Synchronous detection of miRNAs, their targets and downstream proteins in transferred FFPE sections: applications in clinical and basic research.

After discovering new miRNAs, it is often difficult to determine their targets and effects on downstream protein expression. In situ hybridization (ISH) and immunohistochemistry (IHC) are two commonly used methods for clinical diagnosis and basic research. We used an optimized technique that simultaneously detects miRNAs, their binding targets and corresponding proteins on transferred serial formalin fixed paraffin embedded (FFPE) sections from patients. Combined with bioinformatics, this method was used to validate the reciprocal expression of specific miRNAs and targets that were detected by ISH, as well as the expression of downstream proteins that were detected by IHC. A complete analysis was performed using a limited number of transferred serial FFPE sections that had been stored for 1-4 years at room temperature. Some sections had even been previously stained with H&E. We identified a miRNA that regulates epithelial ovarian cancer, along with its candidate target and related downstream protein. These findings were directly validated using sub-cellular components obtained from the same patient sample. In addition, the expression of Nephrin (a podocyte marker) and Stmn1 (a recently identified marker related to glomerular development) were confirmed in transferred FFPE sections of mouse kidney. This procedure may be adapted for clinical diagnosis and basic research, providing a qualitative and efficient method to dissect the detailed spatial expression patterns of miRNA pathways in FFPE tissue, especially in cases where only a small biopsy sample can be obtained.

A quantitative in situ hybridization protocol for formalin-fixed paraffin-embedded archival post-mortem human brain tissue.

The use of radioactive in situ hybridization (ISH) to quantitatively determine low-to-moderate abundant mRNA expression in formalin-fixed, paraffin-embedded archival post-mortem human brain tissue is often limited by non-specific-deposits, visible as speckles. In the present study, optimal hybridization conditions were achieved for quantifying the mRNA expression of histidine decarboxylase (HDC) by a number of alterations in a routine protocol, which included (1) during purification of the oligo-probes, glycogen was omitted as a carrier for precipitation, (2) after precipitation, the labeled probe contained within the pellet was first dissolved in water instead of in hybridization buffer (HBF), (3) during hybridization, the dithiothreitol (DTT) concentration was increased from 200 to 800 mM in HBF, and (4) stringencies during hybridization and post-hybridization washes were increased by increasing the temperature. The effect of the adjustment was quantified on adjacent sections from 18 subjects (9 with Parkinson's disease and 9 controls), by comparing the data from the standard and new protocol. The results showed that the improved protocol brought about significantly clearer background with higher signal-to-noise ratios (p=0.001). We propose that this protocol is also applicable for detection of other lower-abundant genes in human brain tissue and probably in other tissues as well. In the present study, this is not only illustrated for HDC ISH, but also for corticotrophin-releasing hormone mRNA expression in the hypothalamic paraventricular nucleus.

Uncaria rhynchophylla ameliorates unpredictable chronic mild stress-induced depression in mice via activating 5-HT1A receptor: Insights from transcriptomics.

BACKGROUND: Depression is a pervasive or persistent mental disorder that causes mood, cognitive and memory deficits. Uncaria rhynchophylla has been widely used to treat central nervous system diseases for a long history, although its efficacy and potential mechanism are still uncertain. PURPOSE: The present study aimed to investigate anti-depression effect and potential mechanism of U. rhynchophylla extract (URE). STUDY DESIGN AND METHODS: A mouse depression model was established using unpredictable chronic mild stress (UCMS). Effects of URE on depression-like behaviours, neurotransmitters, and neuroendocrine hormones were investigated in UCMS-induced mice. The potential target of URE was analyzed by transcriptomics and bioinformatics methods and validated by RT-PCR and Western blot. The agonistic effect on 5-HT1A receptor was assayed by dual-luciferase reporter system. RESULTS: URE ameliorated depression-like behaviours, and modulated levels of neurotransmitters and neuroendocrine hormones, including 5-hydroxytryptamine (5-HT), 5-hydroxyindole acetic acid (5-HIAA), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), corticosterone (CORT), corticotropin-releasing hormone (CRH), and adrenocorticotropic hormone (ACTH), in UCMS-induced mice. Transcriptomics and bioinformatics results indicated that URE could regulate glutamatergic, cholinergic, serotonergic, and GABAergic systems, especially neuroactive ligand-receptor and cAMP signaling pathways, revealing that Htr1a encoding 5-HT1A receptor was a potential target of URE. The expression levels of downstream proteins of 5-HT1A signaling pathway 5-HT1A, CREB, BDNF, and PKA were increased in UCMS-induced mice after URE administration, and URE also displayed an agonistic effect against 5-HT1A receptor with an EC50 value of 17.42 µg/ml. CONCLUSION: U. rhynchophylla ameliorated depression-like behaviours in UCMS-induced mice through activating 5-HT1A receptor.

Apomorphine-induced turning behavior in 6-hydroxydopamine lesioned rats is increased by histidine and decreased by histidine decarboxylase, histamine H1 and H2 receptor antagonists, and an H3 receptor agonist.

The role of histamine and its receptors in basal ganglia neurocircuitry was assessed in apomorphine-induced turning behavior. Rats with unilateral 6-hydroxydopamine lesions of the substantia nigra pars compacta and medial forebrain bundle were administered histaminergic agents, and apomorphine-induced turning behavior was tested on Days 7 and 14 post-lesion. Compared with saline-treated rats, histidine (500 mg/kg, i.p.), a precursor of histamine, increased turning behavior (p<0.05), while alpha-fluoromethylhistidine (alpha-FMH, 25 microg, i.c.v.), an irreversible inhibitor of histidine decarboxylase, decreased turning behavior (p<0.05) but only on Day 14 post-lesion. Both the histamine H(1) receptor antagonist pyrilamine (10 and 50 microg, i.c.v.) and the H(2) receptor antagonist cimetidine (10 and 50 microg, i.c.v.) significantly decreased turning behavior on Days 7 and 14 post-lesion. The histamine H(3) receptor agonist immepip (10 microg, i.c.v.) decreased turning behavior (p<0.05) on Day 14 post-lesion. The present findings indicate the complex interactions of histamine on basal ganglia function.

[Landscape pattern analysis and optimum design of park green space in Nanchang City, China based on GIS.] / 基于GISçš„å—æ˜Œå¸‚å ¬å›­ç»¿åœ°æ™¯è§‚æ ¼å±€åˆ†æžä¸Žä¼˜åŒ–è®¾è®¡.

Based on the current map data of park green space in the main urban area of Nanchang, the spatial database of park green space was set up with GIS technique, with the corresponding landscape indices being calculated by FRAGSTATS, the software of landscape pattern. Based on analyzing current landscape pattern of green space in Nanchang, the optimization strategy and scheme were proposed and the optimized landscape pattern was evaluated. The results indicated that the spatial distribution of patches in current park green space was uneven and area discrepancy was large, which is especially true in densely populated areas with less patch number of park green space and obviously low available area for disaster shelter. By substantially increasing the quantity and area of patches, improving the inter-patch connectivity, and increasing landscape fragmentation index appropriately, the "point-line-plane" pattern of park green space system in Nanchang would be optimized and the spatial distribution would be more rational, which could effectively enhance its role in biodiversity conservation, disaster prevention, and risk avoidance. The optimized indices of patch diversity, evenness and aggregation would be significantly increased, the dominance index would be reduced, and the landscape diversity would be more abundant.

Involvement of brain endogenous histamine in the degeneration of dopaminergic neurons in 6-hydroxydopamine-lesioned rats.

Previous studies have suggested that brain histamine is involved in the pathogenesis of Parkinson's disease (PD), but the role of endogenous histamine in the degeneration of dopaminergic neurons in the substantia nigra pars compact (SNpc) remains unclear. We aimed to investigate this issue by changing the brain histamine levels by giving histaminergic agents, and administrating histamine receptor antagonists in the PD animal model, i.e. the 6-hydroxydopamine (6-OHDA)-lesioned rat. In saline-treated animals, 6-OHDA infusion produced a progressive increase in apomorphine-induced turning rate and a loss of tyrosine hydroxylase immunoreactive (TH-ir) neurons in the SNpc. Histaminergic agents were given prior and daily for 1, 7 or 14 days after 6-OHDA infusion. Histidine (500 mg/kg, i.p.), a precursor of histamine, increased the turning rate (27% on day 7 and 26% on day 14, respectively; P<0.05) and also the loss of TH-ir neurons, but only on day 1 and 7 (67% vs 47% and 90.4% vs 74% loss, respectively; P<0.05). In contrast, alpha-fluoromethylhistidine (alpha-FMH, 25 microg, i.c.v.), an irreversible inhibitor of histidine decarboxylase (HDC), significantly decreased the turning rate (25% on day 7 and 26% on day 14, respectively; P<0.05) and prevented the loss of TH-ir neurons, also only on day 1 and day 7 (28% vs 47% and 58% vs 74% loss, respectively; P<0.05). In addition, the histamine H(1) receptor antagonist pyrilamine (5 microg, i.c.v.), but not the H(2) receptor antagonist cimetidine (5 microg, i.c.v.), also decreased the turning rate (38% on day 7 and 21% on day 14, respectively; P<0.05) and prevented the loss of TH-ir neurons on day 1 and day 7 (38% vs 51% and 60% vs 78% loss, respectively; P<0.05). On day 14 after 6-OHDA lesion, there were no significant differences in the number of TH-ir neurons among all the different treatment groups. Taken together, these findings indicate that endogenous histamine may accelerate the degeneration of dopaminergic neurons via its H(1) receptor, while attenuation of histamine transmission may play a protective role on it in the early stage of development of 6-OHDA lesioned PD rats.

Neuronal histamine production remains unaltered in Parkinson's disease despite the accumulation of Lewy bodies and Lewy neurites in the tuberomamillary nucleus.

Neuronal histamine production in the hypothalamic tuberomamillary nucleus (TMN) was hypothesized to change significantly in Parkinson's disease (PD) in relation to the accumulation of Lewy bodies/Lewy neurites (LBs/LNs). We measured the messenger ribonucleic acid (mRNA) levels of histidine decarboxylase (HDC), the key enzyme of histamine production, and the amount of LBs/LNs in the TMN by quantitative in situ hybridization and immunocytochemistry in postmortem human brain material of clinical PD (CPD), preclinical PD, and control subjects. No significant difference of histidine decarboxylase mRNA levels was observed among different clinical or Braak-PD stages, in spite of the strong accumulation of LBs/LNs in the TMN of clinical PD patients. We conclude that neuronal histamine production remains largely unaltered in PD despite the abundant LB/LN accumulation in the TMN.

Possible mechanisms of the protection of ginsenoside Re against MPTP-induced apoptosis in substantia nigra neurons of Parkinson's disease mouse model.

We have investigated the role of ginsenoside Re (Re) in preventing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced apoptosis of the substantia nigra neurons in the mouse model of Parkinson's disease (PD). C57BL mice have been administrated i.s.c. with MPTP to establish the PD model. Pretreatment groups were given different doses of Re (6.5, 13, 26 mg kg(-1)) i.g. for 13 days. Transmission electron microscope (TEM), tyrosine hydroxythase (TH) immunostaining and TDT-mediated dUTP nick-end labeling (TUNEL) staining have been used to observe the damage of substantia nigral neurons. To measure the expression of inducible nitric oxide synthase (iNOS), Bcl-2, Bax protein and expression of Bcl-2, Bax gene, immunohistochemistry and in situ hybridization have been explored respectively. Western blot analysis has been performed with anti-caspase-3. Pretreatment with Re (13, 26 mg kg(-1)) markedly increases TH-positive neurons and decreases the TUNEL-positive ratio compared with the MPTP model group. Furthermore, Re could enhance the expression of Bcl-2 protein and Bcl-2 mRNA, but reduce the expression of Bax, Bax mRNA, and iNOS, and weaken the cleavage of caspase-3. In summary, ginsenoside Re showed protection from MPTP-induced apoptosis in the PD model mouse nigral neurons and this effect may be attributable to upregulating the expression of Bcl-2 protein, downregulating the expression of Bax, and iNOS protein, and inhibiting the activation of caspase-3.