G-CSF attenuates neuroinflammation and neuronal apoptosis via the mTOR/p70SK6 signaling pathway in neonatal Hypoxia-Ischemia rat model.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is an important cause of permanent damage to the central nervous system, associated with long-lasting neurological disabilities and neurodevelopmental impairment in neonates. Granulocyte-colony stimulating factor (G-CSF) has been shown to have neuroprotective activity in a variety of experimental brain injury models and G-CSF is a standard treatment in chemotherapeutic-induced neutropenia. The underlying mechanisms are still unclear. The mTOR (mammalian target of rapamycin) signaling pathway is a master regulator of cell growth and proliferation in the nervous system. However, the effects of G-CSF treatment on the mTOR signaling pathway have not been elucidated in neonates with hypoxic-ischemic (HI) brain injury. Our study investigated the neuroprotective effect of G-CSF on neonates with hypoxic-ischemic (HI) brain injury and the possible mechanism involving the mTOR/p70S6K pathway. METHODS: Sprague-Dawley rat pups at postnatal day 7 (P7) were subjected to right unilateral carotid artery ligation followed by hypoxic (8% oxygen and balanced nitrogen) exposure for 2.5 h or sham surgery. Pups received normal saline, G-CSF, G-CSF combined with rapamycin or ethanol (vehicle for rapamycin) intraperitoneally. On postnatal day 9 (P9), TTC staining for infarct volume, and Nissl and TUNEL staining for neuronal cell injury were conducted. Activation of mTOR/p70S6K pathway, cleaved caspase-3 (CC3), Bax and Bcl-2 and cytokine expression levels were determined by western blotting. RESULTS: The G-CSF treated group was associated with significantly reduced infarction volume and decreased TUNEL positive neuronal cells compared to the HI group treated with saline. The expression levels of TNF-α and IL-1ß were significantly decreased in the G-CSF treated group, while IL-10 expression level was increased. The relative immunoreactivity of p-mTOR and p-p70S6K was significantly reduced in the HI group compared to sham. The HI group treated with G-CSF showed significant upregulated protein expression for p-mTOR and p-p70S6K levels compared to the HI group treated with saline. Furthermore, G-CSF treatment increased Bcl-2 expression levels and decreased CC3 and Bax expression levels in the ipsilateral hemispheres of the HI brain. The effects induced by G-CSF were all reversed by rapamycin. CONCLUSION: Treatment with G-CSF decreases inflammatory mediators and apoptotic factors, attenuating neuroinflammation and neuronal apoptosis via the mTOR/p70S6K signalling pathway, which represents a potential target for treating HI induced brain damage in neonatal HIE.

Altered functional connectivity of the marginal division in Parkinson's disease with mild cognitive impairment: A pilot resting-state fMRI study.

BACKGROUND: The marginal division (MrD) is an important subcortical center involved in learning and memory. Mild cognitive impairment (MCI) is commonly seen in patients with Parkinson's disease (PD), but the neurobiological basis is yet to be elucidated. PURPOSE: To use resting-state functional magnetic resonance imaging (rs-fMRI) to explore the altered functional connectivity (FC) of the MrD in patients with PD-MCI. STUDY TYPE: Prospective pilot study. POPULATION: Twenty-five patients with PD-MCI; 25 PD patients and no cognitive impairment (PD-NCI); and 25 healthy control (HC) participants. SEQUENCE: 3.0 T GE Healthcare MRI scanner; three-dimensional T1 -weighted fast spoiled gradient recalled echo (3D T1 -FSPGR); rs-fMRI. ASSESSMENT: The MrD was defined using manual delineation, which was the seed point to compute the FC to examine correlations between low-frequency fMRI signal fluctuations in MrD and the whole brain. STATISTICAL TESTS: Between-group comparisons of the rs-fMRI data were computed using two-sample t-tests in a voxelwise manner after controlling for age and sex, to determine the brain regions that showed significant differences in FC with the bilateral MrDs. Correlation analyses were performed for FC values and cognitive abilities in patients with PD. RESULTS: In the PD-MCI group, compared with the PD-NCI group, we observed lesser FC between the MrD bilaterally and right putamen, left insula, left cerebellum, and left thalamus; greater FC between the MrD bilaterally and left middle cingulate cortex, left middle frontal gyrus, left superior frontal gyrus, left supplementary motor area, and left middle/inferior occipital gyrus. Moreover, the strength of FC between the MrD and regions that showed differences between the PD-MCI and PD-NCI groups was significantly correlated with neuropsychological scores in patients with PD. DATA CONCLUSION: The current study suggests that MrD dysfunction may contribute to MCI in PD. However, the mechanisms underlying this process require further investigation. Level of Evidence 1. Technical Efficacy Stage 2. J. Magn. Reson. Imaging 2019;50:183-192.

New learning and memory related pathways among the hippocampus, the amygdala and the ventromedial region of the striatum in rats.

BACKGROUND: The hippocampus, central amygdaloid nucleus and the ventromedial region (marginal division) of the striatum have been reported to be involved in the mechanism of learning and memory. This study aimed elucidating anatomical and functional connections among these brain areas during learning and memory. RESULTS: In the first part of this study, the c-Fos protein was used to explore functional connections among these structures. Chemical stimulation of either hippocampus or central amygdaloid nucleus results in dense expression of c-Fos protein in nuclei of neurons in the marginal division of the striatum, indicating that the hippocampus and the central amygdaloid nucleus might be functionally connected with the marginal division. In the second part of the study, the cholera toxin subunit B-horseradish peroxidase was injected into the central amygdaloid nucleus to observe anatomical connections among them. The retrogradely transported conjugated horseradish peroxidase was observed in neurons of both the marginal division and dorsal part of the hippocampus following the injection. Hence, neural fibers from both the marginal division and the hippocampus directly projected to the central amygdaloid nucleus. CONCLUSION: The results implicated potential new functional and structural pathways through these brain areas during the process of learning and memory. The pathways ran from ventromedial portion (the marginal division) of the striatum to the central amygdaloid nucleus and then to the hippocampus before going back to the marginal division of the striatum. Two smaller circuits were between the marginal division and the central amygdaloid nucleus, and between the central amygdaloid nucleus and the hippocampus. These connections have added new dimensions of neural networks of learning and memory, and might be involved in the pathogenesis of dementia and Alzheimer disease.

[Influence of stapedectomy on the hearing of guinea pigs].

OBJECTIVE: To compare the influences of stapedectomy and small fenestra stapedotomy on the hearing of guinea pigs. METHODS: Twenty-four (48 ears) guinea pigs were randomized equally into two groups, and the left ears were subjected to stapedectomy and total stapes replacement with a prosthesis, or sham operation (12 ears) to expose the footplate of the stapes and the round window. Each guinea pig was tested by ABR perioperatively. Four guinea pigs were chosen randomly from each group and decapitated for morphological examination by light microscopy and scanning electron microscopy after ABR test. RESULTS: In the sham operation group, the post-operative latencies of each wave, the intervals and the hearing threshold exhibited no significant changes other than prolonged latency of wave I. In stapedectomy group, the hearing threshold increased to 23.75-/+3.77 dBSPL 1 h after operation with significantly prolonged post-operative latencies of all the waves and intervals but for III-IV interval, which was shortened. The latencies of each wave (especially waves I and III) in the stapedectomy group were increased by a greater magnitude than those in the sham operation group, but the intervals were comparable between the two groups. No significant difference was noted in the parameters of ABR either 1 h or 1 day after the operation between the two groups, in which the architecture of cochleas remained intact with similar number of spiral ganglion cells. The stereocilia of the outer hearing cells (OHC) were normal in the sham operation group while in stapedectomy group, slight stereocilia disorder occurred but became normal 1 day after operation. No obvious changes were found in the stereocilia of the inner hearing cell (IHC) in either groups. CONCLUSION: Stapedectomy can induce mild hearing loss without seriously damaging the function of the cochlea in guinea pigs.

[Analysis of auditory steady-state response to multiple simultaneous stimuli and cochlea morphology in guinea pigs with posterior semicircular canal fistula].

OBJECTIVE: To observe the changes of auditory steady-state response (ASSR) threshold in guinea pigs with posterior semicircular canal fistula. METHODS: In 10 guinea pigs, a window was opened in the posterior semicircular canal of the left ear with the right ear serving as the control for testing the ASSR under anesthesia with pentobarbital sodium. RESULTS: The mean and standard deviation of ASSR threshold (dB SPL) at 0.5, 1, 2, and 4 kHz in the left ear was 35.00+/-14.33, 25.50+/-12.37, 20.00+/-9.37 and 20.00+/-9.18, respectively, and was 31.00+/-16.19, 25.50+/-12.34, 18.00+/-6.96 and 18.50+/-6.71 in the right ear, respectively. Paired-sample t test showed no significant difference in the ASSR at the same frequency between the two ears. CONCLUSION: Small fistula by surgery causes no significant hearing loss in guinea pigs.

Expression of c-fos and c-jun proteins in the marginal division of the rat striatum during learning and memory training.

BACKGROUND: A new brain region, the marginal division (MrD), was discovered at the caudal margin of the neostriatum. The MrD was shown to be involved in learning and memory in the rat. The aim of this study was to investigate the expression of the immediate-early genes c-fos and c-jun in the MrD of the striatum during learning and memory processes in the rat, immunocytochemical and Western blot methods were used to examine Y-maze trained rats. METHODS: The rats were divided into three groups, namely the training, pseudotraining, and control groups. After Y-maze training, the expression of the immediate-early genes c-fos and c-jun in the MrD of the rats was investigated using immunocytochemical and Western blot methods. RESULTS: After one hour of Y-maze training, the expression of c-jun and c-fos proteins was significantly enhanced in the MrD; the c-jun protein, in particular, was more intensely expressed in this region than in other parts of the striatum. The expression of these two proteins in the training group was significantly higher than in the pseudotraining and control groups. In addition, positive expression was also found in the hippocampus, cingulum cortex, thalamus, and in other areas. Western blot disclosed two immunoreactive bands for the anti-c-fos antibody (47 kD and 54 kD) and two immunoreactive bands for the anti-c-jun antibody (39 kD and 54 kD). CONCLUSIONS: These results indicate that the immediate-early genes c-fos and c-jun participate in signal transduction during the learning and memory processes associated with Y-maze training in rats.

Marginal division of the neostriatum: a subcortical memory center.

The marginal division (MrD) is a pan-shaped subdivision in the caudal margin of the neostriatum newly discovered in the brains of the rat, cat, monkey and humans. A variety of intensely expressed neuropeptides and monoamines and their receptors were identified in the fibers, terminals and neuronal somata in the MrD with immunohistochemical and patch clamp methods. The MrD was shown to be involved in learning and memory by double-blind studies of Y-maze learning and long-term potentiation in rats. c-Fos expression and tract-tracing techniques with immunoelectronmicroscopy indicated that the MrD is a new component of the limbic system and is a key linking area between the limbic system and the basal nucleus of Meynert. Functional magnetic resonance image (fMRI) studies illustrated that the MrD and the prefrontal cortex are involved in digital working memory in the human brain. A cerebral hemorrhage case report confirmed the findings with fMRI. In conclusion, based on the position of the MrD, its advanced development in higher mammalian brains, abundant blood supply and diverse connections with other memory-related structures, MrD is likely to be an important subcortical center of learning and memory.

[Functional connection between the marginal division and hippocampus in rats].

OBJECTIVE: To investigate the functional connection between the marginal division of the striatum and hippocampus, a brain region that play a vital role in learning and memory. METHODS: Morphological localization of functional activity of the nervous system was employed. Kainic acid (0.01%) was stereotaxically injected into the hippocampus as a chemical stimulus, and immunohistochemistry method was used to show the expression of c-Fos in rat brain. RESULTS: c-Fos was intensely expressed in the hippocampus, amygdaloid nucleus, the bed nucleus of the stria terminals and cerebral cortex; in the striatum, a stretch in the marginal division where c-Fos-positive nuclei congregated was observed, while c-Fos expression was scarcely detectable in the caudate putamen and globus pallidus. CONCLUSION: Functional connection exists between the marginal division and hippocampus in rats.

[Expression of immediate-early genes c-fos and c-jun in the marginal division of striatum during learning and memory].

OBJECTIVE: To study the expression of immediate-early genes c-fos and c-jun in the marginal division (MrD) of rat striatum during learning and memory. METHODS: After Y-maze training in rats, the expression of immediate-early genes c-fos and c-jun in the MrD was investigated immunocytochemically. RESULTS: After 1 h of Y- maze training, the expression of c-Fos and c-Jun proteins was significantly enhanced in the MrD, where c-Jun protein in particular was more intensely expressed than in other parts of the striatum. The training group showed significantly higher expressions of the 2 proteins than pseudotraining group (P<0.01). In addition, positive expression was also observed in the hippocampus, cingulum cortex and other parts of the brain. CONCLUSION: Immediate-early genes c-fos and c-jun in the MrD participate in the signal transduction during learning and memory processes in the courses of Y-maze training of the rats.