| bachgroudCranial irradiation is commonly used in the treatment of primary brain tumors, head and cervical cancers, brain metastasis and Malignant disease that involved central nervous system, such as leukemia and lymphoma, pediatric patients are frequently met in this circumstance. Unfortunately, in both pediatric and adult patients, cranial irradiation may lead to a debilitating cognitive decline. Months to years after treatment, patients who were cured of their initial disease present with progressively severe deficits in the hippocampal- dependent functions ,including learning, memory and spatial information processing. Pediatric patients are afflicted with these toxic response in their late life, which also institute huge burden for their family and society, they usually needs special education or institutionalization. For the adult patients, there is also possibility of developing dementia in long-term survivors. In fact, the severity of cognitive deterioration seemed to depend on the radiation dosage delivered to medial temporal lobes, and radiation brain injury are classically blamed to vascular injury and demyelinative necrosis.howere,Most cognitive deficits occured without morphological changes, such as overt vasculopathy , demyelination or necrosis, especially when relatively low absorbed dose was given, indicating that an insidious pathological process was behind the scences.Recently, It is proposed that arrested neurogenesis plays a role in radiation-induced cognitive dysfunction. The dentate gyrus of the hippocampus is oneof the two regions in the adult mammalian brain-the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus ,which continues to produce new neurons throughout adulthood,even in human aged 70. In the hippocampus of both rodents and primates, adult-generated neuronal cells derive from progenitor cells in the SGZ and migrate into the GCL, develop granule cell morphology and neuronal markers, and extend axons to connect with their target area, CA3. These new cells have passive membrane properties, action potentials, and functional synaptic inputs similar to those found in mature dentate granule cells , these cells become functionally integrated into the hippocampal circuitry, as evidenced by their responsiveness to stimulation of the perforant path. Most importantly, the new neurons are found to involve in synaptic plasticity, which can be considered as a cellular substrate for learning, investigations using a hippocampal slice model showed that radiation-induced reductions in dentate neurogenesis were associated with an inhibition of long-term potentiation(LTP), a type of synaptic plasticity. The newly generated cells may have a function in cognition, manipulations that increase neurogenesis, such as an enriched environment and exercise, are associated with improved memory and enhanced synaptic plasticity. Newly adult-generated neurons ablation using the toxin methylazoxymethanal acetate or irradiation were found to coincide with impaired learning of hippocampal-dependent task, like trace eyeblink conditioning and place-recognition.In young mice, three months after irradiation, Reductions of new neurons were associated with spatial memory retention deficits monitored with Morris water maze,but not all forms of hippocampal-Dependent Learning were related to hippocampal neurogenesis.Ionizing irradiation has long been known to stop cellular proliferation. At appropriate doses, it kills predominantly dividing cells, sparing neighboring non-dividing cells. In the rat, proliferating SGZ precursor cells undergo apoptosisafter irradiation, reductions in precursor cell proliferation are observed months after exposure. A single 10 Gy dose of X-rays to the rat brain almost completely abolishes the production of new neurons, whereas surviving precursor cells adopt a glial phenotype. Further investigation demonstrated the production of new neurons was reduced in a dose-dependent fashion, and no apparent change was observed in the production of astocytes with dose.The process of neurogenesis consists of three distinct developmental processes, including proliferation, survival, and differentiation. It was reported that irradiation didn't lead to an acute ablation of precursor cells, but irradiation decreased the proportion of proliferative cells adopting a neuronal fate, disrupted the neurogenic microenvironment manifested by elevated activated microglial cell and altered neuro-angiogenic relationship in the neurogenic zone. Based on these findings, Restoration strategies for radiation-induced newly-generated neurons depletion should address both precursor cell loss and damaged neurogenic niche, precursor cell transplantation and factors facilitating signal interaction for precursor cell to adoptneuronal fate are choices.The inhibitory amino acid taurine is an osmoregulatator and neuromodulator,it exert neuroprotective actions in neural tissue, whether it has restorative properties for radiation-arrested hippocampal neurogenesis remains unknown.MATERIALS AND METHODSAdult male Wistar rats weighing 220±20g at the beginning of the study were used. They were given free access to ordinary laboratory chow and water.IrradiationAdult wistar rats were anesthetized with 10% Chloral Hydrate (3.5ml /kg) and exposed to cranial irradiation using 6MV X-ray with varian linear accelerator(600C), each rat was irradiated individually in a 3 X 2 cm treatment field with shielding of body, neck, eyes and snout. Only the cranium was unshielded and covered with 2cmthickness plastic material, rats in irradiated groups were placed in sternal recumbancy and given a single lOGy dose. The corrected dose rate was approximately 200 cGy per min at a source to skin distance of 100 cm. Sham-irradiated controls for all experiments received anesthesia only.Tissue preparationAdult male rats were anesthetized with 10% chloral hydrate (3.5ml/kg weight ,i.p.) and were sacrificed by trahscardial perfusion with heparinized 0.9% saline followed by ice-cold 4% paraformaldehyde in PBS (0.1 M, pH7.2-7.4). Brains were removed and immersed in fixative (4°C for 6 hr), transferred to PBS containing 30% sucrose (4°C for 48 hr), and then frozen. Adjacent sections [corresponding to coronal coordinates interaural 4.48-5.86 mm, bregma -4.52 to bregma -3.14 (DG)] were cut on a cryotome ( Leica CM 1850, German) at 10 or 30um and stored at -20 °C till used.BrdU and taurine administrationTo study cytogenesis and neurogenesis, one months after irradiation,the irradiated and control Animals received twice daily injections of Brdu(75 mg/kg weight, i.p.) at 8-h intervals for 2 days consecutively. 4weeks after BrdU injection, the animals were transcardially perfused, taurine were administered once daily (lOOmg/kg weight, i.p.) one week before irradiation and continued for two consecutive weeks.Histological proceduresTUNEL staining TUNEL staining were performed according to the TUNEL Apoptag kit protocol (chemicon) with slight modification. Briefly, lOum coronal sections were microwaved 5 min and brought to room temperature and rehydrated in PBS. Endogenous peroxidase activity was quenched for 10 min in 3% H2O2 in Methanol. Sections were washed two times for 5 min in PBS, then were appliedwith equilibration water for 30 min under room temperature, followed by applying a reaction mix of 55ul/5cm2 of working strength TdT enzyme, and sections were incubated at 37°C for 60 min. After washing in stop/wash buffer for lmin, Slides were incubated in anti-digoxigenin peroxidase conjugate for 30 min at room temperature, and washed in PBS four times for 2 min each. The sections were colorized with 0.02% DAB in 0.1 M PBS, pH 7.4, containing 0.02% H2O2 and then washed in PBS to end the reaction. Sections were washed in water, allowed to dry overnight, counterstained with 0.5%(w:v) methyl green, and coverslipped. Only yellow-staining cells were counted as apoptotic phenotype.BrdU and nestin Immunohistochemisty 30um coronal sections were pretreated with 50% formamidey 280 mM NaCl/30 mM sodium citrate at 65°C for 2 h, incubated in 2 N HC1 at 37°C for 30 min, and rinsed in 0.1 M boric acid (pH 8.5) at room temperature for 10 min. Sections were incubated in 1% H2O2 in PBS for 15 min, in blocking solution (2%goat serum/0.3% Triton X-100/0.1% BSA in PBS) for 2 h at room temperature, and with mouse monoclonal anti-BrdUrd antibody (Roche; 2 mg/ ml) or mouse anti-rat nestin antibody (mouse anti-rat 1:1000 chemicon) at 4°C overnight ? Sections were washed with PBS, incubated with SABC kits(SA1021,Boster,Wuhan,China). The horseradish peroxidase reaction was detected with 0.05% diaminobenzidine (DAB) and 0.03% H2O2. Processing was stopped with H2O; sections were dehydrated through graded alcohols, cleared in xylene, and coverslipped in permanent mounting medium (Vector). Sections were examined with a Olympus BX51 system microscope (Olympus, Japan).Double Immunolabeling Sections were fixed with 4% paraformaldehyde in PBS for 1 h at room temperature, washed twice with PBS, and incubated in 2 N HC1 at 37°C for 1 h. After washing again, sections were incubated with blocking solution, then with primary antibodies at 4°C overnight, and then with secondary antibodies inblocking solution at room temperature for 2 h. The anti-BrdUrd antibodies used were sheep polyclonal anti-BrdUrd (BioDesign, New York; 25 mg/ml),The other primary antibodies were mouse monoclonal anti-NeuN (Chemicon; 1:100 dilution). The secondary antibodies were rhodamine conjugated rat-absorbed donkey anti-sheep IgG (Jackson ImmunoResearch; 1:200 dilution) and HTC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch; 1:200 dilution). Sections were mounted with Moviol 4-88 (sigma). Fluorescence signals were detected with a Olympus BX51 fluorescence microscope (Olympus, Japan) or confocal microscope (ACAS570,Meridian,USA) at excitation/emission wavelengths of 535/565 nm (rhodamine, red) and 470/505 nm (FITC, green). Results were recorded with an IJP70 digital camera (Olympus, Japan). Behavioral testOpen field test this test was conducted in an open wooden box(80X80X 40),its floor was divided into 25 squares by open markings, these squares with side bordering in the walls of the box are termed peripheral squares, the remaining squares are called central squares, between test sessions the apparatus was cleaned thoroughly with disinfectant to remove fecess urines and other scents from previous subject. The enclosure was situated in an isolated room and illuminated with a 40-watt fluorescent lamp.The following behavioral responses were scored within three minutes: number of crossings (i.e. number of floor sections traversed), number of rearings (standing with the forelegs raised in the middle of the arena or against the walls), This test was conducted one week after irradiation,.each rat was tested one time only.Water-maze place learning The water maze (a circular pool measured 180 cm in diameter, with 60 cm high walls made of black plastic) was filled to 35 cm with room-temperature water(24 ± 2°C), The water surface was covered with uniform foam dust. An escape platform was located 2 cm below the water's surface in itssoutheast quadrant., which was invisible to the rat. The platform remained in a fixed position in the center of the quadrant throughout training and could not be seen by the swimming animal. Rats were exposed to eight trials per day for 4 days and an inter-trial interval of 5min. For each trial, the rat was placed in a random quadrant of the tank, facing the tank wall, and provided 120s to locate and mount the platform. Rats were allowed to remain on the platform for 30s once they found it or, if after 120s, a rat failed to find the platform, it was placed on the platform by the experimenter. Latency to reach the platform was recorded. If the rat failed to locate the platform, it was placed there for 30 s. 4 weeks after training, four trial for each rat was repeated to assess its long term spatial memory capacity.Data analysisFor each experiment, the slides were coded before quantitative analysis, and the code was not broken until the analysis was complete. Cell counting was performed at 20x magnification on microscope by an investigator blind to treatment history. For each brain, at least three sections were selected for analysis from middle to caudal dentate gyrus (between levels bregma -4.52 to bregma -3.14). For each selected section, labeled cells was counted in the dentate gyrus including the granule cell layer and hilus.AU parameters are presented as means + S.E.M. Differences between experimental groups were examined performing analysis of variance( ANOVA) followed by Tamhane posthoc test corrected for unequal variances. Statistical significance was assumed for P<0.05. RESULTSApoptotic detectionTwelve hours after 10 Gy cranial irradiation, the number of TUNEL positive cells increased markedly (42.1 ±9.6versus 0.25+0.46 P<0.001). The vast majority of labeled cells localized within the subgranular zone, only isolated positive cellswere seen in the hilus and in the granular layer, coincided with the distribution of neurogenic region. Taurine administration can decrease TUNEL positive cell numbers in irradiated group (P=0.03) . Nestin ImmunohistochemistyNo significant difference was found in nestin positive cells number between irradiated and control groups two months after irradiation (226.0±33.3 versus 195.0 ±39.1, P=0.11) .taurine administration exert no effect on nestin positive cells number.BrdU Immunohistochemisty and Double ImmunolabelingIn non-irradiated group, lots of BrdU positive cells are present mainly within the subgranular zone, two months after irradiation, only sparse BrdU positive cells were scattered in the subgranular zone, difference was drastically significant (27.0+13.6 versus 7.8 + 3.2 P<0.001) ,taurine didn't alter radiation-induced BrdU positive cells reduction, the proportion of proliferative (BrdU) cells that co-express markers for mature neurons (NeuN) were determined by Double Irnmunolabeling, the proportion of mature neurons was significantly decreased by irradiation (12.8 ±5.0% versus 81.3 + 6.6 % ,P < 0.001), while taurine partially reversed radiation-arrested neurogenesis (23.1 + 8.6% versus 12.8 + 5.0%, P=0.006) .Open field testTwo behavioral responses (crossing and rearing) were obtained to Analyze the effect of irradiation on locomotor activity, statistics revealed irradiation only decreased the vertical locomotor activity.Water-maze place learningRats were trained in Morris water maze 4 weeks after irradiation, Records revealed that control group and irradiated group learned the task equally well, latency to find the hidden platform were not significantly different in the same trial betweengroups( P>0.05), and all were shortened with successive days of training.4 weeks after training, Each rat was tested with four trial, significant difference were found in latency to find the hidden platform between groups (P<0.001) .Conclusionsl.lOGy irradiation induced apoptosis predominantly in the subgranular zone of the adult rat hipocampal dentate gyrus, one of the neurogenic zones in adult brain.2.10Gy irradiation didn't ablate neural precursor cell in in the dentate gyrus of the adult rat hippocampus, evidenced by no apparent changes in the number of nestin positive cell two months after irradiation.3.10Gy irradiation inhibited both cytogenesis and neurogenesis in the subgranular zone of the adult rat dentate gyrus,which demonstrated ionizing radiation impaired the proliferation and differentiation of neural precursor cell.4.Open field test revealed irradiation decreased the locomotor activity, corresponding to lethargy manifested by patient after cranial irradiation.5.Cranial irradiation exerted no detrimental effects on spatial learning and short-term spatial memory in rat, but long-term spatial memory deficits were observed four weeks after spatial learning test, indicating neurogenesis may be involved in the forming of long-term spatial memory.6.Taurine can decrease apoptosis in the dentate gyrus of adult rat 12 hours after irradiation, but it didn't enhance cytogenesis two months after irradiation.7.The proportion of proliferative cells adopting neuronal fate was increased after taurine administration, suggesting taurine might have beneficial effects in protecting neurogenic niche.
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