Hypothyroid effects on astrocytes and microglia in the adult rat hippocampus

BACKGROUND： Thyroid hormones modulate proliferation of astrocytes and microglia depending on maturation stage and localization. Studies have demonstrated that triiodothyronine treatment or thyroidectomy during developmental stages results in morphological alterations and changes in the number of astrocytes and microglia. Little is known about the effects of hypothyroidism on astrocytes and microglia in adults. OBJECTIVE： To investigate the effects of hypothyroidism on morphology and number of astrocytes and microglia in the adult rat hippocampus. DESIGN, TIME AND SETTING： A randomized, controlled, neuroendocrinological, animal study was performed at the College of Medicine, Hallym University, South Korea between May 2008 and April 2009. MATERIALS： Methimazole, rabbit anti-glial fibrillary acidic protein （GFAP） antiserum, and rabbit anti-lba-1 antiserum were purchased from Sigma, USA. Rabbit anti-GFAP polyclonal antibody was provided by Chemicon, USA. Rabbit anti-lba-1 polyclonal antibody was purchased from Wako, Japan. Terminal deoxynucleotidyl transferase dUTP-biotin nick-end-labeling （TUNEL） kit was provided by Roche Molecular Biochemicals, Mannheim, Germany. METHODS： Hypothyroidism was induced in Wistar rats via methimazole administration （0.025%） in drinking water for 5 weeks, starting at 6 months of age. MAIN OUTCOME MEASURES： Following methimazole treatment, hippocampai neuronal death was determined using TUNEL staining. The morphology and number of GFAP and lba-1 immunoreactive cells were detected by immunohistochemistry. Hippocampal GFAP and lba-1 protein levels were detected by Western blot analysis. Serum-free triiodothyronine and thyroxine levels were quantified. RESULTS： TUNEL-positive neurons were not observed in the hippocampus of euthyroid and hypothyroid rats. Compared with the euthyroid rats, the number of GFAP immunoreactive astrocytes was decreased, and serum triiodothyronine and thyroxine levels were significantly decreased. In contrast, the number of lba-1 immunoreactive microglia was significantly increased in the hypothyroid rats （P 〈 0.05）. In addition, GFAP immunoreactive astrocytes were morphologically at a resting state, and lba-1 immunoreactive microglia were morphologically hypertrophic. GFAP and IBa-1 protein changes in the hippocampus of euthyroid and hypothyroid rats were in accordance with immunohistochemical data. CONCLUSION： Although methimazole-induced hypothyroidism did not induce neuronal injury in the adult rat hippocampus, it did result in decreased astrocyte numbers and increased microglial hypertrophy.

Time- and cell-type specific changes in iron, ferritin, and transferrin in the gerbil hippocampal CA1 region after transient forebrain ischemia

In the present study, we used immunohistochemistry and western blot analysis to examine changes in the levels and cellular localization of iron, heavy chain ferritin（ferritin-H）, and transferrin in the gerbil hippocampal CA1 region from 30 minutes to 7 days following transient forebrain ischemia. Relative to sham controls, iron reactivity increased significantly in the stratum pyramidale and stratum oriens at 12 hours following ischemic insult, transiently decreased at 1–2 days and then increased once again within the CA1 region at 4–7 days after ischemia. One day after ischemia, ferritin-H immunoreactivity increased significantly in the stratum pyramidale and decreased at 2 days. At 4–7 days after ischemia, ferritin-H immunoreactivity in the glial components in the CA1 region was significantly increased. Transferrin immunoreactivity was increased significantly in the stratum pyramidale at 12 hours, peaked at 1 day, and then decreased significantly at 2 days after ischemia. Seven days after ischemia, Transferrin immunoreactivity in the glial cells of the stratum oriens and radiatum was significantly increased. Western blot analyses supported these results, demonstrating that compared to sham controls, ferritin H and transferrin protein levels in hippocampal homogenates significantly increased at 1 day after ischemia, peaked at 4 days and then decreased. These results suggest that iron overload-induced oxidative stress is most prominent at 12 hours after ischemia in the stratum pyramidale, suggesting that this time window may be the optimal period for therapeutic intervention to protect neurons from ischemia-induced death.

Neuroprotection via maintenance or increase of antioxidants and neurotrophic factors in ischemic gerbil hippocampus treated with tanshinone I

Background Danshen （Radix Salvia miltiorrhizae） has been used as a traditional medicine in Asia for treatment of various microcirculatory disturbance related diseases. Tanshinones are mainly hydrophobic active components, which have been isolated from Danshen and show various biological functions. In this study, we observed the neuroprotective effect of tanshinone I （Tsl） against ischemic damage in the gerbil hippocampal CA1 region （CA1） after transient cerebral ischemia and examined its neuroprotective mechanism. Methods The gerbils were divided into vehicle-treated-sham-group, vehicle-treated-ischemia-group, Tsl-treated-sham- group, and Tsl-treated-ischemia-group. Tsl was administrated intraperitoneally three times （once a day for three days） before ischemia-reperfusion. The neuroprotective affect of Tsl was examined using H＆E staining, neuronal nuclei （NeuN） immunohistochemistry and Fluoro-Jade B staining. To investigate the neuroprotective mechanism of Tsl after ischemia- reperfusion, immunohistochemical （IHC） and Western blotting analyses for Cu, Zn-superoxide dismutase （SOD1）, Mn- superoxide dismutase （SOD2）, brain-derived neurotrophic factor （BDNF） and insulin-like growth factor-I （IGF-I） were performed.Results Treatment with Tsl protected pyramidal neurons from ischemia-induced neuronal death in the CA1 after ischemia-reperfusion. In addition, treatment with Tsl maintained the levels of SOD1 and SOD2 as determined by IHC and Western blotting in the CA1 after ischemia- reperfusion compared with the vehicle-ischemia-group. In addition, treatment with Tsl increased the levels of BDNF and IGF-I determined by IHC and Westem blotting in the Tsl-treated-sham-group compared with the vehicle-treated- sham-group, and their levels were maintained in the stratum pyramidale of the ischemic CA1 in the Tsl-treated- ischemia-group. Conclusion Treatment with Tsl protects pyramidal neurons of the CA1 from ischemic damage induced by transient cerebral ischemia via the maintenance of antioxidants and the increase of neurotrophic factors.

Glehnia fittoralis Extract Promotes Neurogenesis in the Hippocampal Dentate Gyrus of the Adult Mouse through Increasing Expressions of Brain-Derived Neurotrophic Factor and Tropomyosin-Related Kinase B

Background： Glehnia littoralis has been used for traditional Asian medicine, which has diverse therapeutic activities. However, studies regarding neurogenic effects of G. littoralis have not yet been considered. Therefore, in this study, we examined effects of G. littoralis extract on cell proliferation, neuroblast differentiation, and the maturation of newborn neurons in the hippocampus of adult mice. Methods： A total of 39 male ICR mice （12 weeks old） were randomly assigned to vehicle-treated and 100 and 200 mg/kg G. littoralis extract-treated groups （n = 13 in each group）. Vehicle and G. littoralis extract were orally administrated for 28 days. To examine neurogenic effects ofG. litmralis extract, we performed immunohistochemistry tbr 5-bromo-2-deoxyuridine （BrdU, an indicator for cell proliferation） and doublecortin （DCX, an immature neuronal marker） and double immunofluorescence staining for BrdU and neuronal nuclear antigen （NeuN, a mature neuronal marker）. In addition, we examined expressional changes of brain-derived neurotrophic factor （BDNF） and its major receptor tropomyosin-related kinase B （TrkB） using Western blotting analysis. Results： Treatment with 200 mg/kg, not 100 mg/kg, significantly increased number of BrdU-immunoreactive （＋） and DCX＋ cells （48.0 ±3.1and 72.0 ± 3.8 cells/section, respectively） in the subgranular zone （SGZ） of the dentate gyrus （DG） and BrdU＊/NeuN＋ cells （17.0 ±1.5 cells/section） in the granule cell layer as well as in the SGZ. In addition, protein levels of BDNF and YrkB （about 232% and 244% of the vehicle-treated group, respectively） were significantly increased in the DG of the mice treated with 200 mg/kg ofG. littoralis extract. Conclusion： G. littoralis extract promots cell proliferation, neuroblast differentiation, and neuronal maturation in the hippocampal DG, and neurogenic effects might be closely related to increases ofBDN F and TrkB proteins by G. littoralis extract treatment.

Oenanthe Javanica Extract Protects Against Experimentally Induced Ischemic Neuronal Damage via its Antioxidant Effects

Background： Water dropwort （Oenanthejavanica） as a popular traditional medicine in Asia shows various biological properties including antioxidant activity. In this study, we firstly examined the neuroprotective effect of Oenanthejavanica extract （OJE） in the hippocampal comus ammonis 1 region （CA 1 region） of the gerbil subjected to transient cerebral ischemia. Methods： Gerbils were established by the occlusion of common carotid arteries for 5 min. The neuroprotective effect of OJE was estimated by cresyl violet staining. In addition, 4 antioxidants （copper, zinc superoxide dismutase [SOD], manganese SOD, catalase, and glutathione peroxidase） immunoreactivities were investigated by immunohistochemistry. Results： Pyramidal neurons in the CA1 region showed neuronal death at 5 days postischemia; at this point in time, all antioxidants immunoreactivities disappeared in CA1 pyramidal neurons and showed 100 mg/kg, OJE protected CA 1 pyramidal neurons from ischemic damage in many nonpyramidal cells. Treatment with 200 mg/kg, not In addition, 200 mg/kg OJE treatment increased or maintained antioxidants immunoreactivities. Especially, among the antioxidants, glutathione peroxidase immunoreactivity was effectively increased in the CA 1 pyramidal neurons of the OJE-treated sham-operated and ischemia-operated groups. Conclusion： Our present results indicate that treatment with OJE can protect neurons from transient ischemic damage and that the neuroprotective effect may be closely associated with increased or maintained intracellular antioxidant enzymes by OJE.