Paraformaldehyde Research Articles

Subretinal injection of amyloid-β peptide accelerates RPE cell senescence and retinal degeneration.

Drusen are considered a hallmark characteristic of age-related macular degeneration (AMD). In our previous study, we found that amyloid-β (Aβ) peptide, a component of drusen, induced the cells of the retinal pigment epithelium (RPE; RPE cells) to enter senescence; however, its effects in vivo remain unknown. Thus, the present study was carried out to explore the in vivo effects of Aβ peptide on RPE cell senescence and senescence-associated inflammation in C57BL/6 mice. C57BL/6 mice received a subretinal injection of Aβ(1-42) peptide; on day 7 post-injection, the mice were anesthetized and subjected to whole-body perfusion with 4% paraformaldehyde (PFA) in PBS and the whole eyes were then enucleated. Retinal function was assessed by electroretinography (ERG), and the morphological characteristics of the retina were examined by light and electron microscopy. Fundus autofluorescence (FAF) was examined by confocal scanning laser ophthalmoscopy (cSLO). The expression of p16INK4a, a marker of cellular senescence, was examined by immunofluorescence staining and western blot analysis. The RPE-choroid was analyzed for cytokine expression by RT-PCR. In Aβ(1-42)-injected mice, scotopic ERG responses declined. Degenerative alterations, including the disruption of the inner segment (IS)/outer segment (OS) junction and extensive vacuolation and thickness of Bruch's membrane (BrM) were observed under a a light microscope. The accumulation of vacuoles and the loss of basal infoldings in the RPE were identified using an electron microscope. FAF and p16INK4a expression increased in Aβ(1-42)-injected mice. In addition, Aβ(1-42) upregulated interleukin (IL)-6 and IL-8 gene expression in the RPE-choroid. In conclusion, our results confirm the effects of Aβ(1-42) peptide on RPE senescence in vivo. The Aβ-injected mice developed AMD-like ocular pathology. It is thus suggested that RPE cell senescence is a potential mechanistic link between inflammation and retinal degeneration.

Molecular size distribution in synthesis of polyoxymethylene dimethyl ethers and process optimization using response surface methodology

Polyoxymethylene dimethyl ethers (PODEn, CH3O(CH2O)nCH3, where n⩾1) are ideal diesel fuel additives. Among the PODEn compounds, PODE3–5 have the best properties as diesel additives. A theoretical analysis of the molecular size distribution of PODEn synthesized from dimethoxymethane (DMM) and paraformaldehyde (PF) was performed based on a sequential reaction mechanism. The molecular size distribution model follows the Schulz–Flory distribution, and showed a good prediction ability at different reaction temperatures (T) and DMM/CH2O mole ratios (M), which verified the sequential reaction mechanism during the formation of PODEn. The product distribution was optimized using the molecular size distribution model and response surface methodology (RSM). At optimum operating conditions of T=105°C and M=1.1, the conversion of formaldehyde XCH2O has a high value of 92.4%, and the fraction of PODE3–5 in the PODEn mixture is 33.2wt%, while the fractions of PODEn>5 and PODE2 are 9.4wt% and 24.3wt%, respectively.

Ultrastructural evaluation of shrinkage artefacts induced by fixatives and embedding resins on osteocyte processes and pericellular space dimensions.

The integrity of the interface between the osteocyte (Ot) process and the canalicular wall was investigated in terms of change in the lateral dimensions of the Ot process in relation to the canalicular width, i.e., widening of the pericellular space. This has been interpreted as shrinkage of the Ot process relative to the canalicular wall during sample preparation stages of fixation, dehydration, and resin embedding. Sprague-Dawley rat tibial cross-sections were prepared for transmission electron microscopy (TEM). Four different fixative preparations: paraformaldehyde (PF), modified Karnovsky's (MK), glutaraldehyde (GRR) with ruthenium red (GRR), and zinc formalin (ZF); and two different embedding resins: LR Gold (LRG) and Epon812 (Epon) were evaluated. It was found that for LRG embedding, formalin-only fixatives (PF and ZF) induced lower shrinkage than GRR-containing fixatives (MK and GRR). In contrast, for Epon embedding, MK showed the highest shrinkage, while no differences were found between the remaining fixatives (PF, ZF, and GRR). All formalin-containing fixatives (MK, PF, and ZF) induced similar shrinkage in both embedding media. The most dramatic difference was for GRR fixation, which in combination with LRG embedding showed ∼ 62% more shrinkage than with Epon embedding, suggesting that the combination of GRR fixation and LRG embedding synergistically amplifies Ot shrinkage. These differences likely suggest a role of the resin in secondarily influencing the tissue structure following fixation. Further, the work confirms LRG as a poor embedding medium for bone specimens, as it causes large variations in shrinkage depending on fixation.

Effects of varying doses of simvastatin on protein kinase A in immature rabbits with chronic heart failure

Objective To investigate the effects of varying doses of simvastatin (SIM) on protein kinase A(PKA), ventricular remodeling and heart function in immature rabbits with chronic heart failure (CHF), to explore the possible mechanisms for simvastatin on CHF. Methods Sixty male immature rabbits were randomly divided into 5 groups: control (CON) group, CHF model group, small-dose simvastatin (SD-SIM) group[SIM concentration was 0.3 mg/(kg·d)], medium-dose simvastatin (MD-SIM)group[SIM concentration was 1.5 mg/(kg·d)], and the high-dose simvastatin (HD-SIM)group[SIM concentration was 3.0 mg/(kg·d)]. The general condition of immature rabbits in the experiment group was evaluated.Color doppler ultrasonography was used to detect the left ventricular structure and the function.The left ventricular myocardium was taken and fixed in paraformaldehyde (40 g/L). The expression of PKA was evaluated by immunohistochemical assay. Results In CON group, the rabbits were all alive; in the other groups, most immature rabbits were unhairing, with diet reduction, weight lose and sluggish with hyperkeratosis.Overall survival rate was 82%(49/60 cases). The mortality rate in CHF model group was significantly higher than that in the MD-SIM group(41.7% vs 8.3%, P<0.05). Compared with the CON group, left ventricular ejection fraction(LVEF) in the SD-SIM group, HD-SIM group and CHF model group all dropped sharply (all P<0.01), while the left ventricular end diastolic diameter (LVEDd) and left ventricular end systolic diameter (LVESd) increased markedly (all P<0.01). Compared with CHF model group, LVEDd and LVESd decreased and LVEF increased significantly in SD-SIM group, HD-SIM group and MD-SIM group(all P<0.05), the effects of MD-SIM group was the best(P<0.05). LVEF in MD-SIM group increased significantly compared with SD-SIM group, HD-SIM group and CHF model group (all P<0.05). PKA expression in SD-SIM group, MD-SIM group and HD-SIM group increased significantly compared to the CHF model group and CON group (all P<0.01). Compared with the SD-SIM group and HD-SIM group, the expression of MD-SIM group significantly increased (P<0.05). Conclusions Simvastatin can reverse the left ventricular remodeling and improve the heart function of immature rabbits with CHF.The effect of MD-SIM was the best.The mechanism of simvastatin may be correlated to up-regulating the expression of PKA. Key words: Simvastatin; Adriamycin; Chronic heart failure; Protein kinase A

Application of aluminum foil on tissue molding before 4% paraformaldehyde fixation

除了组织标本的取材因素,取材标本的外形构建常常是影响实验结果或病理诊断的重要因素[1-2].良好的组织标本塑形,不但能确保组织学染色效果和标本信息的准确获取,而且能节省研究人员的体力和时间,提高效率,减少标本损耗.但是,许多组织标本,如富含致密结缔组织及水分的组织器官,在早期的化学固定和机器脱水处理之后,质地常常变得硬、脆,甚至卷曲、皱缩,此时进行塑形处理则标本容易受损,甚至碎裂[3-4];即使在随后的石蜡包埋过程中采用热石蜡浸泡,塑形效果仍不理想,且器械的钝性塑形过程常不同程度地破坏标本的组织学结构.因此,在实践中探索一种简单易行的组织标本塑形方法显得尤为重要。

Influence of imidazole and benzotriazole on electroless copper plating

In this study, the effect of imidazole and benzotriazole as primary stabilisers in saccharose and xylitol based copper electroless baths were studied. Copper methane sulphate was used as the complexing agent instead of copper sulphate and para formaldehyde was used as the reducing agent in the bath. The surface morphologies of copper deposits were characterised by SEM, AFM and XRD studies. The electrochemical characteristics were studied by cyclic voltametry to understand the role of the stabilisers in electroless deposition. In the saccharose bath, the imidazole and benzotriazole acted as inhibitors and best deposition was obtained at pH of 12·75. Benzotriazole afforded smoother and shiner deposits than imidazole. In xylitol bath, the stabilizers acted as accelerators and optimum deposition occurred at a pH of 13·25.

Quality improvements of cell membrane chromatographic column

Cell Membrane Chromatography (CMC) is a biological affinity chromatographic method using a silica stationary phase covered with specific cell membrane. However, its short life span and poor quality control was highlighted in a lot of research articles. In this study, special attention has been paid to the disruption, cell load and packing procedure in order to improve the quality of the CMC columns. Hereto, two newly established CMC models, HSC-T6/CMC and SMMC-7721/CMC have been developed and used in this research project. The optimization of the abovementioned parameters resulted in a better reproducibility of the retention time of the compound GFT (RSD<10%) and improved significantly the quality of the CMC columns. 3.5×107cells were the optimal cell load for the preparation of the CMC columns, the disruption condition was optimized to 5 cycles (400W and 20s interval per cycle) by an ultrasonic processor reducing the total time of cell disruption to 1.5min and the packing flow rate was optimized by applying a linear gradient program. Additionally, 4% paraformaldehyde (PFA) was employed to improve the column quality and prolong the column life span. The results showed that the retention time was longer with PFA treated columns than the ones obtained with the control groups

Inside Alzheimer brain with CLARITY: senile plaques, neurofibrillary tangles and axons in 3-D.

Inside Alzheimer brain with CLARITY: senile plaques, neurofibrillary tangles and axons in 3-D.

Effective melanin depigmentation of human and murine ocular tissues: an improved method for paraffin and frozen sections.

PurposeThe removal of excessive melanin pigments that obscure ocular tissue morphology is important to address scientific questions and for differential diagnosis of ocular tumours based on histology. Thus, the goal of the present study was to establish an effective and fast melanin bleaching method for paraffin and frozen mouse and human ocular tissues.MethodsParaffin-embedded and frozen ocular specimens from mice and human donors were subjected to bleaching employing two methods. The first employed potassium permanganate (KMnO4) with oxalic acid, and the second 10% hydrogen peroxide (H2O2). To determine optimal bleaching conditions, depigmentation was carried out at various incubation times. The effect of diluents used for 10% H2O2 was assessed using phosphate-buffered saline (PBS), and deionized water. Three different slide types and two fixatives, which were ice-cold acetone with 80% methanol, and 4% paraformaldehyde (PFA) were used to determine the optimal conditions for better tissue adherence during bleaching. All tissues were stained in hematoxylin and eosin for histological evaluation.ResultsOptimal bleaching was achieved using warm 10% H2O2 diluted in PBS at 65°C for 120 minutes. Chromium-gelatin-coated slides prevented tissue detachment. Adherence of cryosections was also improved with post-fixation using 4% PFA and overnight air-drying at RT after cryosectioning. Tissue morphology was preserved under these conditions. Conversely, tissues bleached in KMnO4/oxalic acid demonstrated poor depigmentation with extensive tissue damage.ConclusionsWarm dilute H2O2 at 65°C for 120 minutes rapidly and effectively bleached both cryo- and paraffin sections of murine and human ocular tissues.

Modified technique of whole retinal capillary network mounting for cells and capillaries counting

Background Previous retinal vascular mounting only obtained part of retinal vessels for the study of retinal diseases,and thus it is difficult to comprehensively assess these diseases.So optimizing the trypsin digestion method to show the complete retinal capillary network is very important for the study of retinal diseases.Objective This study was to modify the preparing way of trypsin digested whole retinal capillary network and offer a basis for a quantitative analysis of cells and capillaries.Methods Thirty Wistar rats were divided into model group (20 rats) and control group (10 rats).Streptozotocinum(STZ) of 1.25％ dissolved in 0.05 mmol/L sodium citratehydrochloric acid buffer was intraperitoneally injected to establish diabetes models in the model group,and the equal volume of solvent was used in the same way in the control group.Eight months after injection,100 ml PBS was injected via ventriculus sinister and released via cut right atrium,and then 100 ml 4％ paraformaldehyde was injected into the ventriculus sinister.The rat retinas containing part of the optic nerve were entirely isolated,and digested by trypsin,and vitreous,inner limiting membrane and neural retinal tissue were removed.The whole retinal capillaries network was mounted on the slide.The ghost pericytes and acellular capillaries in the middle retinal area were identified and counted under the optical microscope.Results The retinal mount exhibited that the retinal vessels were stained by hematoxylin and periodic acid schiff.The vessels network presented with the entire type in shape with the radical central retinal arteries and veins and their branches.The capillary showed the shallow-and deep-layer networks between the small arteries and veins.Pericytes distributed and protruded vessel wall and formed the ghost cells without nuclei.The diameter of acellular capillaries was 20％ or more than that of near capillaries,and no cellular nuclei or ghost cell was found through the vessel.Conclusions The experimental technique for setting-up of cleaned vasculature and mounting vessels on glass microscopic slide provides intact vessels,which is helpful for the evaluation of retinal vascular morphology and quantitative analysis. Key words: Retina; Trypsin digestion method; Rat/Wistar; Retinal vessels/cytology

Laminin α1 Regulates Age-Related Mesangial Cell Proliferation and Mesangial Matrix Accumulation through the TGF-β Pathway

Laminin α1 (LAMA1), a subunit of the laminin-111 basement membrane component, has been implicated in various biological functions in vivo and in vitro. Although LAMA1 is present in kidney, its roles in the kidney are unknown because of early embryonic lethality. Herein, we used a viable conditional knockout mouse model with a deletion of Lama1 in the epiblast lineage (Lama1(CKO)) to study the role of LAMA1 in kidney development and function. Adult Lama1(CKO) mice developed focal glomerulosclerosis and proteinuria with age. In addition, mesangial cell proliferation was increased, and the mesangial matrix, which normally contains laminin-111, was greatly expanded. In vitro, mesangial cells from Lama1(CKO) mice exhibited significantly increased proliferation compared with those from controls. This increased proliferation was inhibited by the addition of exogenous LAMA1-containing laminin-111, but not by laminin-211 or laminin-511, suggesting a specific role for LAMA1 in regulating mesangial cell behavior. Moreover, the absence of LAMA1 increased transforming growth factor (TGF)-β1-induced Smad2 phosphorylation, and inhibitors of TGF-β1 receptor I kinase blocked Smad2 phosphorylation in both control and Lama1(CKO) mesangial cells, indicating that the increased Smad2 phosphorylation occurred in the absence of LAMA1 via the TGF-β1 receptor. These findings suggest that LAMA1 plays a critical role in kidney function and kidney aging by regulating the mesangial cell population and mesangial matrix deposition through TGF-β/Smad signaling.

Nanopodia--thin, fragile membrane projections with roles in cell movement and intercellular interactions.

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 μm diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per μm and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 μm from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 °C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.

Morphological Study of Surgically Obtained Human Cochlear Specimens - Technical Aspects

ObjectTo explore the procedures in per-operative harvesting and management of fresh human cochlear specimens for research. MethodsDuring trans-cochlear surgery to remove large petro-clival meningiomas causing life-threatening compression on the brainstem, cochleae are normally destroyed and drilled away in order to reach the apical petrous and clivus region. Instead the cochlea can be dissected out after ethical permission was obtained from the local ethical committee (EPN) and allowance gained from the patients. Surgery is performed by a team consisting of oto- and neurosurgeons as a two-day procedure with total petrosectomy in combination with an inferior re-routing of the facial nerve. Fixation of the cochleae was done in the operating room as soon as the specimens had been separated from the temporal bones. Decalcification began after hours’ to overnight’s fixation for 4 weeks. Sectioning parallel to the modiolus (mid-modiolus) was performed with a cryostat microtome. The sections were subjected to immunofluorescence (IF). ResultsUsing freshly prepared 4% paraformaldehyde (PFD) solution, adequate fixation of fine inner ear structures was achieved with hours’ immersion of the cochlear specimens. Decalcification in 6.2% ethylene diamine-tetracetic acid (EDTA) solution for 4 weeks yielded a thoroughly decalcified cochlea. Experiences in processing 14 human cochleae and analysing main landmarks in five human inner ear plastic/silicone casts showed that the oval window/stapes footplate are backward tilted, at an angle about 15 degrees, from the plane perpendicular to the modiolar axis. The distance from the modiolar apex to the anterior border of the oval window/footplate in these inner ear casts measured between 4 and 5 mm. High quality IF staining was obtained. ConclusionSurgically obtained human cochlear specimen, when properly processed, contains ideally preserved antigenicity for immunohistochemical study. Adequate orientation during sectioning helps obtain optimal mid-modiolar sections showing fine structures of the cochlea.

Effect of the FKBP51·PHLPP·Akt signal module on the phosphorylation of Akt and hippocampal neuronal injury after the cerebral ischemia/reperfusion

Objective To investigate the effects of the FKBP51·PHLPP·AKT signal module on the phosphorylation of Akt and hippocampal neuronal injury after the cerebral ischemia / reperfusion induced neuronal death in rat hippocampus. Methods Transient(15 min)brain ischemia was induced by the four-vessel occlusion in Sprague-Dawley rats. 6 rats were used in each group. The antisense oligodeoxynucletides(AS ODN)of PHLPP2(PH domain and leucine rich repeat protein phosphatases) was used to suppress the assembly of FKBP51·PHLPP·Akt signal module by intracerebroventricular infusion once per day for 3 days before ischemia. After 6 hours reperfusion, interactions of PHLPP2 and FKBP51(FK506 binding protein 5) with Akt were detected by immunoprecipitation (IP) and the phosphorylation of Akt was detected by western blot(IB). After 5 days reperfusion, rats were perfusion-fixed with paraformaldehyde and Hematoxylin-Eosin staining was used to examine the survival number of CA1 pyramidal cells of hippocampus. Results Compared to PHLPP2 MS ODN group(1.24±0.24, 1.68±0.11, 0.58±0.01), PHLPP2 AS ODN suppressed the assembly of the FKBP51·PHLPP·Akt signaling module(1.06±0.01, 1.04±0.13), and increased the phosphorylation of Akt(0.76±0.02)(P<0.05). Furthermore, compared to PHLPP2 MS ODN group (20.1±2.5), the number of surviving neurons significantly increased in PHLPP2 AS ODN group(88.3±2.7)(P<0.05). Conclusion The increasing assembly of FKBP51·PHLPP·Akt signal module can damage CA1 pyramidal cells of hippocampus by inhibiting the phosphorylation level of Akt. Key words: Global cerebral ischemia/reperfusion; PH domain and leucine rich repeat protein phosphatases; FK506 binding protein 5; Protein kinase B

VGF INVOLVEMENT ON PARKINSON’S DISEASE

Parkinson’s disease (PD) is a neurological disorder characterized by a progressive degeneration of dopaminergic neurons of the substantia nigra, that results in dopamine depletion in the striatum with a deregulation of a number of substances/ neurotrasmitters. A previous study demonstrated a clear-cut decrease of certain peptides derived from the VGF gene in the parietal cortex from patients affected by PD. Hence, we used an animal model of PD, and plasma samples from PD patients and control subjects to investigate the role of VGF in PD. Rats (Sprague Dawley) were treated with injections of 6 hydroxydopamine into the medial forebrain bundle, anesthetized and perfused with 4% paraformaldehyde 3 weeks (group 1: n= 8) or 6 weeks (group 2: n=17) after injection. In addition, subcutaneous injections of L-DOPA (6mg/ Kg) were carried out one week before the sacrifice in rats from group 1 and 2 (n=4 and n=6, respectively). We raised antibodies to the C-terminal end of the human VGF sequence, for immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA, IC50 = 15 pmol/ml). For pilot experiments, human plasma samples were taken from subjects affected by PD (n=13, age range: 51–84 years), and from age matched controls (n=10), to be used for ELISA experiments. We analysed the substantia nigra (SN) of the PD rats using VGF-C-terminus antiserum in double staining with antibodies to glutamate decarboxylase (GAD), tyrosine hydroxylase (TH), serotonin and Substance P. In the control side, VGF-C-terminus peptides were found in neurone terminals/axons within the pars compacta (SNC) and reticulata (SNR) of the substantia nigra, co-localised with GAD and substance P (but not with serotonin or TH) and in closed connection with TH cell bodies of the SNC. In the PD side, while the GAD staining remains unaffected, VGF-C-terminus and substance P immnureactivities disappeared (compared to the control side) in a large population of neurone terminals of both SNC and SNR, and in relation with the TH decrease. When L-DOPA was applied to the animals, VGF-C-terminus immunoreactivity on the PD side returned comparable to the control side. By ELISA, high concentrations of proVGF, in the order of 600-2000 pmol/ml were shown in the plasma from controls with a significant decrease in PD samples (p=0,0029). In conclusion, using the animal models, we showed here for the first time that VGF is involved into nigro-striatal circuits and down-regulated in PD. Besides, of a great interest, is the finding that a deregulation of VGF can occur also in humans.

The effect of paraformaldehyde fixation and sucrose cryoprotection on metal concentration in murine neurological tissue

Aldehyde preservation of tissue and cryoprotection by sugars and polyols has traditionally been used in histology to preserve structural integrity of samples. The effect of paraformaldehyde (PFA) fixation and sucrose cryoprotection on murine brain metals was studied by examining the changes in total brain metal levels throughout a complete fixation/cryoprotection protocol. Blank-corrected metal levels in 4% PFA and two changes of 30% sucrose solutions following brain immersion were determined by ICP-MS. Fixed and cryoprotected tissue was lyophilised, digested in HNO3/H2O2 and analysed using the same ICP-MS protocol. The sum of metal loss or gain in each solution and total digest was used to determine the percentage of total brain metal present in fixed, cryoprotected tissue. Almost all K (99.78%) and Mg (78.01%) leached from brain tissue during fixation and cryoprotection, whilst Ca and Sr either retained or absorbed additional metal ions from the sucrose solution. Less water-soluble transition metals displayed smaller degrees of metal leaching, with Fe, Cu and Zn losing 26.61–31.62% of their total metal content during preparation for cryosectioning. These results suggest that metal leaching is highly metal specific, and that preparatory steps for cryosectioning of brain tissue should be approached according to this specificity.

Subcellular distribution of melatonin receptors in rat parotid salivary glands: effect of melatonin intravenous infusion

The ultrastructural localization of melatonin receptors Mt1 and Mt2 in human salivary glands has previously been reported (Isola el al, 2013). In the acinar cells, the intragranular distribution of these receptors suggested to us that they may serve as a transcytotic system for melatonin. In an attempt to verify our working-hypothesis we currently exposed anaesthetized rats to the intravenous infusion of melatonin for 2 hrs. After another 30 min, the parotid gland on the right side was removed to be compared with prestimulation removed pieces of the left parotid gland serving as control specimens. Parotid gland tissues from 5 rats were cut into small pieces and fixed with a mixture of paraformaldehyde and glutaraldehyde. They were dehydrated, and embedded in Epon Resin. The ultrathin sections were collected on nickel grids and incubated overnight at 4oC with an antibody specific for Mt1 and Mt2, respectively. The grids were then incubated for 1 hr with the appropriate secondary antibody conjugated to gold particles and, finally examined using electron transmission microscopy. In all sections, the immunoreactivity to Mt2 and, with less intensity, to Mt1 was principally expressed in secretory granules of acinar cells. Occasionally, plasma membranes were also stained, although slightly. Statistical analysis showed that, in response to melatonin, the immunogold particles reactive for Mt1, but not those for Mt2, increased. To conclude, in line with our hypothesis, melatonin stimulates an acinar carrier system, which seems predominantly to involve the Mt1 receptor, in order to delivery this hormone into saliva.

Cardiac mitochondria alteration and peripheral vessel morphology in female diabetes

One of diabetes complications is chronic cardiomyopathty and fibrotic alteration of aorta and peripheral vessels. Diabetes has been correlated to ROS superproduction (Lashin et al., 2006) and an alteration of mitochondrial chain complexes function in the whole heart (Herelin et al., 2011). Despite diabetic cardiomyopathy is most frequent in women, to date studies on female experimental diabetic models are lacking. Our aim was to investigate on heart mitochondrial oxygen phosphorylation (OXPHOS), and on aorta and portal vein morphology in female Wistar rats, after 4 and half months of streptozotocin-induced (65 mg/kg) diabetes. Mitochondrial OXPHOS was assessed by means of a Clark-type electrode on the following isolated mitochondrial subpopulations: left and right ventricles subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria. Morphology and extracellular matrix composition of aorta and portal vein were investigated in light microscopy on paraformaldehyde fixed samples, stained with Masson Trichrome method (for collagen fibers) and Weigert’s stain (for elastic fibers). Evaluation of OXPHOS revealed an impairment of complex II in mitochondrial diabetic female rats in left and right IFM, but not in SSM. Interestingly, administration of the substrate glutamate resulted in an improvement of complex I efficiency in left IFM only, while association of complex I and II substrates displayed a reduced efficiency both in left and right IFM. Neither administration of glucidic substrates on SSM or of lipidic substrates on both SSM and IFM resulted in any change of mitochondrial OXPHOS efficiency. The study of connective fibrous composition in aorta and vena porta revealed a slight more abundant collagen production in the aorta’s wall and a disorganized and fragmented aspect of elastic bundles in the portal vein. Taken together, these data suggest a peculiar unknown development of diabetic cardiopathy in female rats.

PFA-fixed Hsp60sp-loaded dendritic cells as a vaccine for the control of mouse experimental allergic encephalomyelitis

We have shown that Hsp60sp-loaded immature dendritic cells (DC/sp) can protect mice from the induction of experimental allergic encephalomyelitis (EAE) by inducing Qa-1-restricted CD8(+) T regulatory (Treg) cells. The binding half-life between Qa-1 and Hsp60sp is particularly short and leads to an unstable Qa-1/peptide complex that significantly decreases the efficacy of this vaccination. To prevent Qa-1/Hsp60sp complex dissociation, we utilized paraformaldehyde (PFA) fixation to stabilize the formation of the Qa-1/Hsp60sp complex and maximize the function of DC/sp as a vaccine to control autoimmune diseases. Compared with the non-fixed DC/sp, the fixed DC/sp (FDC/sp) showed an enhanced ability to activate Qa-1-restricted Hsp60sp-specific CD8(+)T cells in vitro and prevented EAE in vivo. Importantly, the FDC/sp maintained immune activity following cryopreservation for 1 week or after storage for 72h at 4°C. These results indicate that PFA fixation can sustain or increase the efficacy of DC/sp by improving the stability of the Qa-1/Hsp60sp complex on the surface of the DC/sp. In addition, PFA fixation creates a time window for DC/sp storage, transport and application. Our data suggest a potential clinical use of FDC/sp as a vaccine for the prevention and treatment of autoimmune disease.

Bromodeoxyuridine (BrdU)-label-retaining cells in mouse terminal bronchioles.

Adult male mice were continuously treated with bromodeoxyuridine (BrdU) for 1, 2, or 4 weeks by an osmotic pump. To detect BrdU-label-retaining cells (LRCs), putative progenitor/stem cells, other animals were continuously treated with BrdU for 2 weeks, and were then kept without any treatments for 2, 6, or 18 months. The lungs were fixed with 4% paraformaldehyde, and were paraffin-embedded. We observed terminal bronchioles with BrdU immunostaining alone or with BrdU immunostaining accompanying immunostaining for Clara cell secretory protein (CCSP), forkhead box protein J1 (FoxJ1), or calcitonin gene-related peptide (CGRP). The average incidences of BrdU-incorporated cells in the terminal bronchioles after 1, 2, and 4 weeks of continuous BrdU infusion were 6.2%, 11.9%, and 23.1%, respectively. Most BrdU-incorporated cells in these periods were CCSP-immunoreactive (91.7%, 91.3%, and 88.2%, respectively), which means progenitor function of Clara cells. FoxJ1-immunoreactive BrdU-incorporated cells were fewer (5.4%, 3.0%, 2.7%, respectively). The average incidences of BrdU-LRCs in the terminal bronchioles after 2, 6, and 18 months were 7.2%, 4.3, and 2.7%, respectively. Most BrdU-LRCs were CCSP-immunoreactive (91.0%, 92.7%, and 89.6%, respectively), and FoxJ1-immunoreactive BrdU-LRCs were fewer (6.0%, 5.7%, and 2.1%, respectively). CGRP-positive BrdU-incorporated cells were occasional. CGRP-positive BrdU-LRCs were detected in 17.6% of neuroepithelial bodies (NEBs) at 2 months, but disappeared at 6 months. BrdU-positive stem cell candidates, which locate at the brochiolo-alveolar duct junction or cover NEB, were few throughout this study. In conclusion, in the lungs treated only with BrdU, CCSP-immunoreactive cells are important to maintain homeostasis in the terminal bronchiolar epithelium.