Neuroprotection of N-benzylcinnamide on scopolamine-induced cholinergic dysfunction in human SH-SY5Y neuroblastoma cells

Alzheimer＇s disease, a progressive neurodegenerative disease, affects learning and memory resulting from cholinergic dysfunction. Scopolamine has been employed to induce Alzheimer＇s disease-like pathology in vivo and in vitro through alteration of cholinergic system. N-benzylcinnamide （PT-3）, purified from Piper submultinerve, has been shown to exhibit neuroprotective properties against amyloid-β-induced neuronal toxicity in rat cortical primary cell culture and to improve spatial learning and memory of aged rats through alleviating oxidative stress. We proposed a hypothesis that PT3 has a neuroprotective effect against scopolamine-induced cholinergic dysfunction. PT-3 （125-200 nM） pretreatment was performed in human neuroblastoma SH-SY5Y cell line following scopolamine induction. PT-3 （125-200 nM） inhibited scopolamine （2 mM）-induced generation of reactive oxygen species, cellular apoptosis, upregutation of ace- tylcholinesterase activity, downregulation of choline acetyltransferase level, and activation of p38 and JNK signalling pathways. These findings revealed the underlying mechanisms of scopolamine-induced Alzheimer＇s disease-like cellular dysfunctions, which provide evidence for developing drugs for the treatment of this de- bilitating disease.

THE EFFECT OF LIGUSTRAZINE ON NEUROGENESIS IN CORTEX AFTER FOCAL CEREBRAL ISCHEMIA IN RATS

Objective To explore the effect of Ligustrazine on neurogenesis in cortex after focal cerebral ischemia in rats. Methods Focal cerebral ischemia was induced by left middle cerebral artery occlusion with a suture. Two hours later, injection of Ligustrazine (80mg/kg, 1 time/d) was performed peritoneally. Four hours after the ischemia, 5-bromodeoxyuridine (BrdU) (50mg/kg, 1 time/d) was injected peritoneally. At 7d, 14d and 21d after ischemia, BrdU positive cells in the cortex were observed by immunohistochemical staining. Results In ischemic model group, at 7 day, sparsely-distributed BrdU positive cells were observed in the Ⅱ-Ⅵ layers of the ipsilateral cortex, with a band-like distribution in ischemic penumbra. With the prolongation of ischemia, the number of BrdU positive cells increased. In Ligustrazine group, BrdU positive cells were also observed in the Ⅱ-Ⅵ layers of the cortex, with an intense distribution in ischemic penumbra. The numbers of BrdU positive cells at 7d, 14d and 21d were more than those in ischemic model group respectively. Conclusion Ligustrazine increases the proliferated cells in cortex after focal cerebral ischemia in rats. The results suggest that it may be useful for promoting self-repair after ischemia.

A simple and sensitive method to assess ischemia occurrence in the setting of focal cerebral ischemia in rat: A comparative study

Objective: Neurological evaluation is commonly applied to identify ischemia in focal cerebral ischemia model though it might not be sensitive. In present study, we hired sleeping time to assess ischemia occurrence. Methods: Permanent middle cerebral artery occlusion was induced in Sprague-Dawley rats under pentobarbital and ketamine anesthesia respectively. Sleeping time was recorded. Neurological evaluation was conducted by modified Bederson’s scoring system at 4 h and histopathological evaluation was performed at 3 d after middle cerebral artery occlusion. Results: Slices of brain stained by TTC, H&E and hoechst 33258 revealed extensive lesion in the two ischemic groups. The sensitivity to identify ischemia by neurological evaluation was 62.5%, but it was 81.3% and 80% respectively when evaluating by sleeping time (pentobarbital: ≥90.7 min, ketamine: ≥36.1 min). The sensitivity to identify ischemia by sleeping time was significantly higher than that by neurological evaluation (P<0.05). Conclusion: Our results suggested that to identify ischemia by sleeping time is a simple and sensitive method in the setting of focal cerebral ischemia in rat.

PROPERTIES OF PROLIFERATION AND DIFFERENTIATION OF NEONATAL RAT RETINAL PROGENITOR CELLS IN VITRO

Objective To investigate the properties of proliferation and differentiation of neonatal rat retinal progenitor cells (RPCs) in vitro. Methods RPCs were isolated from neonatal SD rats neural retina and cultured in DMEM/F12+N2 with EGF and bFGF (suspension medium )or 10%FBS without EGF and bFGF (differentiation medium). The cells grew as suspended spheres or adherent monolayers, depending on different culture conditions. The neural stem cells or retinal progenitors, neurons, astrocytes, retinal ganglion cells, rod photoreceptors and the proliferating cells were evaluated with immunofluorescence analysis by Nestin or Pax6, Map2, GFAP, Thy-1, Rhodopsin and BrdU antibodies respectively. Results RPCs could propagate and differentiate in suspension or differentiation medium and express the markers of Nestin (92.86%) or Pax6 (86.75%), Map2 (38.54%), GFAP (20.93%), Thy-1 (27.66%) and Rhodopsin(13.33%)in suspension medium; however, Nestin (60.27%), Pax6 (52%), Map2 (34.94%), GFAP (38.17%), Thy-1(30.84%) and Rhodopsin (34.67%) in differentiation medium. 96.4% of the population in the neurospheres was BrdU-positive cells. The cells could spontaneously adherent forming some subspheres and retinal specific cell types. Conclusion Neonatal rat RPCs possess the high degree of proliferation and can differentiate into neurons, astrocytes, retinal ganglion cells and rod photoreceptors in vitro. There are different proportions for RPCs to differentiate into specific cell types.