Summary:
Objective To investigate the relationship between leptin and the occurrence and development of temporal lobe epilepsy.
Methods After injection of C57BL/6J mice by exogenous Leptin, 200 ng of kainic acid (KA) was injected into the right hippocampus to induce temporal lobe epilepsy. The epilepsy scores of the mice were recorded. The pathological changes of hippocampus were observed after 1 week. The expression level of Bax was detected by Western blot. Nissl staining was used to observe the loss of neurons in the hippocampus Hilus region. Immunofluorescence was used to detect the activation and proliferation of astrocytes in Hilus region. Results Based on the 200 ng KA-induced temporal lobe epilepsy model, after pretreatment with 10 mg/kg exogenous Leptin, the epilepsy score increased by about 52%, and Bax expression increased by about 75% (P < 0.05). Hippocampal Hilus region nerves Meta-density is severe and astrocytes are over-activated.
Conclusion Leptin increases KA-induced neuronal apoptosis and aggravates KA-induced neuropathological processes.
Materials and Method
1 Materials C57BL/6J mice (SPF grade, 60), 8 weeks old, male, body weight 20 ~ 22 g, purchased from the Medical Laboratory Animal Center of the PLA General Hospital; standard feed (10% KJ%fat, feed); Kainic acid (KA) (Sigma Chemical Co. St Louis, MO, USA); human recombinant Leptin (Peprotech); Tris (base) (Beijing Yili Fine Chemicals Co., Ltd.); Brain stereotaxic instrument (ZS-B/C, Beijing Zhongshi Di Chuang Technology Development Co., Ltd.); 5μl micro-injector Microliter Syringes (Shanghai Gaoge Industry and Trade Co., Ltd.); Ether (Beijing Yili Fine Chemicals Co., Ltd.) Toluidine blue (Sigma Chemical Co. St Louis, MO, USA); RIPA lysate, BCA protein quantification kit, Western Blot hypersensitive luminescent solution (Beijing Plylai Gene Technology); primary antibody: Bax (Epitomics) ), β-actin (Santa Cruz) glial fibrillary acidic protein (GFAPCell Signaling Technology Inc. #3670), leptin receptor (ObR, Santa Cruz Biotechnology); secondary antibody: goat anti-mouse Alexa Fluor (R) 488, goat Anti-rabbit Alexa Fluor(R) 555 (1: 400, Cell Signaling T Echnology Inc.), goat anti-rabbit IgG/horseradishase marker (Beijing Zhongzhe Jinqiao Co., Ltd.); Hochest 33342 (Sigma); citrate buffer (Beijing Pulilai Gene Technology Co., Ltd.).
2 Establishment of mouse model of temporal lobe epilepsy [5] C57BL/6J mice were divided into 0.9% sodium chloride injection group (control group, 10 rats), and Leptin placed nitrocellulose membrane into 5% skim milk powder (TBST). Dissolve) In a plastic bag, place in a shaker at 37 ° C for 1 h, and dilute the primary antibody, Bax (1 : 1 000), β-actin (1: 500), and block at 4 ° C overnight. After taking out, let room temperature for 30 min, TBS/T rinse film 3 times × 10 min, add diluted secondary antibody (1: 3 000), incubate at 37 °C for 30 min, TBS/T rinse nitrocellulose membrane 3 times × 10 min , adding luminescent liquid, dark room exposure. After scanning, the gray value was analyzed using Image Plus 6.0 software, and averaged by 3 independent experiments.
1. Nissl staining 4 μm paraffin section was dewaxed to water, washed with phosphate buffer solution for 2 min, placed in 1% toluidine blue solution at room temperature for 20 min, dehydrated with gradient alcohol, and the result was nibble It is dark blue in color, the nucleus is light blue, and the background is basically colorless.
1. Immunofluorescence double staining method was used to observe the co-expression of ObR and GFAP. The coronal paraffin sections (4 μm) of mice injected with kainate were dewaxed to water, citrate buffer solution was repaired for 5 min, and 5% goat serum was blocked. Add anti-leptin receptor (ObR) and glial fibrillary acidic protein at 4 ° C overnight, rinse, add secondary antibody Alexa Fluor (R) 488, Alexa Fluor (R) 555, incubate for 40 min at 37 ° C, Hochest 33342 stained nucleus , placed under a laser confocal microscope to observe the photo,
Epilepsy is the second largest disease in the nervous system after cerebrovascular disease. Most of them can be controlled by drugs. 30% of patients with epilepsy develop refractory epilepsy, and temporal lobe epilepsy accounts for a large part. Common pathological changes in temporal lobe epilepsy are hippocampal sclerosis, atrophy, loss of neurons in specific parts of the hippocampus, and gliosis [7]. Mossy fiber sprouting is a typical pathological feature of human temporal lobe epilepsy. The mossy fiber is the axon of dentate gyrus granule cells. Normally, the mossy fiber is associated with the dendrites of the hippocampal Hilus region and the CA3 pyramidal cells. In the case of acid-induced injury, the neurons in the Hilus region and the CA3 region are significantly lost, and the moss fibers are out of contact with the target cells, triggering the abnormal sprouting of the mossy fibers and forming excitatory synapses with self-reporting and dendrites, and the inner molecules. The afferent fibers of the layer start from the Hilus region neurons, and the loss of neurons in the Hilus region causes the mossy fibers to sprout into a new afferent pathway, forming abnormal self-feedback and playing an important role in recurrent seizures [8-10] The hippocampal Hilus region also plays an important role in the onset of epilepsy. In this study, a mouse model of temporal lobe epilepsy induced by kainate was used to focus on the pathological changes in the hippocampal Hilus region of mice.
Leptin is a peptide hormone secreted by fat cells and released into the blood, which regulates energy metabolism through the blood-brain barrier in the central nervous system [11]. In recent years, most studies have shown that leptin has a protective effect on nerve damage, such as cerebral ischemia, Alzheimer's disease, Parkinson's disease, and glutamate-induced epilepsy [12-14]. A small number of studies have shown that high leptin levels have a positive effect on the occurrence of epilepsy [15], but the specific reasons are still unclear. According to a study by Lynch et al [15], this article explores the role of leptin (10 mg/kg) leptin in the model of kainic acid-induced temporal lobe epilepsy and its possible underlying mechanisms.
This study investigated the changes of neurons and astrocytes in the Hilus region in hippocampal injury of temporal lobe epilepsy mice. On the basis of kainate-induced temporal lobe epilepsy, the seizure behavior scores of mice after leptin pretreatment increased, and pathologically showed that neuronal loss in hippocampal Hilus region was aggravated, probably due to the significant increase of Bax expression in the hippocampus of mice. Lead to decreased anti-apoptotic ability of neurons. Neurons and glial cells are the main components of the human brain. The number of glial cells is 5-10 times that of neurons. The most abundant ones are astrocytes. Astrocytes are tiled and tightly wrapped. Neurons and blood vessels have long been thought to play a major role in the nutrition and support of neurons, and play a crucial role in maintaining the microenvironment outside the neurons [16]. Activation of astrocytes is associated with the severity of pathological stimuli. Glial acidic proteins are specific markers of astrocytes and are significantly increased in activated astrocytes and are commonly used to identify areas of brain damage. Astrocytes with non-lesional areas [17]. After the loss of neurons, astrocyte proliferation and activation can both fill and lose the loss, but excessively activated astrocytes form scars, astrocyte glutamate Decreased synthase content is also an important factor in recurrent seizures and difficult to cure [18-19].
This study found that after leptin preconditioning, the expression of GFAP in the hippocampal Hilus region of mice with temporal lobe epilepsy increased, and astrocytes were over-activated, showing that reactive astrocyte morphology mainly manifested as cell proliferation and cell morphogenesis. Including the cell body thickening, the protrusions increased, prolonged, gathered into clusters. Exogenous administration of a certain amount of leptin may aggravate the pathological damage induced by kainate, which may be caused by reducing the anti-apoptotic ability of neurons and over-activating astrocytes. Leptin may be a new entry point for studying temporal lobe epilepsy.
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