Use of RT-PCR and DNA microarrays to characterize RNA recovered by non-invasive tape harvesting of normal and inflamed skin

We describe a non-invasive approach for recovering RNA from the surface of skin via a simple tape stripping procedure that permits a direct quantitative and qualitative assessment of pathologic and physiologic biomarkers. Using semi-quantitative RT-PCR we show that tape-harvested RNA is comparable in quality and utility to RNA recovered by biopsy. It is likely that tape-harvested RNA is derived from epidermal cells residing close to the surface and includes adnexal structures and present data showing that tape and biopsy likely recover different cell populations. We report the successful amplification of tape-harvested RNA for hybridization to DNA microarrays. These experiments showed no significant gene expression level differences between replicate sites on a subject and minimal differences between a male and female subject. We also compared the array generated RNA profiles between normal and 24 h 1% SLS-occluded skin and observed that SLS treatment resulted in statistically significant changes in the expression levels of more than 1,700 genes. These data establish the utility of tape harvesting as a non-invasive method for capturing RNA from human skin and support the hypothesis that tape harvesting is an efficient method for sampling the epidermis and identifying select differentially regulated epidermal biomarkers.     

Developmental regulation of 5-HT1A receptor mRNA in the fetal limbic system: response to antenatal glucocorticoid

The developmental changes in 5-HT1A receptor mRNA expression associated with advancing gestational age were examined in the fetal guinea pig hippocampus and dentate gyrus (DG) by in situ hybridization. We found that 5-HT1A receptor mRNA was present in the hippocampal CA1 subfield and dentate gyrus (DG), and was significantly (P < 0.05) elevated in the DG during the period of rapid brain growth [gestational day (gd) 50; term = 70 days]. Glucocorticoids have been shown to alter 5-HT1A receptor mRNA expression in the adult, but nothing is known about their impact on the developing fetal brain. Expression of 5-HT1A receptor mRNA in the fetal hippocampus was measured following repeated maternal administration (gd40, 41, 50, 51, 60 and 61) of synthetic glucocorticoid (dexamethasone; 1 and 10 mg/kg). Levels of 5-HT1A receptor mRNA were significantly (P < 0.005) elevated in CA1 and DG following repeated exposure to high-dose glucocorticoid (10 mg/kg) in male, but not in female fetuses. Because fetal exposure to glucocorticoids programs hypothalamo-pituitary-adrenal (HPA) function, and hippocampal serotonin is known to influence glucocorticoid receptor (GR) expression, the glucocorticoid-mediated changes in 5-HT1A receptor mRNA may play a role in the programming of HPA function.     

Motor overflow in schizophrenia

The occurrence of motor dysfunction as a sign of schizophrenia, in addition to being a side effect of medication, has received considerable support in recent years. The current study aimed to systematically investigate both the presence and pattern of one such motor dysfunction, motor overflow. It was hypothesised that patients with schizophrenia would show significantly greater motor overflow than controls, and that the pattern of motor overflow occurrence would also vary significantly between the groups. A finger flexion task was used to examine the presence and pattern of motor overflow. Subjects were asked to maintain target forces, using either their index or small finger, representing 25, 50 or 75% of the maximum strength capacity for whichever finger was performing the task. Patients were found to exhibit significantly greater motor overflow than controls. There were also significant findings with respect to the patterns of motor overflow produced, specifically in regards to fine motor control and performance variability. In summary, patients differed significantly from controls in both the degree and pattern of overflow exhibited.     

Carotid endarterectomy: a review

BACKGROUND: Since the validation of carotid endarterectomy (CEA) as an effective means of stroke prevention, there has been renewed interest in its best indications and methods, as well as in how it compares to carotid angioplasty and stenting (CAS). This review examines these topics, as well as the investigation of carotid stenosis and the role of auditing and reporting CEA results. INVESTIGATION: Brain imaging with CT or MRI should be obtained in patients considered for CEA, in order to document infarction and rule out mass lesions. Carotid investigation begins with ultrasound and, if results agree with subsequent, good-quality MRA or CT angiography, treatment can be planned and catheter angiography avoided. An equally acceptable approach is to proceed directly from ultrasound to catheter angiography, which is still the gold-standard in carotid artery assessment. INDICATIONS: Appropriate patients for CEA are those symptomatic with transient ischemic attacks or nondisabling stroke due to 70-99% carotid stenosis; the maximum allowable stroke and death rate being 6%. Uncertain candidates for CEA are those with 50-69% symptomatic stenosis, and those with asymptomatic stenosis > or = 60% but, if selected carefully on the basis of additional risk factors (related to both the carotid plaque and certain patient characteristics), some will benefit from surgery. Asymptomatic patients will only benefit if surgery can be provided with exceptionally low major complication rates (3% or less). Inappropriate patients are those with less than 50% symptomatic or 60% asymptomatic stenosis, and those with unstable medical or neurological conditions. TECHNIQUES: Carotid endarterectomy can be performed with either regional or general anaesthesia and, for the latter, there are a number of monitoring techniques available to assess cerebral perfusion during carotid cross-clamping. While monitoring cannot be considered mandatory and no single monitoring technique has emerged as being clearly superior, EEG is most commonly used. "Eversion" endarterectomy is a variation in surgical technique, and there is some evidence that more widely practiced patch closure may reduce the acute risk of operative stroke and the longer-term risk of recurrent stenosis. CAROTID ANGIOPLASTY AND STENTING: Experience with this endovascular and less invasive procedure grows, and its technology continues to evolve. Some experienced therapists have reported excellent results in case series and a number of randomized trials are now underway comparing CAS to CEA. However, at this time it is premature to incorporate CAS into routine practice replacing CEA. AUDITING: It has been shown that auditing of CEA indications and results with regular feed-back to the operating surgeons can significantly improve the performance of this operation. Carotid endarterectomy auditing is recommended on both local and regional levels.     

Progressive multifocal leukoencephalopathy successfully treated with highly active antiretroviral therapy and cidofovir in an adolescent infected with perinatal human immunodeficiency virus (HIV)

Progressive multifocal leukoencephalopathy is an infectious demyelinating brain disease caused by the JC virus that is associated with significant morbidity and mortality in the immunocompromised host. We report a case of progressive multifocal leukoencephalopathy successfully treated with highly active antiretroviral therapy and cidofovir in an adolescent patient perinatally infected with human immunodeficiency virus (HIV).     

Effects of diabetes mellitus on astrocyte GFAP and glutamate transporters in the CNS

Diabetes mellitus increases the risk of central nervous system (CNS) disorders such as stroke, seizures, dementia, and cognitive impairment. The cellular mechanisms responsible for the increased risk of these disorders are incompletely understood. Astrocytes are proving critical for normal CNS function, and alterations in their activity could contribute to diabetes-related disturbances in the brain. We examined the effects of streptozotocin (STZ)-induced diabetes in rats on the level of the astrocyte intermediate filament protein, glial fibrillary acidic protein (GFAP), number of astrocytes, and levels of the astrocyte glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate/aspartate transporter (GLAST), in the cerebral cortex, hippocampus, and cerebellum by Western blotting (WB) and immunohistochemistry (IH). Studies were carried out at 4 and 8 weeks of diabetes duration. Diabetes resulted in a significant decrease in GFAP protein levels (WB) in the hippocampus and cerebellum at 4 weeks and in the cerebral cortex, hippocampus and cerebellum by 8 weeks. Attenuated GFAP immunoreactivity (IH) was evident in the hippocampus, cerebellum and white matter regions such as the corpus callosum and external capsule at both 4 and 8 weeks of diabetes. Astrocyte cell counts of adjacent sections immunoreactive for S-100B were not different between control and diabetic animals. No significant differences were noted in astrocyte glutamate transporter levels in the cerebral cortex, hippocampus, or cerebellum at either time period (WB, IH). With the expanding list of astrocyte functions in the CNS, the role of astrocytes in diabetes-induced CNS disorders clearly warrants further investigation.     

CREB deficient mice show inhibition and low activity in novel environments without changes in stress reactivity

The ability to respond to unexpected or novel stimuli is critical for survival. Determining that a stimulus is indeed novel requires memory to ascertain its lack of familiarity. As the long-term synaptic changes involved in memory formation require the cAMP response element binding protein (CREB), we examined the extent to which CREB is involved in responses to novel environments. These environments typically trigger an endocrine stress response. Thus, we measured behavioural and stress hormone responses to three novel and one familiar environment in mice with a targeted disruption of the alpha and delta isoforms of the CREB gene (CREB(alphadelta-) deficient mice). We found CREB(alphadelta-) deficient mice to be less active and more inhibited in the elevated plus maze, open field, and light/dark box, without showing differences in anxiety-like behaviour. This inhibition is unique to novel environments because these mice display a normal phenotype in the home cage, a familiar environment. Although CREB(alphadelta-) deficient mice exhibit altered behaviour in novel environments, they show normal reactivity to mild and moderate stress as both basal and stress levels of corticosterone are similar to those of wild-type controls. This is the first report of CREB(alphadelta-) deficient mice to: (i) show altered behaviour, not related to learning and memory-associated behaviours, upon initial exposure to environments and (ii) serve as an animal model that can dissociate locomotor activity from anxiety-like behaviour in novel environments.     

Lysergic acid diethylamide and [-]-2,5-dimethoxy-4-methylamphetamine increase extracellular glutamate in rat prefrontal cortex

The ability of hallucinogens to increase extracellular glutamate in the prefrontal cortex (PFC) was assessed by in vivo microdialysis. The hallucinogen lysergic acid diethylamide (LSD; 0.1 mg/kg, i.p.) caused a time-dependent increase in PFC glutamate that was blocked by the 5-HT(2A) antagonist M100907 (0.05 mg/kg, i.p.). Similarly, the 5-HT(2A/C) agonist [-]-2,5-dimethoxy-4-methylamphetamine (DOM; 0.6 mg/kg, i.p.), which is a phenethylamine hallucinogen, increased glutamate to 206% above saline-treated controls. When LSD (10 microM) was directly applied to the PFC by reverse dialysis, a rapid increase in PFC glutamate levels was observed. Glutamate levels in the PFC remained elevated after the drug infusion was discontinued. These data provide direct evidence in vivo for the hypothesis that an enhanced release of glutamate is a common mechanism in the action of hallucinogens.     

Benign focal epileptiform discharges of childhood and hippocampal sclerosis

PURPOSE: Benign focal epileptiform discharges of childhood (BFEDCs) are common EEG findings between ages 4 and 14 years. This epoch of maturational development overlaps with the age at presentation of temporal lobe epilepsy (TLE) due to hippocampal sclerosis (HS) in children. METHODS: From our series of 17 preadolescent children who eventually underwent anteromesial temporal resection for medically refractory TLE due to HS, we identified two children, plus one thereafter, who were initially dismissed as candidates for epilepsy surgery because of abundant extratemporal sharp waves, which were bilateral in two cases. The sharp waves had the distinctive morphology, distribution, and sleep activation suggestive of BFEDCs, but the medical intractability and seizure symptoms were unusual for benign focal epilepsy of childhood. RESULTS: In each case, surgical candidacy was clarified when magnetic resonance imaging (MRI) showed unilateral HS and video-EEG demonstrated seizure onset in the ipsilateral anteromesial temporal region. The postoperative freedom from seizures in each case (follow-up, 2 to 4 years) confirmed that HS was the primary epileptogenic process, and that the BFEDCs were incidental or an atypical secondary manifestation. CONCLUSIONS: These cases illustrate the need for more extensive study of children with BFEDCs when medical intractability and seizure symptoms speak against a simple diagnosis of benign focal epilepsy of childhood. In addition, we observed that the BFEDCs in two of our children had an unusual bilateral occipitofrontal distribution, and we speculate that the coexistence of the BFEDCs in children with HS may not be an incidental finding.     

Nicotinic acetylcholine receptor immunohistochemistry in Alzheimer's disease and dementia with Lewy bodies: differential neuronal and astroglial pathology

Alzheimer's disease (AD) and dementia with Lewy bodies (DLB) are common forms of dementia in the elderly. The neuropathology of AD and DLB is related to cholinergic dysfunctions, and both alpha4 and alpha7 nicotinic acetylcholine receptor (nAChR) subunits are decreased in several brain areas in both diseases. In this immunohistochemical study, we compared neuronal and astroglial alpha4 and alpha7 subunits in AD, DLB and age-matched controls in the hippocampal formation. The numbers of alpha4 reactive neurons were decreased in layer 3 of the entorhinal cortex of AD and DLB, whereas those of alpha7 reactive neurons were decreased in layer 2 of the subiculum of AD and DLB and in layer 3 of the entorhinal cortex of DLB. In contrast, the intensity of alpha7 reactive neuropil was significantly higher in AD than in controls or DLB in a number of areas of the hippocampus (CA3/4 and stratum granulosum), subiculum and entorhinal cortex. An increase in alpha7 immunoreactivity in AD was also associated with astrocytes. The number of astrocytes double-labelled with alpha7 and glial fibrillary acidic protein (GFAP) antibodies was increased in most areas of the hippocampus and entorhinal cortex in AD compared with controls and DLB. Increased astrocyte alpha7 nAChRs in AD may be associated with inflammatory mechanisms related to degenerative processes specific to this disease.