The effect of opioid dependence on conventional and novel biochemical parameters of bone metabolism.

BACKGROUND: Opioids influence bone metabolism in several ways and osteoporosis associated with the long-term use of opioids is believed to be multifactorial. OBJECTIVES: To investigate the effect of opioid dependence on conventional and novel biochemical parameters of bone metabolism. To evaluate whether the concomitant HCV infection affects these parameters. METHODS: Fifty-nine opioid-dependent subjects and 23 healthy volunteers participated in the study. Parameters of bone metabolism were determined in serum. The determined parameters were procollagen type I N-terminal propeptide (PINP), serum Beta-Crosslaps Ι (ß-CTX), total calcium (Ca), inorganic phosphorus (P), parathormone (PTH) and alkaline phosphatase bone isoenzyme (ALP). RESULTS: The results of our study show that opioid-dependent subjects exhibit higher values in those biochemical markers that are indicative of increased osteoclast activity, such as ß-CTX and ALP, compared to healthy subjects. Furthermore, in opioid-dependent subjects the values of PTH were lower, while those of PINP were higher, in comparison to healthy individuals. No significant difference in the studied parameters was found when opioid-dependent subjects positive for anti-HCV antibodies were compared with opioid-dependent subjects negative for anti-HCV antibodies. CONCLUSION: Our findings show that there is increased bone turnover (bone metabolism) in opioid-dependent subjects, compared to healthy individuals. Future research on bone mineral density in these patients will help us evaluate whether the bone remodeling process is balanced or not.

Alterations in serum MMP and TIMP concentrations following chronic heroin abuse.

UNLABELLED: Abstract Context: Although opiate abuse is known to affect matrix metalloproteinases (MMPs), data on these enzymes and their tissue inhibitors in heroin addicts are scarce. OBJECTIVE: In the present study, we determined serum concentrations of MMP-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in heroin users, and compared them with healthy individuals. We evaluated whether 21 d of abstinence are adequate to reverse the effect of opiates and we compared seropositive with seronegative, for anti-HCV antibodies, heroin users. MATERIALS AND METHODS: Twenty-six heroin-dependent male volunteers and an equal number of healthy individuals participated in this study. ELISA was used to assess the serum levels of MMP-2, MMP-9, TIMP-1 and TIMP-2. Heroin users were assessed both upon admission and upon completion of a 21-d detoxification program. RESULTS: Serum TIMP-1 concentrations were significantly lower and the ratios MMP-2/TIMP-1, MMP-9/TIMP-1 and MMP-2/TIMP-2 were significantly higher in heroin users compared to healthy individuals. Heroin users who were seropositive had lower MMP concentrations, as well as lower MMP/TIMP ratios, compared to those who were seronegative. DISCUSSION: Our results showed that in heroin-addicted individuals, and especially those who are positive for anti-HCV antibodies, the balance between MMPs and TIMPs in serum is disrupted and this disruption cannot be restored within 21 d of abstinence. CONCLUSION: Chronic heroin abuse disrupts the balance between MMPs and TIMPs in serum and this effect is not reversible within 21 d of abstinence.

Dendritic and spinal pathology of the Purkinje cells from the human cerebellar vermis in Alzheimer's disease.

BACKGROUND: Alzheimer's disease constitutes one of the main causes of dementia. It is clinically characterized by memory impairment, deterioration of intellectual faculties and loss of professional skills. Furthermore changes in equilibrium and limb coordination are clinically demonstrable in persons with Alzheimer's disease. In the present study we tried to figure out possible changes of the Purkinje cells in Alzheimer's disease brains. SUBJECTS AND METHODS: We studied the Purkinje cells from the vermis of the cerebellum in 5 Alzheimer' disease brains Golgi technique. RESULTS: In the Purkinje cells from the inferior surface of the cerebellar hemispheres severe dendritic and spinal pathology consisting of loss of distal dendritic segments and alterations of dendritic spine morphology can be noticed in Alzheimer's disease brains. CONCLUSIONS: The morphological and morphometric estimation of the dendrites and the dendritic spines of the Purkinje cells from the inferior surface of the cerebellar hemispheres in Alzheimer's disease brains revealed substantial alterations of the dendritic arborization and marked loss of the dendritic spines, which may be related to cognitive impairment and motor deficits in Alzheimer's disease.

A simple HPLC method for the simultaneous determination of two selective serotonin reuptake inhibitors and two serotonin-norepinephrine reuptake inhibitors in hair, nail clippings, and cerebrospinal fluid.

A novel and simple high-performance liquid chromatography method has been developed for the simultaneous determination of two selective serotonin reuptake inhibitors (fluoxetine and paroxetine) and two serotonin-norepinephrine reuptake inhibitors (venlafaxine and duloxetine) in alternative samples of toxicological interest such as hair, nail clippings, and cerebrospinal fluid (CSF). The separation was achieved on a Hichrom Kromasil 100-5C(18) (250 × 4.6 mm) 5 µm column by using ammonium acetate (0.05 M)-acetonitrile (59:41% v/v) as the mobile phase, delivered isocratically at a flow rate of 1.3 mL/min, within ca. 10 min. Ultraviolet detection at 235 nm was used for monitoring the eluting analytes. Validation was performed in terms of linearity, selectivity, accuracy, precision, and stability. Correlation coefficients were greater than 0.9954. The limits of quantitation ranged between 0.3 and 2.1 ng/µL for all analytes in the liquid matrix (CSF), while the respective values were in the range of 0.3-3.6 ng/mg for solid matrices (hair and nail clippings), with an injection volume of 20 µL. Repeatability and intermediate precision (relative standard deviation, RSD%) were less than 16.6%. The method was successfully applied to actual hair and nail samples from a patient under fluoxetine treatment.

Morphological and morphometric changes in the Purkinje cells of patients with essential tremor.

Essential tremor (ET) is a progressive neurological syndrome characterised by involuntary tremors of the hands or arms, head, jaw and voice. The pathophysiology of ET is not clearly understood yet. However, previous studies have reported several changes in the brain of patients with ET. One of the brain areas extensively investigated is the cerebellum. In the present study, a morphometric analysis of Purkinje cells in patients with ET and ET-plus was performed, and subsequently compared with normal controls using the Golgi silver staining method and 3D neuronal reconstruction. Substantial morphological changes were uncovered in the Purkinje cells of patients with ET compared with normal controls, including a decreased dendritic length and field density, an overall loss of terminal branches and a decreased density of dendritic spines.

Disposable pipette extraction for gas chromatographic determination of codeine, morphine, and 6-monoacetylmorphine in vitreous humor.

The availability of a sensitive and rapid analytical method for the determination of opiates, and other substances of forensic interest, in a variety of biological specimens is of utmost importance to forensic laboratories. Solid-phase extraction is very popular in the pre-treatment of forensic samples. Nevertheless, a new approach, disposable pipette extraction (DPX), is gaining increasing interest in sample preparation. DPX has already been applied to the analysis of drugs of abuse in common biological matrices, such as urine and blood, but has not yet been evaluated on alternative biological samples, such as vitreous humor. The objective of this study was to evaluate the applicability of DPX on the analysis of opiates in vitreous humor. The currently developed method is fast, reliable, and easy to perform. The sensitivity, precision, and accuracy are satisfactory. Recoveries obtained are within the range of 72-91%, whereas the sample volume of vitreous humor required is only 100 µL.

Epigenetic mechanisms in metal toxicity.

The true understanding of epigenetics evolved over time as our knowledge on DNA methylation and chromatin modifications and their effects on gene expression increased. The current flurry of research on epigenetics and the increasing documentation of the effects of various environmental factors on DNA methylation, chromatin modification, as well as on the expression of small non-coding RNAs (ncRNAs) have expanded the scope of research on the etiology of various diseases including cancer. The current review briefly discusses various molecular mechanisms of epigenetic regulation of gene expression, and expands the discussion with examples of heavy metal-induced alterations of gene expression and the associated epigenetic changes.

Morphological alterations of the pyramidal and stellate cells of the visual cortex in schizophrenia.

Schizophrenia is a severe brain disorder characterized by certain types of delusion, hallucination and thought disorder. Studies have revealed impaired synaptic plasticity and reduced gamma-aminobutyric acid levels of the visual cortex in patients with schizophrenia. While previous work established a critical role for interneurons and cortical connectivity in the generation of hallucinations, the present study set out to examine the morphology of pyramidal cells and interneurons from layers 3 and 4 in the primary visual cortex from schizophrenic brains and to identify any dendritic and spinal alterations in comparison to normal control brains. The morphological and morphometric changes of the pyramidal cells and the interneurons of the visual cortices of 10 brains obtained from patients with schizophrenia, in comparison to 10 age-matched controls, were studied using the Golgi method and 3D neuronal reconstruction techniques. Analysis using the Golgi impregnation technique revealed a significant loss of distal dendritic segments, tortuous branches and varicosities and an overall restriction of the dendritic field in the brains of schizophrenic patients in both pyramidal cells and in aspiny interneurons. The present results may explain certain clinical phenomena associated with the visual cortex usually encountered in schizophrenia.

Isoprostane as a marker of oxidative stress in chronic heroin users: correlation with duration of heroin use or concomitant hepatitis C infection.

BACKGROUND: Although chronic heroin abuse has been extensively linked to oxidative stress, and while plasma 15-F(2t)-IsoP is considered a good indicator of oxidative stress, there remain few references in the literature about the plasma concentration of this marker in heroin dependent subjects. OBJECTIVES: To determine plasma 15-F(2t)-IsoP, as a marker of oxidative stress, in chronic heroin users, and to examine whether the values of this marker correlate with the duration of heroin use or with the presence of anti-HCV antibodies. METHODS: Forty-two chronic heroin users and twenty two healthy control subjects were recruited for this study. An enzyme-immunoassay method was used for the determination of 15-F(2t)-IsoP in plasma. RESULTS: Plasma 15-F(2t)-IsoP values were significantly higher in chronic heroin users compared to healthy controls. No correlation was found between the values of plasma 15-F(2t)-IsoP and the duration of heroin use. Heroin dependent subjects positive for anti-HCV antibodies had significantly lower values of plasma 15-F(2t)-IsoP as compared to those without a history of HCV infection. CONCLUSIONS: The elevated plasma 15-F(2t)-IsoP values in heroin dependent subjects, compared to healthy individuals, indicate a shift of the balance between oxidants and antioxidants towards the former and suggest that heroin dependent subjects could benefit from an antioxidant therapy.

Evaluation of prooxidant-antioxidant balance in chronic heroin users in a single assay: an identification criterion for antioxidant supplementation.

BACKGROUND: Opiate abuse has been linked to oxidative stress, through the separate evaluation of oxidants and antioxidants. OBJECTIVES: To determine prooxidant-antioxidant balance (PAB) in chronic heroin users in a single assay, easily applied in a clinical setting. Specifically, to examine whether PAB values correlate with the duration of abuse or with the presence of anti-HCV antibodies. METHODS: Sixty-four chronic heroin users - 34 cases and 30 controls - participated in this study. PAB was determined by an Enzyme-linked immunosorbent assay (ELISA) method, developed by members of the study group. RESULTS: In heroin users, oxidative balance was disrupted in favor of prooxidants. There was no correlation of PAB values with the duration of abuse or with the presence of anti-Hepatitis C virus (HCV) antibodies. CONCLUSIONS: Chronic heroin users can benefit from an antioxidant therapy, and the method currently presented can be used as an identification criterion.

The clockwise rotation of myocardial fiber orientation from epicardial to endocardial surface in left ventricular free wall in a post-mortem human heart.

Following fixation and MRI imaging a post-mortem human heart was sliced at the sagittal plane. Each anatomical section was then cut into smaller segments and each one was objected to classical histology process. The resulting microscopy slides were digitalized with a scanner. The histological section reconstruction was achieved using Adobe Photoshop CS2(R). Using specific software, called FiberCad, the user can define and draw (with the assistance of optical microscope) those fibers that are parallel and those fibers that are vertical to the slides plane. To better align the histological 3D reconstruction, the software is equipped with an option that allows the user to make best possible fit between histological and MRI slices. We present the consequent sagittal sections of LV free wall (from epicardial to endocardial surface), whereby the clockwise rotation of the mean orientation of the fibers that are on the plane of sectioning is clearly evident. We present a post mortem analysis of the complete LV free wall of a human heart.

Purkinje Cells Pathology in Alzheimer's Disease.

Alzheimer's disease (AD) is one of the main causes of dementia in senium and presenium. It is clinically characterized by memory impairment, deterioration of intellectual faculties, and loss of professional skills. The cerebellum is a critical part in the distributed neural circuits participating not only in motor function but also in autonomic, limbic, and cognitive behaviors. In present study, we aim to investigate the morphological changes in the Purkinje cells in different cerebellar regions in AD and to correlate them with the underlying AD pathology. Purkinje cells exhibit significant morphometric alterations in AD and prominently in the anterior lobe which is related to higher cognitive functions. The present study gives new insights into the cerebellar pathology in AD and confirms that Purkinje cells pathology is a key finding in AD brains and that AD is characterized by regional-specific atrophy picked in the anterior cerebellar lobe.

Purkinje cells pathology in schizophrenia. A morphometric approach.

OBJECTIVES: Schizophrenia is a brain disorder that affects more than 21 million people worldwide. Ventricle enlargement and reduction in the volume of the temporal lobe overall and in medial temporal structures constitutes the main macroscopic findings, whilst synaptic and spinal changes as well as gliosis in the hippocampal formation, the prefrontal and the entorhinal cortex stand among cardinal microscopic findings in the schizophrenic brains. In recent years, accumulated evidence comes to light about the role of cerebellum in the pathophysiology of schizophrenia. MATERIALS AND METHODS: The present study is based on the morphological analysis and 3D neuronal reconstruction of the Purkinje cells from 10 schizophrenic brains and 10 normal controls. RESULTS: Significant morphological alterations such as loss of distal and terminal dendritic branches and decrease of the density of the dendritic spines constitute the main morphological findings found in the present study. CONCLUSIONS: The present findings may be added to accumulated evidence on macroscopic and microscopic pathology of the cerebellum in schizophrenia. Morphological alterations of Purkinje cells seem to be a central feature of neuropathology of schizophrenia, reflecting to impairment of neuronal connectivity and functionality, and related to motor and cognitive symptoms.

Age-related dendritic and spinal alterations of pyramidal cells of the human visual cortex.

INTRODUCTION: Normal aging is characterized by deterioration of visual abilities, affecting mainly visual acuity, contrast and wavelength sensitivity. In the present study we attempted to describe the morphological and morphometric alterations of the dendrites and the dendritic spines of the pyramidal cells of the visual cortex during normal aging, in order to approach the visual impairment of aged individuals from a neuropathological point of view. MATERIAL AND METHODS: We studied the visual cortex in 20 brains using the Golgi technique. RESULTS: In pyramidal cells, which represent the majority of cortical neurons, age-related pathology can be observed in cell somata as well as, most importantly, in dendrite number and morphology. The apical dendrites of some pyramidal cells are distorted and tortuous. Horizontal dendritic arborization is also severely decreased. These alterations were more prominent in the corticocortical pyramidal neurons of the 5th layer. CONCLUSIONS: The morphological and morphometric assessment of the dendrites and the dendritic spines in the visual cortex in normal aging revealed substantial alterations of the dendritic arborization and marked loss of the dendritic spines, which may be related to visual impairment even in normal aging.

Dendritic and spinal alterations of neurons from Edinger-Westphal nucleus in Alzheimer's disease.

Alzheimer's disease (AD) is a heterogeneous neurodegenerative disorder, causing a progressive decline of intellectual faculties, impairment of behavior and social performance, and impairment of speech eloquence, associated with various neurological manifestations based on a variable neuropathological background. Edinger-Westphal nucleus is a selective target of Alzheimer pathology early in the course of the disease. We attempted to determine the morphological alterations of the dendrites and the dendritic spines in Edinger-Westphal nucleus of 7 cases that fulfilled the diagnostic criteria for Alzheimer's disease. For the histological study, we applied (a) routine neuropathological techniques and (b) rapid Golgi method. We proceeded to 3D neuronal reconstruction for the estimation of dendritic and spinal changes in Alzheimer's disease. The morphological and morphometric analysis revealed a substantial neuronal loss and synaptic alterations in Edinger-Westphal nucleus in all the cases of Alzheimer's disease. Distal dendritic branches are prominently affected. The neuronal loss and alteration of the spines in Edinger-Westphal nucleus in Alzheimer's disease may be related to the exaggerated pupillary reaction to cholinergic antagonists. Furthermore, the vulnerability of distal branches to Alzheimer's disease might be related to neuroplasticity impairment.

Disposable pipette extraction for the simultaneous determination of biperiden and three antipsychotic drugs in human urine by GC-nitrogen phosphorus detection.

BACKGROUND: Disposable pipette extraction with reversed-phase sorbent is proposed for the fast and simple GC determination of the antipsychotic drugs chlorpromazine, olanzapine, clozapine and biperiden - an anticholinergic drug - in human urine. The method was validated and successfully applied to postmortem urine samples. The analytical run was 11 min and lidocaine was used as the internal standard. RESULTS: The developed method showed good linearity, over the range of 0.34 to 5 ng/µl, for all compounds. The within-day and between-day precision and accuracy assays revealed values ≤15%, while the recoveries ranged from 85 to 120%. LOD and LOQ ranged from 0.28 to 0.42 ng/µl and from 0.85 to 3.58 ng/µl, respectively. CONCLUSION: The developed method is a user-friendly technique, which is simple and fast.

Recent advances in the bioanalysis of modified nucleotides in epigenetic studies.

Epigenetic alterations, such as DNA methylation, are involved in the pathogenesis of various diseases, the toxicity of diverse agents, the process of aging, the development of stem cells and numerous other mechanisms. DNA methylation is one of the most well-studied epigenetic alterations in mammals. Nevertheless, the scientific interest is now focusing on novel modified nucleotides with potential regulatory roles, such as 5-hydroxymethylcytosine. We currently present and discuss novel bioanalytical strategies developed for the determination of various modified nucleotides in epigenetic studies.

Effect of chronic heroin and cocaine administration on global DNA methylation in brain and liver.

Drug abuse is associated with epigenetic changes, such as histone modifications and DNA methylation. The purpose of the present study was to examine the effect of chronic cocaine and heroin administration on global DNA methylation in brain and liver. Male, 8 week old, C57BL/6J mice received heroin in a chronic 'intermittent' escalating dose paradigm, or cocaine in a chronic escalating dose 'binge' paradigm, which mimic the human pattern of opioid or cocaine abuse respectively. Following sacrifice, livers and brains were removed and DNA was extracted from them. The extracted DNA was hydrolyzed and 2'-deoxycytidine and 5-methyl-2'-deoxycytidine were determined by HPLC-UV. The % 5-methyl-2'-deoxycytidine content of DNA was significantly higher in the brain compared to the liver. There were no differences between the control animals and the cocaine or heroin treated animals in neither of the tissues examined, which is surprising since cocaine administration induced gross morphological changes in the liver. Moreover, there was no difference in the % 5-methyl-2'-deoxycytidine content of DNA between the cocaine and the heroin treated animals. The global DNA methylation status in the brain and liver of mice chronically treated with cocaine or heroin remains unaffected, but this finding cannot exclude the existence of anatomical region or gene-specific methylation differences. This is the first time that global DNA methylation in the liver and whole brain has been studied following chronic cocaine or heroin treatment.

Glutamate receptors in human caudate nucleus in normal aging and Alzheimer's disease.

Neostriatum is one of the brain areas that are not primarily affected in Alzheimer's disease, according to classic regard of the disease. However, recent data emphasize the involvement of neostriatum, especially the head of the caudate nucleus, in the emergence of characteristic symptoms of the disease. Glutamatergic neurotransmission is a key component of striatal pathways. The present study is focused on glutamate receptors of striatal neurons on human caudate nucleus in normal aging and Alzheimer's disease. Immunohistochemical studies were carried out for N-methyl-D-aspartate receptor subunit 1 (NMDAR1), α -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit 2 (GluR2) and metabotropic glutamate receptor 5 (mGluR5). Ionotropic receptors (NMDAR1 and GluR2) were found to be expressed by 82% - 93% of striatal neurons with no significant alterations in aging and Alzheimer's disease. On the other hand, metabotropic receptor mGluR5 was found to be expressed by just 40% of striatal neurons in young individuals, with significant intensity variations among the neurons. This percent was increased in elderly individuals and Alzheimer's disease patients to 80% and 92% of striatal neurons, respectively. The up-regulation of mGluR5 both in normal aging and Alzheimer's disease is possibly associated with reorganization of neuronal connections, indicates the complexity of this receptor function and renders quite unpredictable the intervention and treatment of dementia with mGluR5 inhibitors or modulators.

Dendritic, axonal, and spinal pathology of the Purkinje cells and the neurons of the dentate nucleus after long-term phenytoin administration: a case report.

Phenytoin is a commonly prescribed anticonvulsant drug; however, there is evidence that long-term administration is related to cerebellar ataxia, cerebellar atrophy, loss of Purkinje cells, and hyperplasia of Bergman glia cells. The aim of the present study was to detect and describe any possible alterations of the Purkinje cells, and neurons of the dentate nucleus, as those can be seen with the use of silver impregnation techniques, such as Golgi and Nauta method. The study was performed on a 7-year-old boy who was under phenytoin treatment for more than 3.5 years and had clinical manifestations of cerebellar ataxia. Golgi silver impregnation technique revealed substantial loss of dendritic spines and tertiary dendritic branches, both on the Purkinje cells and the neurons of the dentate nucleus, whereas the Nauta method demonstrated swollen and degenerated axons of Purkinje cells.