Alterations in hippocampal GABA
A/central benzodiazepine receptor (GABA
A/cBZR) expression and mossy fibre sprouting (MFS) may have aetiological significance in temporal lobe epileptogenesis. Their ...relationship with each other is also unknown. We utilised
3H-flumazenil autoradiography to quantify changes in GABA
A/cBZR density and affinity in all hippocampal laminae, and Timm's staining for MFS at different stages of epileptogenesis in the amygdala kindling rat model (after 24 stimulations, 48 stimulations and two weeks post-kindling). During kindling, receptor density was significantly elevated within the dentate stratum moleculare and granulosum, but decreased within the stratum radiatum of CA3 and CA2. Two weeks post-kindling, receptor density remained upregulated in the dentate stratum moleculare and was also upregulated in CA3 stratum oriens and CA1 stratum moleculare. MFS was significantly increased in the dentate stratum moleculare at two weeks post-kindling, with a strong inverse correlation between MFS and GABA
A/cBZR density in this region. No changes in GABA
A/cBZR binding affinity were detected for any hippocampal subregion at any time point. Our results demonstrate that changes in hippocampal GABA
A/cBZR expression are lamina- and time-specific. Within the dentate gyrus, receptor density is upregulated throughout epileptogenesis, whilst within the hippocampus proper, receptor density is downregulated early in epileptogenesis but upregulated at the chronic phase. A novel association between MFS and GABA
A/cBZR density has been demonstrated by this study, which could represent an important compensatory or pathological mechanism associated with epileptogenesis.
Purpose
11
CFlumazenil shows promise as a clinical and research PET radiotracer to image changes in GABA
A
central benzodiazepine receptor (cBZR), but its widespread use has been limited by ...practical limitations of
11
C. This study evaluated the imaging characteristics of two fluorinated PET radiotracers in rats in vivo:
18
Ffluoroflumazenil (
18
FFFMZ) and
18
Fflumazenil (
18
FFMZ).
Methods
PET acquisitions were performed on a small-animal scanner following injection of
18
FFFMZ in nine rats and
18
FFMZ in eight rats. The following treatments were investigated: (1) injection of the tracer dose, (2) presaturation then injection of the tracer dose, and (3) injection of the tracer dose followed by a displacement injection. Unchanged tracer was measured in plasma and brain structures in four animals 10 and 30 min after injection, and ex-vivo autoradiography was also performed.
Results
For both
18
FFFMZ and
18
FFMZ maximal brain activity peaked rapidly, and was highest in the hippocampus (1.12±0.06 SUV, 1.24±0.10 SUV, respectively), and lowest in the pons (1.00±0.07 SUV, 1.03±0.09 SUV, respectively). By 50 min after injection, maximal uptake for
18
FFFMZ and
18
FFMZ had decreased in the hippocampus to 18±3% and 80±1% (
p
<0.01), respectively. The presaturation and displacement studies showed a higher nonspecific component for
18
FFFMZ than for
18
FFMZ. Metabolite studies showed that at 30 min only 10% of the signal was from
18
FFFMZ in the brain. This nonspecific binding was apparent on autoradiography. In contrast,
18
FFMZ accounted for >70% of the signal in the brain, which resulted in well-defined regional binding on autoradiography.
Conclusion
These results demonstrate that
18
FFMZ is a superior radiotracer to
18
FFFMZ for in-vivo PET imaging of the GABA
A
/cBZR, having slower metabolism and leading to lower concentrations of metabolites in the brain that results in a substantially better signal-to-noise ratio.
Summary
1. Using synthetic proteinase‐activated receptor‐2 (PAR2)‐activating peptides (PAR2APs) corresponding to the tethered ligand domain of the extracellular N‐terminus of PAR2 to mimic the ...actions of activating proteinases and using primary cultures of calvarial osteoblasts derived from both wild‐type (WT) and PAR2‐null (KO) mice, we investigated the potential role of PAR2 in regulating osteoblast function.
2. Primary calvarial osteoblasts from WT and KO mice were evaluated for their growth kinetics and mineralization in the absence of PAR2 agonists and for their responses in a variety of functional assays to the PAR2APs Ser‐Leu‐Ile‐Gly‐Arg‐Leu‐amide (SLIGRL‐NH2) and 2‐furoyl‐Leu‐Ile‐Gly‐Arg‐Leu‐Orn‐amide (2‐fLIGRLO‐NH2), as well as to trypsin.
3. In contrast with WT cells, PAR2‐KO osteoblasts did not exhibit increased collagen Type I mRNA expression in response to SLIGRL‐NH2. When grown in serum‐containing medium, KO cells increased in number more rapidly than WT cells, an effect that could be attributed to decreased apoptosis rather than increased proliferation. Surprisingly, in both WT and KO osteoblasts, the two PAR2APs induced mobilization of intracellular calcium stores. Similarly, the PAR2APs inhibited serum deprivation‐induced apoptosis and parathyroid hormone‐, 1,25‐dihydroxyvitamin D3‐ or interleukin‐11‐induced mineralization in WT and KO cells.
4. We conclude that PAR2 plays a role in osteoblast survival and collagen Type I mRNA induction and that osteoblasts can respond to the PAR2APs via both PAR2‐dependent and ‐independent mechanisms.
Contributing to bone loss with aging is a progressive reduction in osteoblast number and function leading to decreased bone formation. In aging bone, mesenchymal stem cells decrease in number and ...their differentiation potential into osteoblasts is reduced. Instead, there is a shift towards adipogenic differentiation and increased lipid accumulation in the marrow of osteoporotic bones. Bone marrow adipocytes produce palmitic acid (PA), a saturated fatty acid, which is toxic to osteoblasts in vitro. Vitamin D (1,25(OH)
D
) stimulates osteoblastogenesis and has known anti-apoptotic effects on osteoblasts, as such it may protect human primary osteoblasts from PA-induced lipotoxicity. Here, the effects of PA (250 μM) or 1,25(OH)
D
(10
M), alone or in combination, on osteoblast differentiation and mineralization, viability and autophagy were investigated. In PA-treated osteoblasts, 1,25(OH)
D
ameliorated the decrease in the mRNA transcript abundance of representative palmitoylation (ZDHHC1, ZDHHC2 and ZDHHC12) and osteogenic (alkaline phosphatase and osteocalcin) genes. Collectively these gene regulate signaling pathways pertinent to osteoblastogenesis. In osteoblasts treated with PA and 1,25(OH)
D
, the capacity to undergo differentiation and mineralization was recovered and cell viability was increased when compared to osteoblasts treated with PA alone. 1,25(OH)
D
, irrespective of PA treatment, increased the expression of key osteogenic signaling proteins; specifically, SMAD1-3,5, Runx2 and β-catenin. 1,25(OH)
D
also attenuated the high level of impaired autophagy induced by PA and potentiated a shift towards activated, functional autophagy and increased flux through autolysosomes. Altogether, these findings provide in vitro evidence regarding the potential of 1,25(OH)
D
to protect osteoblasts from lipotoxicity by modulating autophagy and facilitating cell differentiation, which may enhance bone formation in an osteoporotic microenvironment with a high level of marrow adipose tissue.
Recently, a high affinity amylin binding site was identified in the
mouse α-TSH thyrotroph cell line. In this study, we have
characterized binding sites for 125I-salmon calcitonin
(125I-sCT), ...125I-rat α-calcitonin
gene-related peptide (125I-CGRP), and 125I-rat
amylin in α-TSH cells. Using 125I-CGRP or
125I-rat amylin, equilibrium was rapidly reached, and
binding was fully reversible. Competition binding revealed the relative
potency of peptides was sCT>amylin, CGRP≫rCT, which is similar to
the specificity profile of amylin receptors characterized in rat brain.
Furthermore, specific binding of 125I-rat amylin and
125I-CGRP to membrane preparations was reduced by 52% and
39%, respectively, in the presence of 20 μm GTP-γ-s,
indicating a requirement of G protein coupling for high affinity
binding. In contrast, 125I-sCT binding reached equilibrium
more slowly, was essentially irreversible, and was unaltered by
GTP-γ-s. Competition binding studies using 125I-sCT as
radioligand demonstrated only weak interaction by CGRP or amylin,
consistent with other described CT receptors. Assessment of
ligand-induced cAMP accumulation and intracellular calcium signaling
revealed a relative specificity profile of sCT>rCT with little or no
second messenger signaling stimulated by amylin or CGRP, consistent
with a C1-CT receptor phenotype. RT-PCR amplification of messenger RNA
indicated that the predominant isoform was the C1a CT receptor. In
cross-linking studies, 125I-rat amylin and
125I-CGRP specifically labeled a major band of relative
molecular mass (Mr) approximately 80K, being approximately
10 kDa higher than the major 125I-sCT binding protein. Full
deglycosylation of N-linked carbohydrates with endoglycosidase F
reduced the Mr of each of the labeled proteins to
approximately 50K. Cross-linked amylin or CT receptors were
immunoprecipitated with C-terminally directed antimouse or antirat CT
receptor antibodies but were not immunoprecipitated with nonimmune sera
or antihuman CT receptor antibodies. The current data demonstrate
expression of two biochemically distinct receptor phenotypes in mouseα
-TSH cells, a CT receptor phenotype and an amylin receptor phenotype
that have highly similar protein backbones.
Imbalance of inhibitory GABAergic neurotransmission has been proposed to play a role in the pathogenesis of temporal lobe epilepsy (TLE). This study aimed to investigate whether .sup.18 F-flumazenil ...(.sup.18 F-FMZ) PET could be used to non-invasively characterise GABA.sub.A /central benzodiazepine receptor (GABA.sub.A /cBZR) density and affinity in vivo in the post-kainic acid status epilepticus (SE) model of TLE. Dynamic .sup.18 F-FMZ -PET scans using a multi-injection protocol were acquired in four male wistar rats for validation of the partial saturation model (PSM). SE was induced in eight male Wistar rats (10 weeks of age) by i.p. injection of kainic acid (7.5-25 mg/kg), while control rats (n = 7) received saline injections. Five weeks post-SE, an anatomic MRI scan was acquired and the following week an .sup.18 F-FMZ PET scan (3.6-4.6 nmol). The PET data was co-registered to the MRI and regions of interest drawn on the MRI for selected structures. A PSM was used to derive receptor density and apparent affinity from the .sup.18 F-FMZ PET data. Alterations to hippocampal GABA.sub.A /cBZR density and affinity in the post-kainic acid SE model of TLE are detectable in vivo with .sup.18 F-FMZ PET and a PSM. These changes are independent from hippocampal cell and volume loss. .sup.18 F-FMZ PET is useful for investigating the role that changes GABA.sub.A /cBZR density and binding affinity play in the pathogenesis of TLE.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Studies report that ...C-flumazenil (FMZ) PET more specifically localizes the epileptogenic zone in patients with medically refractory focal epilepsy than ...F-FDG PET. However, practical aspects of ......C use limit clinical application. We report a phase I/IIa study assessing the clinical use of ...F-FMZ PET for the localization of the epileptogenic zone in patients with drug-resistant temporal lobe epilepsy (TLE). Receptor binding was quantified using kinetic modeling that did not require arterial sampling. Dynamic ...F-FMZ PET and static interictal ...F-FDG PET scans were compared in healthy controls (n = 17 for ...F-FMZ and n = 20 for ...F-FDG) and TLE patients with mesial temporal sclerosis on MR imaging (MTS, n = 12) and with normal MR imaging (NL TLE, n = 19). Masked visual assessment of images was undertaken. Parametric images of ...F-FMZ binding potential (BP...) were generated using the simplified reference tissue model. Region-of-interest analysis on coregistered MR images and statistical parametric mapping were used to quantify ...F-FMZ BP... and ...F-FDG uptake in the temporal lobe. The visual assessment of static standardized uptake value images showed ...F-FMZ PET to have high specificity (16/17 94%) and moderate sensitivity (21/31 68%) for the localization of the epileptogenic zone, with a more restricted abnormality than ...F-FDG PET. However, the ...F-FMZ standardized uptake value images were falsely localizing in 3 of 31 patients (10%). Region-of-interest analysis demonstrated reductions in ipsilateral hippocampal ...F-FMZ BP... in patients with either MTS or NL TLE, compared with controls subjects. Ipsilateral hippocampal ...F-FMZ BP... was independent of both hippocampal volume and ...F-FDG uptake, whereas ipsilateral hippocampal volume was correlated with ...F-FDG uptake (r... = 0.69, P < 0.0001). Statistical parametric mapping analysis demonstrated decreased uptake in 14 of 31 (45%) cases with ...F-FMZ PET and 18 of 29 (62%) with ...F-FDG PET. Cluster size was significantly smaller on ...F-FMZ than ...F-FDG images (37 vs. 160 voxels, P < 0.01). ...F-FMZ PET has potential as a clinical tool for the localization of the epileptogenic zone in the presurgical evaluation of drug-resistant TLE, providing information complementary to ...F-FDG PET, with a more restricted region of abnormality. (ProQuest: ... denotes formulae/symbols omitted.)
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