Cell proliferation and apoptosis in optic nerve and brain integration centers of adult trout Oncorhynchus mykiss after optic nerve injury

Fishes have remarkable ability to effectively rebuild the structure of nerve cells and nerve fibers after central nervous system injury.However,the underlying mechanism is poorly understood.In order to address this issue,we investigated the proliferation and apoptosis of cells in contralateral and ipsilateral optic nerves,after stab wound injury to the eye of an adult trout Oncorhynchus mykiss.Heterogenous population of proliferating cells was investigated at 1 week after injury.TUNEL labeling gave a qualitative and quantitative assessment of apoptosis in the cells of optic nerve of trout 2 days after injury.After optic nerve injury,apoptotic response was investigated,and mass patterns of cell migration were found.The maximal concentration of apoptotic bodies was detected in the areas of mass clumps of cells.It is probably indicative of massive cell death in the area of high phagocytic activity of macrophages/microglia.At 1 week after optic nerve injury,we observed nerve cell proliferation in the trout brain integration centers：the cerebellum and the optic tectum.In the optic tectum,proliferating cell nuclear antigen（PCNA）-immunopositive radial glia-like cells were identified.Proliferative activity of nerve cells was detected in the dorsal proliferative（matrix） area of the cerebellum and in parenchymal cells of the molecular and granular layers whereas local clusters of undifferentiated cells which formed neurogenic niches were observed in both the optic tectum and cerebellum after optic nerve injury.In vitro analysis of brain cells of trout showed that suspension cells compared with monolayer cells retain higher proliferative activity,as evidenced by PCNA immunolabeling.Phase contrast observation showed mitosis in individual cells and the formation of neurospheres which gradually increased during 1–4 days of culture.The present findings suggest that trout can be used as a novel model for studying neuronal regeneration.

Effect of ketamine on aquaporin-4 expression and neuronal apoptosis in brain tissues following brain injury in rats

BACKGROUND： Aquaporin-4 （AQP-4） is closely related to the formation of brain edema. Neuronal apoptosis plays an important part in the conversion of swelled neuron following traumatic brain injury. At present, the studies on the protective effect of ketamine on brain have involved in its effect on aquaporin-4 expression and neuronal apoptosis in the brain tissues following brain injury in rats. OBJECTIVE： To observe the effect of ketamine on AQP-4 expression and neuronal apoptosis in the brain tissue following rat brain injury, and analyze the time-dependence of ketamine in the treatment of brain injury.DESIGN： Randomized grouping design, controlled animal tria SETTING ： Department of Anesthesiology, the Medical School Hospital of Qingdao University MATERIALS： Totally 150 rats of clean grade, aged 3 months, were involved and randomized into control group and ketamine-treated group, with 75 rats in each. Each group was divided into 5 subgroups separately at 6,12, 24, 48 and 72 hours after injury, with 15 rats at each time point. Main instruments and reagents： homemade beat machine, ketamine hydrochloride （Hengrui Pharmaceutical Factory, Jiangsu）, rabbit anti-rat AQP-4 polyclonal antibody, SABC immunohistochemical reagent kit and TUNEL reagent kit （Boster Co.,Ltd., Wuhan）. METHODS： This trial was carried out in the Institute of Cerebrovascular Disease, Medical College of Qingdao University during March 2005 to February 2006. A weight-dropping rat model of brain injury was created with Feeney method. The rats in the ketamine-treated group were intraperitoneally administered with 50 g/L ketamine （120 mg/kg） one hour after injury, but ketamine was replaced by normal saline in the control group. In each subgroup, the water content of cerebral hemisphere was measured in 5 rats chosen randomly. The left 10 rats in each subgroup were transcardiacally perfused with ketamine, then the brain tissue was made into paraffin sections and stained by haematoxylin and eosin. Neuronal morphology was observed. AQP-4 expression and neuronal apoptosis were measured with immunohistochemical method and TUNEL method respectively. MAIN OUTCOME MEASURES： Water content in brain tissue, neuronal morphology, the number of AQP-4 positive neurons and TUNEL positive neurons in rats of two groups at each time point after injury. RESULTS： Totally 150 rats entered the stage of result analysis. （1） Water content of brain tissue： The water content of brain tissue at each time point after injury in the ketamine-treated group was lower than that in the control group. There were very significant differences in water content at 12 and 24 hours after injury respectively between ketamine-treated group and control group [（77.34±2.35）% vs. （82.31 ±1.48）%; （78.01 ±2.21 ）% vs. （83.86±2.37）%, t=-4.001 6,4.036 7, both P 〈 0.01]. （2） Neuronal morphology： Pathological changes in traumatic region and peripheral region of injury in the ketamine-treated group were significantly lessened, and necrotic and apoptotic cells in the ketamine-treated group were also significantly reduced as compared with control group. （3） AQP-4 expression： AQP-4 positive neurons at each time point in the ketamine-treated group were significantly less than those in the control group. There were very significant differences in AQP-4 expression at 12 and 24 hours after injury between ketamine-treated group and control group [（34.17±4.74） /visual field vs. （43.42±5.65） /visual field;（40.83±3.17） /visual field vs. （58.88±6.23） /visual field,t=3.966 3,8.165 7, both P〈 0.01]. （4） Neuronal apoptosis： TUNEL positive neurons at each time point in the ketamine-treated group were less than those in the control group. There were very significant differences in the neuronal apoptosis at 12 and 24 hours after injury between ketamine-treated group and control group [（26.25±3.04） /visual field vs. （32.75±4.39） /visual field; （29.33± 4.02） /visual field vs. （39.83±5.61） /visual field,t=-3.849 3,5.169 2,both P 〈 0.01]. CONCLUSION： Ketamine can reduce brain edema, AQP-4 expression and neuronal apoptosis following brain injury in rats, and has obvious therapeutic effect on brain injury, especially at 12 and 24 hours after injury.

Amentoflavone protects hippocampal neurons: anti-inflammatory, antioxidative, and antiapoptotic effects

Amentoflavone is a natural biflavone compound with many biological properties, including anti-inflammatory, antioxidative, and neuroprotective effects. We presumed that amentoflavone exerts a neuroprotective effect in epilepsy models. Prior to model establishment, mice were intragastrically administered 25 mg/kg amentoflavone for 3 consecutive days. Amentoflavone effectively prevented pilocarpine-induced epilepsy in a mouse kindling model, suppressed nuclear factor-κB activation and expression, inhibited excessive discharge of hippocampal neurons resulting in a reduction in epileptic seizures, shortened attack time, and diminished loss and apoptosis of hippocampal neurons. Results suggested that amentoflavone protected hippocampal neurons in epilepsy mice via anti-inflammation, antioxidation, and antiapoptosis, and then effectively prevented the occurrence of seizures.

Loss of micro RNA-124 expression in neurons in the peri-lesion area in mice with spinal cord injury

Micro RNA-124（mi R-124） is abundantly expressed in neurons in the mammalian central nervous system, and plays critical roles in the regulation of gene expression during embryonic neurogenesis and postnatal neural differentiation. However, the expression profile of mi R-124 after spinal cord injury and the underlying regulatory mechanisms are not well understood. In the present study, we examined the expression of mi R-124 in mouse brain and spinal cord after spinal cord injury using in situ hybridization. Furthermore, the expression of mi R-124 was examined with quantitative RT-PCR at 1, 3 and 7 days after spinal cord injury. The mi R-124 expression in neurons at the site of injury was evaluated by in situ hybridization combined with Neu N immunohistochemical staining. The mi R-124 was mainly expressed in neurons throughout the brain and spinal cord. The expression of mi R-124 in neurons significantly decreased within 7 days after spinal cord injury. Some of the neurons in the peri-lesion area were Neu N＋/mi R-124-. Moreover, the neurons distal to the peri-lesion site were Neu N＋/mi R-124＋. These findings indicate that mi R-124 expression in neurons is reduced after spinal cord injury, and may reflect the severity of spinal cord injury.

Alterations of bcl-2, bcl x and bax protein expressions in area CA_3 of rat hippocampus following fluid percussion brain injury

Objective: To investigate the alterations of bcl 2 gene family in the area of CA 3 in rats and the molecular mechanism of neuronal apoptosis following traumatic brain injury. Methods: Male Sprague Dawley rats were subjected to lateral fluid percussion brain injury of moderate severity. bcl 2, bcl x, and bax protein expressions were detected by immunohistochemistry. Results: The immunoreactivity of bcl 2 and bcl x proteins decreased in the hippocampus ipsilateral impact site at 6 hours after injury, and this was the main cause of down regulation of the value of (bcl 2 +bcl x)/ bax. During the period of 1～3 days after injury, bax protein expression increased significantly, while bcl 2 and bcl x protein expressions decreased relatively slowly. The decreased value of (bcl 2+bcl x)/ bax was mainly due to the bax up regulation. Conclusions: The bcl 2 gene family is involved in neuronal apoptosis after traumatic brain injury, and the protein expression alterations of the bcl 2 gene family members lead to apoptosis of the neuronal cells.