Histological examination of the effects of epidermal growth factor on regeneration of acute peripheral nerve injuries on rabbit model.

BACKGROUND: Peripheral nerve injuries are one of the most common and costly injuries especially in the young population. In this study, it is aimed to determine the histological role of epidermal growth factor (EGF) in nerve regeneration with an acute damage made on sciatic nerve in the rabbit model. METHODS: We used 18 New Zealand rabbits (nine in control group and nine in experimental group). Each group was divided into two groups consisting of five rabbits planned for diameter measurement and four rabbits planned for spatial measurement. The sciatic nerve exploration in the right flank of each animal, full-thickness nerve damage, and then epineural repair was made by a single researcher. 10 µg/kg EGF was given to the repair area of the experimental group and five more EGF injections were given to the experimental group every other day postoperatively. In the control group, we used saline solution. Rabbits were observed for 8 weeks. During follow-up, two rabbits died. At the end of 8 weeks, the nerve tissue of each animal was evaluated histologically and morphologically. RESULTS: In the experimental group consisting of five rabbits, the mean thickness of connective tissue (epineurium+ mesoneurium) was 156,867 µm; while, in the control group, the thickness was 25,170 µm. In the other groups, the numerical increase in epineurium and mesoneurium areas was detected in the EGF (+) group as a result of the comparative spatial measurements. Epineurium and mesoneurium enlargement was observed in the EGF-given group. Adipocyte and capillary increase was observed in connective tissue. CONCLUSION: EGF increases epineurium and mesoneurium diameters in peripheral connective tissue in acute peripheral nerve injury regeneration. However, further studies are needed to understand this effect clinically and physiologically.

Modelling cognitive deficits in Parkinson's disease: Is CA2 a gateway for hippocampal synucleinopathy?

BACKGROUND: Cognitive dysfunction is one of the most disabling non-motor symptoms of Parkinson's disease (PD), though its pathological correlates still remain elusive. Hippocampal Lewy pathology has recently been correlated by compelling evidence from post-mortem and imaging studies. Animal models recapitulating cognitive impairment in PD are essential to better understand the underlying pathophysiology. To investigate the hippocampal involvement in cognitive dysfunction of PD, we generated an experimental model by inducing midbrain and hippocampal α-synuclein pathology simultaneously. METHODS: Rats were injected either with human α-synuclein or green fluorescent protein (GFP) expressing adeno-associated viral vectors (AAV), or saline bilaterally into substantia nigra (SN) and dentate gyrus (DG). A group of untreated animals were used as naïve controls. Cognitive and behavioral changes were evaluated with tests probing for spatial learning, short-term memory, anxiety and hedonistic behavior. Immunohistochemical staining, immunoblotting and stereological analysis were performed for pathological characterization. RESULTS: Bilateral α-synuclein overexpression in SN and DG led to mild but significant motor impairment as well as dysfunctions in short-term memory and spatial learning. There was no hedonistic deficit, whereas a hypo-anxious state was induced. While stereological analysis revealed no significant neuronal loss in any sectors of cornu ammonis, there was considerable decrease (43%) in TH+-neurons in SN pars compacta supporting the well-known vulnerability of nigral dopaminergic neurons to α-synuclein mediated neurodegeneration. On the other hand, synaptophysin levels decreased in similar amounts both in striatum and hippocampus, suggesting comparable synaptic loss in target areas. Interestingly, phosphorylated-S129-α-synuclein staining revealed significant expression in CA2 characterized by more mature and dense cellular accumulations compared to CA1-CA3 sub-regions displaying more diffuse grain-like aggregates, suggesting preferential susceptibility of CA2 to produce α-synuclein induced pathology. CONCLUSION: Bilateral α-synuclein overexpression in DG and SN reproduced partial motor and hippocampus related cognitive deficits. Using this model, we showed a predisposition of CA2 for pathological α-synuclein accumulation, which may provide further insights for future experimental and clinical studies.

Development of an Anti-Inflammatory Drug-Incorporated Biomimetic Scaffold for Corneal Tissue Engineering.

Purpose: The aim of this study was to design naproxen sodium (NS)-containing, biomimetic, porous poly(lactide-co-glycolide) (PLGA) scaffolds for regeneration of damaged corneal epithelium. Methods: NS-incorporated PLGA scaffolds were prepared using the emulsion freeze-drying method and then coated with collagen or poly-l-lysine. Porosity measurements of the scaffolds were performed by the gas adsorption/desorption method and the scaffolds demonstrated highly porous, open-cellular pore structures with pore sizes from 150 to 200 µm. Results: The drug loading efficiency of scaffolds was found to be higher than 84%, and about 90%-98% of NS was released at the end of 7 days with a fast drug release rate at the initial period of time and then in a slow and sustained manner. The corneal epithelial cells were isolated from New Zealand white rabbits. The obtained cells were seeded onto scaffolds and continued to increase during the time period of the study, indicating that the scaffolds might promote corneal epithelial cell proliferation without causing toxic effects for at least 10 days. Conclusions: The NS-loaded PLGA scaffolds exhibited a combination of controlled drug release and biomimetic properties that might be attractive for use in treatment of corneal damage both for controlled release and biomedical applications.

Alterations of Tear Film and Ocular Surface in Children with Type 1 Diabetes Mellitus.

Purpose: To investigate whether diabetes mellitus (DM) affects ocular surface of children with well-controlled type 1 DM.Methods: Sixty-five diabetic patients and 55 age-matched controls enrolled to study. Detailed ocular surface assessment including, ocular surface disease index (OSDI) questionnaire, tear film break-up time (TBUT) analysis, Schirmer test, and conjunctival impression cytologic analysis were performed.Results: Schirmer test and TBUT results were significantly lower in DM group than controls (p = 0.001, for all). OSDI scores of all participants were within normal range. Impression cytology analysis showed grade 0 changes in all participants and there was no difference between groups for goblet cell density (p > 0.05). The TBUT results were significantly associated with duration of DM (r = -0.309, p = 0.036).Conclusion: Diabetic children without symptoms, signs, and definite diagnosis of dry eye still had lower TBUT and Schirmer test results than controls; however, impression cytology analysis was similar in both groups.

Effects of prenatal binge-like ethanol exposure and maternal stress on postnatal morphological development of hippocampal neurons in rats.

BACKGROUND: Alcohol is one of the most commonly used drugs of abuse negatively affecting human health and it is known as a potent teratogen responsible for fetal alcohol syndrome (FAS), which is characterized by cognitive deficits especially pronounced in juveniles but ameliorating in adults. Searching for the potential morphological correlates of these effects, in this study, we compared the course of developmental changes in the morphology of principal hippocampal neurons in fetal-alcohol (A group), intubated control (IC group), and intact control male rats (C group) over a protracted period of the first two postnatal months. METHODS: Ethanol was administered to the pregnant Wistar dams intragastrically, throughout gestation days (GD) 7-20, at a total dose of 6g/kg/day resulting in the mean blood alcohol concentration (BAC) of 246.6±40.9mg/dl. Ten morphometric parameters of Golgi-stained hippocampal neurons (pyramidal and granule) from CA1, CA3, and DG areas were examined at critical postnatal days (PD): at birth (PD1), at the end of the brain growth spurt period (PD10), in juveniles (PD30), and in young adults (PD60). RESULTS: During postnatal development, the temporal pattern of morphometric changes was shown to be region-dependent with most significant alterations observed between PD1-30 in the CA region and between PD10-30 in the DG region. It was also parameter-dependent with the soma size (except for CA3 pyramids), number of primary dendrites, dendrite diameter, dendritic tortuosity and the branch angle demonstrating little changes, while the total dendritic field area, dendritic length, number of dendritic bifurcations, and spine density being highly increased in all hippocampal regions during the first postnatal month. Moderate ethanol intoxication and the maternal intubation stress during gestation, showed similar, transient effects on the neuron development manifested as a smaller soma size in granule cells, reduced dendritic parameters and lower spine density in pyramidal neurons at PD1. Full recovery from these effects took place within the first 10 postnatal days. CONCLUSIONS: This study showed regional and temporal differences in the development of different morphometric features of principal hippocampal neurons in intact subjects over a protracted 2-months postnatal period. It also demonstrated an overlap in the effects of a moderate fetal ethanol intoxication and a mild maternal stress produced by the intragastric intubation, a commonly used method of ethanol administration to the pregnant dams. Fast recovery from the adverse effects on the soma size, dendritic arborization and spines density observed at birth indicates towards the fetal ethanol/stress induced developmental retardation.

Efficacy of Combined Use of Different Nickel-Titanium Files on Removing Root Canal Filling Materials.

INTRODUCTION: The aim of this study was to estimate the remaining amount of residual filling materials in root canals after retreatment using ProTaper Universal Retreatment (PTUR) files (Dentsply Maillefer, Ballaigues, Switzerland) alone or with the additional use of the Self-Adjusting File (SAF; ReDent-Nova, Ra'anana, Israel), Reciproc (VDW, Munich, Germany), or Hedström-files (H-file; VDW, Antaeos, Munich, Germany) with volumetric estimation using the stereologic method via cone-beam computed tomographic images. METHODS: Forty-eight mandibular premolars with single canals were used. The canals were instrumented with ProTaper rotary instruments up to F4 and filled with gutta-percha and AH26 sealer (Dentsply De Trey, Johnson City, TN). All the samples were placed into the silicone models. Samples were scanned with cone-beam computed tomographic imaging and assigned into 4 groups (n = 12) according to retreatment files: the PTUR system group, the PTUR system plus SAF group, the PTUR system plus Reciproc group, and the PTUR system plus H-file group. The specimens were rescanned after retreatment procedures, and the volume estimations of the remaining filling materials were performed using the stereologic method. Data were analyzed using Kruskal-Wallis and Dunn tests. RESULTS: There was no significant difference among the groups regarding mean percentage volumes of the filling materials before retreatment procedures (P > .05). None of the retreatment procedures provided complete removal of the filling materials. The additional use of the SAF did not significantly improve the removal of filling materials when compared with the PTUR system alone (P > .05). However, the additional use of Reciproc or hand H-files significantly improved the removal of filling materials when compared with the PTUR system alone (P < .05). CONCLUSIONS: The additional use of files with different motion kinetics improved the removal of root canal fillings; however, none of the systems completely removed the root canal filling material from the root canals.

Radiological, stereological, and microscopic evaluation of the quality of canal fillings in oval-shaped root canals prepared with self-adjusting file.

The aim of this study was to evaluate the quality of the canal fillings performed by thermoplastic technique (Herofill) and cold lateral condensation (CLC) techniques in oval shaped canals following self-adjusting file (SAF) preparation using radiological, stereological, and microscopic evaluation methods. Twelve pairs of oval-shaped root canals were prepared with SAF and filled using CLC (n = 12) or the Herofill system (n = 12). The quality of the root canal filling was evaluated by (i) scoring bidirectional storage phosphor plate images, (ii) stereological (volumetric) analysis of cone beam computerized tomography (CBCT) images, and (iii) microscopic analysis of the cross-sectioned area of the canal and gutta-percha via image analysis software. Chi-square test, t-test, and Mann-Whitney U-tests were used respectively (p < 0.05) for the aforementioned evaluations. 58.33% of the fillings (total n = 24) that were found to be sufficient in a bucco-lingual (BL) projection were considered insufficient in the mesio-distal (MD) projection according to radiological scoring. The difference in the findings between the two types of projections (BL and MD) was significant (p < 0.05). The results of the stereological evaluation of CBCT images and image analysis of cross-sections showed no statistically significant difference between the CLC and Herofill (p > 0.05). Herofill was not superior to cold lateral compaction. Stereology can be used to obtain unbiased information about the volume of root canal fillings. Both filling techniques failed to achieve the desirable goal of 0% empty volume in all of the cases and all evaluation systems confirmed this result. SCANNING 38:427-433, 2016. © 2015 Wiley Periodicals, Inc.

Investigation into the effects of prenatal alcohol exposure on postnatal spine development and expression of synaptophysin and PSD95 in rat hippocampus.

Ethanol is known as a potent teratogen responsible for the fetal alcohol syndrome characterized by cognitive deficits especially pronounced in juveniles but ameliorating in adults. Since the mechanisms of these deficits and following partial recovery are not fully elucidated, the aim of the present study was to investigate the process of synaptogenesis in the hippocampus over the first two months of life in control and fetal-alcohol rats. Ethanol was delivered to the pregnant dams by intragastric intubation throughout 7-21 gestation days at the daily dose of 6g/kg generating a mean blood alcohol level of 246.6±40.9mg/dl on gestation day 20. The spine densities as well as the expression of pre- and postsynaptic proteins, synaptophysin (SYP) and PSD-95 protein, were evaluated for three distinct hippocampal regions: CA1, CA2+3, and DG and four postnatal days: PD1, PD10, PD30 and PD60, independently. Our results confirmed an intensive synaptogenesis within the brain spurt period (first 10 postnatal days), however, the temporal pattern of changes in the SYP and PSD-95 expression was different. The ethanol exposure during half of the 1st and the whole 2nd human trimester equivalent resulted in an overall trend toward lower values of synaptic indices at PD1 with a fast recovery from these deficits observed already at PD10. At PD30, around the age when the most pronounced behavioral deficits have been previously reported in juvenile fetal-alcohol subjects, no significant changes were found in either the hippocampal levels of synaptic proteins or in the spine density in principal hippocampal neurons.

A comprehensive characterization study of human bone marrow mscs with an emphasis on molecular and ultrastructural properties.

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) continue to draw attention of researchers in the fields of basic science and medicine due to their indispensible regenerative, reparative, angiogenic, anti-apoptotic, and immunosuppressive properties, all of which collectively point out their enormous therapeutic potential. There is still, however, a need for further investigation of their characteristics to broaden their field of use and learn much more about how to control their fate and improve their therapeutic effectiveness. hBM-MSCs were extensively characterized in terms of their growth characteristics, genetic stability, and differentiation capability to the mesodermal and ectodermal cell lineages; a special emphasis was given to their phenotypic and ultrastructural properties. Expression of embryonic stem cell markers Oct4, Rex-1, FoxD-3, Sox2, and Nanog was shown with real-time PCR. Transmission electron microscopy revealed the ultrastructural characteristics of hBM-MSCs; they had pale, irregularly shaped and large euchromatic nuclei, and two distinct areas in their cytoplasm: an intensely stained inner zone rich in mitochondria and rough endoplasmic reticulum (rER) with dilated cisternae and a relatively peripheral zone poor in organelles. hBM-MSCs expressed adipogenic (adipophilin and PPARγ), myogenic (desmin, myogenin, α-SMA), neurogenic (γ-enolase, MAP2a,b, c-fos, nestin, NF-H, NF-L, GFAP, ß3-tubulin), osteogenic (osteonectin, osteocalcin, osteopontin, Runx-2, type I collagen), and chondrogenic (type II collagen, SOX9) markers either at RNA or protein level even under basal conditions, without any stimulation towards differentiation. The differentiation potential of hBM-MSCs to adipogenic, osteogenic, and neurogenic lineages was shown by using the relevant differentiation factors.

Stereological analysis of sciatic nerve in chickens following neonatal pinealectomy: an experimental study.

BACKGROUND: Although the injury to the peripheral nervous system is a common clinical problem, understanding of the role of melatonin in nerve degeneration and regeneration is incomplete. METHODS: The current study investigated the effects of neonatal pinealectomy on the sciatic nerve microarchitecture in the chicken. The chickens were divided into two equal groups: unpinealectomized controls and pinealectomized chickens. At the end of the study, biochemical examination of 10 sciatic nerve samples from both groups was performed and a quantitative stereological evaluation of 10 animals in each group was performed. The results were compared using Mann-Whitney test. RESULTS: In this study, the results of axon number and thickness of the myelin sheath of a nerve fiber in newly hatched pinealectomy group were higher than those in control group. Similarly, surgical pinealectomy group had significantly larger axonal cross-sectional area than the control group (p < 0.05). In addition, the average hydroxyproline content of the nerve tissue in neonatal pinealectomy group was higher than those found in control group. Our results suggest that melatonin may play a role on the morphologic features of the peripheral nerve tissue and that melatonin deficiency might be a pathophysiological mechanism in some degenerative diseases of peripheral nerves. The changes demonstrated by quantitative morphometric methods and biochemical analysis has been interpreted as a reflection of the effects of melatonin upon nerve tissue. CONCLUSION: In the light of these results from present animal study, changes in sciatic nerve morphometry may be indicative of neuroprotective feature of melatonin, but this suggestion need to be validated in the human setting.

Moderate exercise delays the motor performance decline in a transgenic model of ALS.

The relationship between exercise and amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by motor neuron loss, rapidly progressive weakness and early death has been controversial. We studied the effect of a high (HEX) and moderate-level exercise (MEX) on body weight, motor performance and motor neuron counts in the ventral horn of spinal cords in a transgenic mouse model of ALS (G93A-SOD1) that overexpresses a mutated form of the human SOD1 gene that is a cause of familial ALS. These transgenic mice show several similarities to the human disease, including rapid progressive motor weakness from 100 days of age and premature death at around 135 days of age. Mice were exposed to high or mid-level exercise of left sedentary (SED). At 70, 95 and 120 days of age, spinal cords were processed following euthanasia. Motor neurons larger than 15 mum in diameter were counted with a design-based stereological protocol using an optical fractionator probe in the ventral horn of different regions of the cord and compared to wild-type littermates. Moderate exercise delayed the onset of motor deficit by over a week. High exercise slightly but significantly hastened the onset of motor performance deficits. Motor neuron density in the lumbar cord was significantly higher in MEX group compared to SED at 95 days of age. These results show the beneficial effects of moderate exercise on the preservation of motor performance that correlates with higher motor neuron density in the ventral horn of the lumbar spinal cord in G93A mice.

Isolation and in vitro characterisation of dental pulp stem cells from natal teeth.

Dental pulp stem cells were primarily derived from the pulp tissues of exfoliated deciduous teeth, primary incisors and permanent third molar teeth. The aim of this study was to isolate and extensively characterise SCs derived from human natal dental pulp (hNDP). For characterisation, proliferation capacity, phenotypic properties, ultrastructural and differentiation characteristics and gene expression profiles were utilised. A comparison was done between the properties of NDP-SCs and the properties of mesenchymal stem cells (MSCs) from bone marrow (BM) of the human. Stem cells isolated from hNDP and hBM were analysed by flow cytometry, reverse transcriptase-PCR, Real Time-PCR, and immunocytochemistry. Both cell lines were directionally differentiated towards adipogenic, osteogenic chondrogenic, myogenic and neurogenic lineages. hNDP-SCs and hBM-MSCs expressed CD13, CD44, CD90, CD146 and CD166, but not CD3, CD8, CD11b, CD14, CD15, CD19, CD33, CD34, CD45, CD117, and HLA-DR. Ultrastructural characteristics of hNDP-SCs showed more developed and metabolically active cells. hNDP-SCs and hBM-MSCs expressed some adipogenic (leptin, adipophilin and PPARgamma), myogenic (desmin, myogenin, myosinIIa, and alpha-SMA), neurogenic (gamma-enolase, MAP2a,b, c-fos, nestin, NF-H, NF-L, GFAP and betaIII tubulin), osteogenic (osteonectin, osteocalcin, osteopontin, Runx-2, and type I collagen) and chondrogenic (type II collagen, SOX9) markers without any stimulation towards differentiation under basal conditions. Embryonic stem cell markers Oct4, Rex-1, FoxD-3, Sox2, and Nanog were also identified. The differentiation potential of hNDP-SCs and hBM-MSCs to adipogenic, osteogenic, chondrogenic, myogenic and neurogenic was shown. This report described the first successful isolation and characterisation of hNDP-SCs.

Volumetric assessment of lateral pterygoid muscle in unilateral chewing: a stereologic study.

Unilateral chewing habit is common in dentate populations. It may initiate a hypertrophy process in the lateral pterygoid muscles compared with the nonused sides. We hypothesized that the lateral pterygoid muscles of the affected sides would be larger in volume than those of the nonaffected sides in subjects with unilateral chewing habit. Because analysis of medical images in accordance with the rules of modern stereology enables precise unbiased estimates of muscle volume, and magnetic resonance imaging (MRI) is a criterion standard as an imaging modality for the analysis of muscles because of its excellent soft-tissue resolution, we estimated lateral pterygoid muscle volume both at right and left sides using stereologic techniques (Cavalieri principle) by means of MRI. The MRI-estimated volumes of both the inferior and the superior heads of the lateral pterygoid muscle at the chewing side were significantly greater than the opposite side when compared. This finding may assist in the interpretation of lateral pterygoid muscle and temporomandibular joint-related problems in maxillofacial surgery clinics. This investigation has also demonstrated that systematically sampled MRI slices through the lateral pterygoid muscle can be used to obtain unbiased estimates.

Structural and immunophenotypic characterization of high endothelial venules in rat and human tissue.

OBJECTIVE: To present additional data on high endothelial venule (HEV) structure and immunophenotype. METHODS: We used the zinc iodide-osmium tetroxide technique (ZIO), which is a metallophilic fixation and staining technique to examine HEVs at light and electron microscopic levels as this technique was previously reported to be reactive with cells in HEVs. Tonsils and lymph nodes were obtained from the Surgery and Otorhinolaryngology Departments, Hacettepe University Hospital, Ankara, Turkey during 2002 and 2003. An indirect immunohistochemical technique was used to examine frozen human tissue samples. RESULTS: Organelle rich high endothelial cells, sheet-like processes of pericytes surrounding HEVs, structural relation of pericyte processes with fibroblastic reticular cells, an unusual multivesicular body-like organelle within high endothelial cells were presented. Expression of a large panel of defined and yet non-defined antigens on HEVs are also presented using an indirect immunoperoxidase technique. CONCLUSION: Presence of some of these antigens on HEVs was previously reported while no previous report is available for others. Significance of the expression of these antigens in HEVs, structural hints for trans endothelial migration of lymphocytes and their travel along the reticular cell meshwork is briefly discussed.

Immunohistochemical analysis of CD31, CD36, and CD44 antigens in human omentum.

OBJECTIVE: Milky spots in the human omental tissue are known to be consisting of lymphocytes, macrophages and mast cells. Our goal was to evaluate the relationship of lymphoid cells and macrophages with vasculature and stromal components. METHODS: In this study we examined the biopsy specimens obtained from the adult patients whom were operated for different purposes in the General Surgery Department of Dicle University Hospital, Ankara, Turkey. We used CD31 as an endothelial cell marker, CD36 which is known to react with microvascular endothelium and adipocytes, and CD44 which is a hyaluronic acid receptor using an indirect immunoperoxidase technique. RESULTS: We observed that CD31 was mainly reactive with vascular endothelial cells and platelets, CD36 was reactive with microvascular endothelium and adipocytes and CD44 was mainly expressed by the endothelial cells of high endothelial venules, fibroblasts in stromal compartments and by large mononuclear cells. CONCLUSION: We determined the structural and immunophenotypic features of omental lymphoid tissue components stressing vascular and stromal elements, and we briefly discussed the significance of the expression of these molecules in the determined locations.