Paul Harrison

Platelet degranulation and monocyte-platelet complex formation are increased in the acute and convalescent phases after ischaemic stroke or transient ischaemic attack.

Flow cytometric studies suggest that platelets are activated in ischaemic stroke or transient ischaemic attack (TIA). However, few studies have measured circulating leucocyte–platelet complexes in this patient population. Whole blood flow cytometry was used to quantify the expression of CD62P‐, CD63‐, and PAC1‐binding, and the percentages of leucocyte–platelet complexes in acute (1–27u2003d, nu2003=u200379) and convalescent (79–725u2003d, nu2003=u200370) ischaemic cerebrovascular disease (CVD) patients compared with controls without CVD (nu2003=u200327). We performed a full blood count, and measured plasma levels of soluble P‐selectin, soluble E‐selectin, and von Willebrand factor antigen (VWF:Ag) as additional markers of platelet and/or endothelial cell activation. The median percentage CD62P expression and the median percentage monocyte–platelet complexes were higher in both acute and convalescent CVD patients than controls (Pu2003≤u20030·02). The mean white cell count and mean VWF:Ag levels were significantly elevated in the acute and convalescent phases after ischaemic stroke or TIA (Pu2003≤u20030·02). Otherwise, there was no significant increase in any other marker of platelet or endothelial activation in CVD patients. There was a positive correlation between the percentage expression of CD62P and the percentages of both neutrophil–platelet and monocyte–platelet complexes in the acute phase, and the percentages of all leucocyte–platelet complexes in the convalescent phase after ischaemic CVD. This study provides evidence for ongoing excessive platelet and/or endothelial activation in ischaemic CVD patients despite treatment with antithrombotic therapy.

Distribution of messenger RNAs encoding the enzymes glutaminase, aspartate aminotransferase and glutamic acid decarboxylase in rat brain.

In situ hybridization histochemistry (ISHH) using synthetic oligonucleotide probes has been used to identify cells containing the mRNAs coding for glutaminase (GluT), aspartate aminotransferase (AspT) and glutamic acid decarboxylase (GAD). The distribution of GAD mRNA confirms previous descriptions and matches the distribution of GAD detected using specific antibodies. AspT mRNA is widely distributed in the brain, but is present at high levels in GABAergic neuronal populations, some that may be glutamatergic, and in a subset of neurons which do not contain significant levels of either GAD or GluT mRNA. Particularly prominent are the neurons of the magnocellular division of the red nucleus, the large cells in the deep cerebellar nuclei and the vestibular nuclei and neurons of the lateral superior olivary nucleus. GluT mRNA does not appear to be present at high levels in all GAD-containing neurons, but is seen prominently in many neuronal populations that may use glutamate as a neurotransmitter, such as neocortical and hippocampal pyramidal cells, the granule cells of the cerebellum and neurons of the dentate gyrus of the hippocampus. The heaviest labelling of GluT mRNA is seen in the lateral reticular nucleus of the medulla. ISHH using probes directed against the mRNAs encoding these enzymes may be an important technique for identifying glutamate and aspartate using neuronal populations and for examining their regulation in a variety of experimental and pathological circumstances.

Circulating reticulated platelets in the early and late phases after ischaemic stroke and transient ischaemic attack.

The percentage of reticulated platelets (% RP) could be a useful marker of increased platelet production and/or turnover in patients with increased platelet activation, but few flow cytometric studies have measured the % RP in patients with ischaemic cerebrovascular disease (CVD). Whole blood flow cytometry using thiazole orange was performed to compare the % RP in patients in the early (1–27u2003d, nu2003=u200379) and late phases (79–725u2003d, nu2003=u200370) after ischaemic stroke or transient ischaemic attack (TIA) with controls without CVD (nu2003=u200327). The impact of aspirin dose escalation (75–300u2003mg/d) on the % RP was investigated in 10 patients in the late phase after stroke/TIA. The platelet count and mean platelet volume (MPV) were similar in CVD patients and controls. Compared with controls, the unadjusted % RP was not significantly higher in early or late phase CVD patients (Pu2003≤u20030·3). However, having adjusted for age, the % RP was higher in early (Pu2003=u20030·047) and late phase CVD patients (Pu2003=u20030·01). There was a positive correlation between % RP and MPV in EDTA‐ and citrate‐anticoagulated blood in both early and late phase CVD patients (P≤ 0·01). The % RP was not significantly influenced by aspirin dose. These data do not convincingly support an excessive stimulus to platelet production in the early or late phases after ischaemic stroke/TIA, but are consistent with the hypothesis that reticulated platelets are larger than more mature ‘non‐reticulated’ platelets in ischaemic CVD.

An Interlaboratory Study of a Candidate Reference Method for Platelet Counting

A multinational interlaboratory task force explored the important variables of platelet reference counting and developed a candidate flow cytometric reference method based on the RBC/platelet ratio. A multicenter comparison was performed to determine whether the method met the necessary criteria and was precise enough to be recommended as a new reference method. Each laboratory analyzed serial dilutions of normal specimens, stabilized material, and at least 60 patient specimens with a range of platelet counts from 1 to 400 x 10(3)/microL (1-400 x 10(9)/L). Pooled analysis of the serial dilutions showed that RBC-platelet and RBC-RBC coincidence events became negligible at sufficiently high dilutions (i.e., > 1:1,000). All laboratories demonstrated excellent intra-assay and acceptable interlaboratory precision. Two antibodies (CD61 and CD41) were used for identifying platelets and individually gave acceptable results, but in a minority of samples, staining differences were observed. The optimum method thus uses a double-labeling procedure with a final dilution factor of 1:1,000. The study demonstrated that this method meets the criteria for a reference platelet count.

Terminal coma affects messenger RNA detection in post mortem human temporal cortex

In situ hybridization histochemistry has been used to study the amount of M1 muscarinic receptor mRNA in temporal cortex from subjects with Alzheimers disease and other neurodegenerative disorders, where the duration of terminal coma was known. Total polyadenylated mRNA and glutamate decarboxylase activity were also measured. Both muscarinic receptor mRNA and enzyme activity showed a significant decline with increasing duration of terminal coma, but were not related to diagnosis. Polyadenylated mRNA signal did not show an association with coma. These data indicate the need to consider the nature of the terminal illness in post mortem studies of mRNA as well as for neurochemical research.

Increased muscarinic receptor messenger RNA in Alzheimer's disease temporal cortex demonstrated by in situ hybridization histochemistry

A 35S-labelled synthetic oligonucleotide directed against part of the mRNA coding for the M1 subtype muscarinic receptor was used for in situ hybridization histochemistry in sections of human temporal cortex. M1 receptor mRNA was found in cell populations throughout the grey matter, especially in pyramidal cells. Quantitative densitometric analysis of autoradiograms was used to compare levels of this mRNA between Alzheimers disease and controls. A significant (2.7-fold) increase in hybridization signal was found in Alzheimers disease cases, both in absolute terms and relative to total polyadenylated mRNA as determined by hybridization with an oligodeoxythymidine probe. Elevated levels of muscarinic receptor mRNA may reflect up-regulation of transcription of this gene in response to the cholinergic deficits occurring in the disease.

Alzheimer's disease: specific increases in a G protein subunit (GSα) mRNA in hippocampal and cortical neurons

The GTP binding protein, Gs, activates adenyl cyclase in direct response to stimulation of several neurotransmitter receptors. In situ hybridization histochemistry (ISHH) with a 35S-labelled oligonucleotide has been used to detect the mRNA encoding the alpha subunit of Gs (Gs alpha) in human hippocampus, temporal and visual cortices and cerebellum, and its level has been compared between Alzheimers disease (AD) and control brains. A marked regional increase was found in the hippocampus of AD cases. Analysis of levels of Gs alpha mRNA in individual constituent pyramidal cells confirmed this increase (3 to 4-fold in densitometric units) in hippocampal fields CA1, CA3 and CA4, as well as in temporal cortex. Levels of Gs alpha mRNA were also determined relative to total poly(A)+ mRNA in the same cell populations in each case. Gene-specific elevation of Gs alpha mRNA was thereby confirmed in hippocampal fields, and also in temporal cortex. No changes were seen in visual cortex. The increase in Gs alpha mRNA may represent a response by AD neurons in affected areas to receptor alterations, or to an abnormality in receptor-G protein coupling. Alternatively, altered G protein gene expression might be a pathogenic event underlying changes in linked receptor populations.

Messenger RNA encoding the D2 dopaminergic receptor detected by in situ hybridization histochemistry in rat brain.

A 30 base synthetic oligonucleotide probe was used to detect the mRNA encoding the rat D2 dopaminergic receptor. On Northern analysis, the probe identified a single species of mRNA of approximately 2.9 kb, present at highest levels in the striatum but also found in the brainstem, neocortex and diencephalon. On sections, neurons containing high levels of the mRNA were detected in the striatum, the substantia nigra pars compacta and the ventral tegmental area. Lower levels of signal were seen over neurons in the hypothalamus, the frontal neocortex, and the globus pallidus.

The expression of prion protein by endothelial cells: a source of the plasma form of prion protein?

Summary. The neuronal prion protein (PrPC) is also expressed within peripheral tissues including human blood. The majority of blood PrPC is found within the plasma fraction. We hypothesized that the vascular endothelium could be a source of this PrPC. Reverse transcription polymerase chain reaction demonstrated that both human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC‐1) expressed PrPC mRNA. Flow cytometry confirmed PrPC expression on HMEC‐1s and HUVECs (120 900 ± 15 058 and 58 327 ± 4577 molecules PrPC/cell respectively), with no upregulation following cellular activation. Confocal immunofluorescence microscopy confirmed that HMEC‐1s and HUVECs were positive for PrPC on the plasma membrane. Time‐resolved dissociation‐enhanced fluoroimmunoassay (DELFIA®) analysis of cell culture medium demonstrated a slow constitutive release of soluble PrPC not associated with activation. In contrast to von Willebrand factor antigen, PrPC plasma levels in vivo decrease following desmopressin therapy in patients with von Willebrand disease. Measurement of PrPC plasma levels in patients with varying blood counts demonstrated no association between cell count and PrPC concentration. However, there was a higher level of PrPC in plasma from patients with end‐stage renal failure. In conclusion, endothelial cells of both macrovascular and microvascular origin expressed high levels of PrPC which can be constitutively released into the cell culture medium.

Increased platelet count and leucocyte–platelet complex formation in acute symptomatic compared with asymptomatic severe carotid stenosis

Objective: The risk of stroke in patients with recently symptomatic carotid stenosis is considerably higher than in patients with asymptomatic stenosis. In the present study it was hypothesised that excessive platelet activation might partly contribute to this difference. Methods: A full blood count was done and whole blood flow cytometry used to measure platelet surface expression of CD62P, CD63, and PAC1 binding and the percentage of leucocyte–platelet complexes in patients with acute (0–21 days, nu200a=u200a19) and convalescent (79–365 days) symptomatic (nu200a=u200a16) and asymptomatic (nu200a=u200a16) severe (⩾70%) carotid stenosis. Most patients were treated with aspirin (37.5–300 mg daily) although alternative antithrombotic regimens were more commonly used in the symptomatic group. Results: The mean platelet count was higher in patients with acute and convalescent symptomatic compared with asymptomatic carotid stenosis. There were no significant differences in the median percentage expression of CD62P and CD63, or PAC1 binding between the acute or convalescent symptomatic and asymptomatic patients. The median percentages of neutrophil–platelet (pu200a=u200a0.004), monocyte–platelet (pu200a=u200a0.046), and lymphocyte–platelet complexes (pu200a=u200a0.02) were higher in acute symptomatic than in asymptomatic patients. In patients on aspirin monotherapy, the percentages of neutrophil–platelet and monocyte–platelet complexes (pu200a=u200a0.03) were higher in acute symptomatic (nu200a=u200a11) than asymptomatic patients (nu200a=u200a14). In the convalescent phase, the median percentages of all leucocyte–platelet complexes in the symptomatic group dropped to levels similar to those found in the asymptomatic group. Conclusion: Increased platelet count and leucocyte–platelet complex formation may contribute to the early excess risk of stroke in patients with recently symptomatic carotid stenosis.