the typical course of seizures, with a gradual increase of intensity to SE. No differences in behavioral seizures were noted in the mice pretreated with or without FK506. Control animals did not exhibit any behavioral seizure activity.2.MRI scans of all groups at the level of the hippocampus were performed. The focal brain edema was observed in hippocampus at 24h after SE, but disappeared at 7d after SE. FK506 markedly attenuated brain focal edema at 24h after SE and rapamycin did not show any inhibitory effect on brain focal edema.3.Brain water content increased significantly at 24h, and returned to normal level at 7d after SE. FK506, but not rapamycin, markedly decreased brain water content at 24h induced by SE.4.The total CaN protein levels in hippocampus at 24h,72h and 7d after SE were measured using Western blot analysis. Immunoblots indicated a significant increase in CaN protein expression at 24h after SE, which remained until 7d. Taken together with the SE-induced CaN in time course, it is suggested that the CaN protein expression is involved in brain focal edema.5.Glial fibrillary acidic protein(GFAP) is a cytoskeletal protein present in astroglial somata and projections, and is used as specific immunomarker of astrocytes. In morphometrical studies, astroglial reaction may be demonstrated by the increase in cell area of GFAP immunolabeled astrogliosis. Therefore, we used immunohistochemistry to detect astrocytes. The results showed that GF^P expression was appeared at 72h after SE, and obviously increased at 7d after SE. FK506 significantly attenuated reactive astrogliosis. Conclusion1.MRI scans and brain water content showed the brain edema was observed in hippocampus at 24h after SE, but disappeared at 7d after SE. FK506 markedly attenuated brain focal edema. It suggests that FK506 plays a role in protecting hippocampal neurons after SE.2. Immunoblots indicated a significant increase in CaN protein expression at 24h after SE, which remained until 7d. Taken together with the SE-induced CaN in time course, it is suggested that the CaN protein expression is involved in brain focal edema. FK506 prevents brain focal edema by inhibiting calcineurin.3. GFAP expression was appeared at 72h after SE, which indicated astrocyte activation. It is suggested that the CaN protein expression is involved in astroglial reaction after SE. FK506 prevents astrocyte activation by inhibiting calcineurin.In the present study, we established lithium chloride-pilocarpin-induced SE (LPCSE) models to investigate the alteration of behavior, evaluate the ability of spatial learning and memory, brain focal edema by non-invasive Magnetic resonance imaging (MRI-7T), calcineurin expression by immunoblotting, and astrocyte expression by immunohistochemistry. We found that FK506 inhibited the development of epilepsy, significantly prevented cognitive impairment. The T2 high-signal-intensity of MRI was observed in hippocampus at 24h after SE, which reflects focal brain edema.FK506 markedly attenuated brain focal edema at 24h after SE and rapamycin did not show any inhibitory effect on brain focal edema. FK506, a CaN inhibitor, significantly attenuated GFAP expression and reactive astrogliosis. GFAP expression with rapamycin was slightly lower than with pilocarpine alone, but higher than with FK506, suggesting that rapamycin was significantly less effective than FK506 at reducing GFAP expression after status. This suggested that the effects of FK506 on reactive astrogliosis were partially mediated by its ability to inhibit CaN protein expression. Our data suggested that calcineurin plays a pivotal role in cognitive function, brain focal edema and astrocyte expression. FK506 displayed the effect of antiepileptic and brain protection, indicated that FK506 may have clinical potential in the treatment of status epilepticus.