Avoiding artefacts during electron microscopy of silver nanomaterials exposed to biological environments.

Electron microscopy has been applied widely to study the interaction of nanomaterials with proteins, cells and tissues at nanometre scale. Biological material is most commonly embedded in thermoset resins to make it compatible with the high vacuum in the electron microscope. Room temperature sample preparation protocols developed over decades provide contrast by staining cell organelles, and aim to preserve the native cell structure. However, the effect of these complex protocols on the nanomaterials in the system is seldom considered. Any artefacts generated during sample preparation may ultimately interfere with the accurate prediction of the stability and reactivity of the nanomaterials. As a case study, we review steps in the room temperature preparation of cells exposed to silver nanomaterials (AgNMs) for transmission electron microscopy imaging and analysis. In particular, embedding and staining protocols, which can alter the physicochemical properties of AgNMs and introduce artefacts thereby leading to a misinterpretation of silver bioreactivity, are scrutinized. Recommendations are given for the application of cryogenic sample preparation protocols, which simultaneously fix both particles and diffusible ions. By being aware of the advantages and limitations of different sample preparation methods, compromises or selection of different correlative techniques can be made to draw more accurate conclusions about the data.

Experimental heart failure modelled by the cardiomyocyte-specific loss of an epigenome modifier, DNMT3B.

Differential DNA methylation exists in the epigenome of end-stage failing human hearts but whether it contributes to disease progression is presently unknown. Here, we report that cardiac specific deletion of Dnmt3b, the predominant DNA methyltransferase in adult mouse hearts, leads to an accelerated progression to severe systolic insufficiency and myocardial thinning without a preceding hypertrophic response. This was accompanied by widespread myocardial interstitial fibrosis and myo-sarcomeric disarray. By targeted candidate gene quantitative RT-PCR, we discovered an over-activity of cryptic splice sites in the sarcomeric gene Myh7, resulting in a transcript with 8 exons missing. Moreover, a region of differential methylation overlies the splice site locus in the hearts of the cardiac-specific conditional knockout (CKO) mice. Although abundant and complex forms of alternative splice variants have been reported in diseased hearts and the contribution of each remains to be understood in further detail, our results demonstrate for the first time that a link may exist between alternative splicing and the cardiac epigenome. In particular, this gives the novel evidence whereby the loss of an epigenome modifier promotes the development and progression of heart disease.

Enhanced peroxynitrite formation is associated with vascular aging.

Vascular aging is mainly characterized by endothelial dysfunction. We found decreased free nitric oxide (NO) levels in aged rat aortas, in conjunction with a sevenfold higher expression and activity of endothelial NO synthase (eNOS). This is shown to be a consequence of age-associated enhanced superoxide (.O(2)(-)) production with concomitant quenching of NO by the formation of peroxynitrite leading to nitrotyrosilation of mitochondrial manganese superoxide dismutase (MnSOD), a molecular footprint of increased peroxynitrite levels, which also increased with age. Thus, vascular aging appears to be initiated by augmented.O(2)(-) release, trapping of vasorelaxant NO, and subsequent peroxynitrite formation, followed by the nitration and inhibition of MnSOD. Increased eNOS expression and activity is a compensatory, but eventually futile, mechanism to counter regulate the loss of NO. The ultrastructural distribution of 3-nitrotyrosyl suggests that mitochondrial dysfunction plays a major role in the vascular aging process.

Application of environmental scanning electron microscopy to determine biological surface structure.

The use of environmental scanning electron microscopy in biology is growing as more becomes understood about the advantages and limitations of the technique. These are discussed and we include new evidence about the effect of environmental scanning electron microscopy imaging on the viability of mammalian cells. We show that although specimen preparation for high-vacuum scanning electron microscopy introduces some artefacts, there are also challenges in the use of environmental scanning electron microscopy, particularly at higher resolutions. This suggests the two technologies are best used in combination. We have used human monocyte-derived macrophages as a test sample, imaging their complicated and delicate membrane ruffles and protrusions. We have also explored the possibility of using environmental scanning electron microscopy for dynamic experiments, finding that mammalian cells cannot be imaged and kept alive in the environmental scanning electron microscopy. The dehydration step in which the cell surface is exposed causes irreversible damage, probably via loss of membrane integrity during liquid removal in the specimen chamber. Therefore, mammalian cells should be imaged after fixation where possible to protect against damage as a result of chamber conditions.

Post-translational modifications regulate matrix Gla protein function: importance for inhibition of vascular smooth muscle cell calcification.

BACKGROUND: Matrix Gla protein (MGP) is a small vitamin K-dependent protein containing five gamma-carboxyglutamic acid (Gla) residues that are believed to be important in binding Ca(2+), calcium crystals and bone morphogenetic protein. In addition, MGP contains phosphorylated serine residues that may further regulate its activity. In vivo, MGP has been shown to be a potent inhibitor of vascular calcification; however, the precise molecular mechanism underlying the function of MGP is not yet fully understood. METHODS AND RESULTS: We investigated the effects of MGP in human vascular smooth muscle cell (VSMC) monolayers that undergo calcification after exposure to an increase in Ca(2+) concentration. Increased calcium salt deposition was found in cells treated with the vitamin K antagonist warfarin as compared to controls, whereas cells treated with vitamin K(1) showed decreased calcification as compared to controls. With conformation-specific antibodies, it was confirmed that warfarin treatment of VSMCs resulted in uncarboxylated (Gla-deficient) MGP. To specifically test the effects of MGP on VSMC calcification, we used full-length synthetic MGP and MGP-derived peptides representing various domains in MGP. Full length MGP, the gamma-carboxylated motif (Gla) (amino acids 35-54) and the phosphorylated serine motif (amino acids 3-15) inhibited calcification. Furthermore, we showed that the peptides were not taken up by VSMCs but bound to the cell surface and to vesicle-like structures. CONCLUSIONS: These data demonstrate that both gamma-glutamyl carboxylation and serine phosphorylation of MGP contribute to its function as a calcification inhibitor and that MGP may inhibit calcification via binding to VSMC-derived vesicles.

Characterization of the snake venom ligand [125I]-DNP binding to natriuretic peptide receptor-A in human artery and potent DNP mediated vasodilatation.

BACKGROUND AND PURPOSE: The natriuretic peptides, ANP and BNP, modulate vascular smooth muscle tone in human conduit arteries. Surprisingly, the natriuretic peptide receptor-A (NPR-A) has not been visualized using radioligand binding in these vessels. A new member of this peptide family, Dendroaspis natriuretic peptide (DNP) identified from snake venom, has been proposed to be present in human plasma and endothelial cells. Also, recently a novel radioligand, [(125)I]-DNP, has been characterized as selective for NPR-A in human heart. EXPERIMENTAL APPROACH: Our aims were to investigate expression and function of NPR-A receptors in human mammary artery using [(125)I]-DNP to quantify receptor density, immunocytochemistry to delineate the cellular distribution of the receptor and in vitro pharmacology to compare DNP induced vasodilatation to that of ANP. KEY RESULTS: Saturable, sub-nanomolar affinity [(125)I]-DNP binding was detected to smooth muscle of mammary artery, with receptor density of approximately 2 fmol mg(-1) protein, comparable to that of other vasoactive peptides. NPR-A immunoreactivity was localised to vascular smooth muscle cells and this was confirmed with fluorescence dual labelling. NPR-A expression was not detected in the endothelium. Like ANP, DNP fully reversed the constrictor response to ET-1 in endothelium intact or denuded mammary artery, with comparable nanomolar potencies. CONCLUSIONS AND IMPLICATIONS: This is the first characterization of NPR-A in human mammary artery using [(125)I]-DNP and we provide evidence for the presence of receptor protein on vascular smooth muscle cells, but not endothelial cells. This implies that the observed vasodilatation is predominantly mediated via direct activation of smooth muscle NPR-A.

Apoptosis regulates human vascular calcification in vitro: evidence for initiation of vascular calcification by apoptotic bodies.

The mechanisms involved in the initiation of vascular calcification are not known, but matrix vesicles, the nucleation sites for calcium crystal formation in bone, are likely candidates, because similar structures have been found in calcified arteries. The regulation of matrix vesicle production is poorly understood but is thought to be associated with apoptotic cell death. In the present study, we investigated the role of apoptosis in vascular calcification. We report that apoptosis occurs in a human vascular calcification model in which postconfluent vascular smooth muscle cell (VSMC) cultures form nodules spontaneously and calcify after approximately 28 days. Apoptosis occurred before the onset of calcification in VSMC nodules and was detected by several methods, including nuclear morphology, the TUNEL technique, and external display of phosphatidyl serine. Inhibition of apoptosis with the caspase inhibitor ZVAD.fmk reduced calcification in nodules by approximately 40%, as measured by the cresolphthalein method and alizarin red staining. In addition, when apoptosis was stimulated in nodular cultures with anti-Fas IgM, there was a 10-fold increase in calcification. Furthermore, incubation of VSMC-derived apoptotic bodies with (45)Ca demonstrated that, like matrix vesicles, they can concentrate calcium. These observations provide evidence that apoptosis precedes VSMC calcification and that apoptotic bodies derived from VSMCs may act as nucleating structures for calcium crystal formation.

Acetylated low-density lipoprotein stimulates human vascular smooth muscle cell calcification by promoting osteoblastic differentiation and inhibiting phagocytosis.

BACKGROUND: Vascular smooth muscle cells (VSMCs) in atherosclerotic lesions display an osteogenic phenotype, and calcification commonly occurs in association with lipid. We therefore tested the hypothesis that lipid components in atherosclerotic lesions influenced VSMC phenotype and calcification using an in vitro model of calcification. METHODS AND RESULTS: In situ hybridization of human atherosclerotic plaques (n=10) collected from patients undergoing carotid endarterectomy demonstrated that subsets of lipid-filled VSMCs adjacent to sites of calcification expressed alkaline phosphatase, bone Gla protein, and bone sialoprotein, suggesting an osteogenic phenotype. Treatment of VSMCs in culture with acetylated low-density lipoprotein (acLDL) or lipoprotein-deficient serum altered the time course of bone-associated protein gene expression and calcification. AcLDL increased nodule calcification 3-fold, whereas lipoprotein-deficient serum significantly inhibited it. Reverse transcriptase-polymerase chain reaction and Western analysis demonstrated the presence of the acLDL receptor, SRA1, exclusively in calcifying nodular VSMCs, and blockade of SRA with polyinosinic acid inhibited acLDL-induced calcification. Because apoptotic bodies can serve as nucleation sites for calcification, we investigated whether acLDL could stimulate apoptosis in nodules. Apoptosis of nodular VSMCs was unaltered, but the number of apoptotic bodies per nodule increased approximately 3-fold, implying a defect in phagocytosis. Consistent with these observations, binding of apoptotic bodies to VSMCs was decreased in the presence of acLDL. CONCLUSIONS: These studies suggest that modified lipoproteins stimulate calcification by enhancing osteogenic differentiation of VSMCs and by a novel mechanism whereby acLDL interacts with SRA on VSMCs and blocks phagocytic removal of apoptotic bodies.

Immunoelectron microscopical localization of human papillomavirus type 16 L1 and E4 proteins in cervical keratinocytes cultured in vivo.

The human papillomavirus (HPV) causes warts, but is also associated with the development of squamous cell dysplasia and carcinoma. The virus is host and tissue specific and the numerous HPV types show predilection for different body sites. Experimental production of HPV 16 particles is at present only possible using in vivo culture of keratinocytes containing episomal viral DNA. Using immunoelectron microscopy, we have investigated the localization of HPV 16 E4 and L1 proteins in a keratinized epithelium formed by grafting HPV 16-containing cervical keratinocytes onto the athymic mouse. New viral progeny are produced in this system, as confirmed by labeling of intranuclear particles with a mouse monoclonal antibody against the HPV 16 major capsid (L1) protein. The role of the E4 protein is not yet clear, although it is believed to be important for the later stages of the virus life cycle. Here we confirm its cytoplasmic localization in the cells of the spinous and granular layers and demonstrate co-localization with keratin tonofilaments.

Susceptibility of human placental syncytiotrophoblastic mitochondria to oxygen-mediated damage in relation to gestational age.

When maintaining first trimester placental villi in organ culture under conventional normoxic conditions, we have observed widespread degeneration of the syncytiotrophoblast within 24 h despite excellent viability for the cytotrophoblastic and stromal cell types. Here we identify loss of mitochondrial activity as an early event in this process. In the light of proposals that the early part of gestation occurs in a low oxygen environment and also reported associations between mitochondrial disruption and oxidative stress, we cultured first trimester villi under low oxygen conditions (2.5%). Mitochondrial superoxide dismutase (MnSOD) localization and activity at different gestational ages were also determined. It was found that syncytiotrophoblastic and mitochondrial morphology improved, and mitochondrial activity was retained for 6 h and more if 8- to 10-week-old tissue was placed into a low oxygen environment immediately after removal from the uterus. The effect of oxygen concentration was less marked when using tissue of 14 weeks or more gestational age, which showed good survival and retention of mitochondrial activity under both low and ambient oxygen conditions. This correlated with our finding that placental MnSOD activity increased significantly between 8 and 14 weeks of gestation. Immunohistochemistry demonstrated that at 11 weeks; MnSOD was localized predominantly within the cytotrophoblast cells, whereas by 16 weeks it was found in the syncytiotrophoblast also. These results indicate an acute sensitivity of first trimester placenta syncytiotrophoblast to oxygen-mediated damage.

Postnatal development of a sexually dimorphic, hypothalamic nucleus in gerbils: a stereological study of neuronal number and apoptosis.

Steroid-sensitive, vocal courtship behavior is a function of a specific, hypothalamic nucleus, the sexually dimorphic area pars compacta (SDApc) in the male adult gerbil. Gender-related differences in the number of neurons in this nucleus are evident immediately after birth. By using unbiased stereological estimates of cell numbers in Nissl-stained, paraffin-wax sections of brain, we investigated the mechanisms differentiating cell number between the sexes in the SDApc on postnatal days 0, 3, 6, and 15. Cell death, identified by pyknosis, was greatest in the SDApc between days 0-3 in males, whereas in females, maximum values were reached between days 3-6. Similarly, the ratio of pyknotic to normal neurons peaked between days 0-3 in males and 3-6 in females but then declined in both sexes. Pyknotic cells were seldom seen in either sex by day 15. Morphological characteristics of apoptosis including chromatin condensation, cell fragmentation, and ingestion of apoptic bodies by macrophages were all demonstrated by transmission electron microscopy. Macrophages showed specific morphological characteristics of microglia. Cell division (mitosis) was identified in the SDApc during postnatal days 0, 3, and 6 but the numbers of mitotic figures were low, negligible on day 15, and similar between the sexes. These results demonstrate that cell death and proliferation occur simultaneously in the neonatal gerbil brain. The stereological estimates of cell death in the developing SDApc indicated a lower incidence of neuronal death occurring earlier in males than in females.

The effect of concurrent administration of isradipine on the development of fatty streaks in the cholesterol-fed rabbit: a morphometric study.

Calcium antagonists attenuate the development of aortic lesions in cholesterol-fed rabbits. This study was undertaken to examine the influence of isradipine (dose: 0.3 mg/kg per day orally) on the histological components of these lesions in New Zealand White rabbits (age: 12 weeks, weight: 2-2.5 kg). Five groups of animals were fed standard chow with the following supplements for 3 weeks: Group 1, no supplements; Group 2, 40 g cholesterol; Group 3, 60 g cholesterol; Group 4, 40 g cholesterol + isradipine; Group 5, 60 g cholesterol + isradipine. After 3 weeks, the animals were killed and the aorta prepared for morphometry. The volume of intima/cm aorta was estimated and the volume fraction (Vv) of the intima occupied by components of the lesions was estimated by point counting. By integrating these two measurements the volume/unit length (mm3/cm) of the following components of the aorta were estimated: intima, non-cellular components, endothelial cells, myointimal cells, lipid accumulating myointimal cells and foam cells. Cholesterol feeding for 3 weeks was associated with significant increases in the volume of non-cellular components of the intima, endothelial cells, myointimal cells, lipid accumulating myointimal cells and foam cells. Administration of isradipine significantly reduced all these parameters. It is concluded that isradipine attenuates cellular hyperplasia and accumulation of non-cellular components of lesions in cholesterol fed rabbits.

The effect of isradipine administration on existing fatty streaks in the cholesterol-fed rabbit: a morphometric study.

Previous studies have shown that the administration of certain calcium channel blocking drugs (at an appropriate time point) can reduce the severity of atherosclerotic lesion formation. This study was undertaken to determine if the administration of isradipine would reverse established lesions produced by feeding rabbits an atherogenic diet. Rabbits were fed cholesterol for three weeks and examined directly, or after being left for a four week washout period, with or without a daily oral supplement of isradipine. Fatty streaks were well established after three weeks of cholesterol feeding and were more extensive at the end of the washout period, as indicated by gross changes in the volume of the intima per unit length of aorta. When isradipine was administered during the washout period, the volume of the intima per unit length of aorta fell to levels below those produced by cholesterol feeding for three weeks alone. The major components of the lesions affected to accommodate these changes were the foam cells and myointimal cells; these were examined in detail using morphometry and lipid cytochemistry. The mean volume of intima/cm of aorta occupied by foam cells and myointimal cells both fell by more than 60% to levels lower than those found after three weeks of cholesterol feeding alone. The volume of the extracellular space of the intima occupied by cytochemically demonstrable unesterified and esterified cholesterol was reduced by isradipine administration as was that of foam cells, all to levels lower than those found after three weeks of cholesterol feeding alone. These data indicate that the administration of isradipine during a washout period, after cholesterol feeding, can promote the regression of fatty streak lesions.

Changes in vimentin in human macrophages during apoptosis induced by oxidised low density lipoprotein.

Macrophage apoptosis contributes to the development of human atherosclerotic lesions. Oxidised LDL may be involved in macrophage death in vivo. We examined morphological and biochemical changes to the vimentin filament network during apoptosis of human macrophages. Only oxidised LDL, but not native or acetylated LDL, induced apoptosis, wherein vimentin was cleaved into fragments of 48-50, 46, 29 and 26 kDa. The use of caspase inhibitors suggested that caspase-6 mediates the formation of the 26 and 46 kDa fragments of vimentin. We were unable to demonstrate any significant involvement of caspase-3 in vimentin cleavage. However, caspase-3 was clearly activated during apoptosis whilst caspase-6 expression in macrophages was minimal. Vimentin filament breakdown occurred early during apoptosis and vimentin immunoreactivity was present in apoptotic bodies. However, the application of caspase inhibitors had no effect on the morphology of the vimentin network in apoptotic cells, suggesting that filament breakdown is not mediated by caspase proteolysis. Similar changes in vimentin were also seen in gliotoxin-induced apoptosis.

Evidence that the death of macrophage foam cells contributes to the lipid core of atheroma.

Sections of human atherosclerotic lesions of different stages show that, in early lesions, the acellular lipid core is usually immediately adjacent to the deepest edge of a collection of macrophage foam cells. Advanced lesions with a large lipid core have variable numbers of macrophage foam cells, close to the lateral edges, or shoulders, of the core. In both early and advanced lesions, some of the macrophages nearest the core appear to be dying. Lipid cores contain two materials which in earlier lesions are found only in macrophages, namely ceroid and CD68 antigen, but do not contain recognisable smooth muscle cell actin. It is concluded that death of macrophage foam cells contributes to the origin and slow enlargement of the lipid core. The cause of macrophage death is not yet certain, but is under investigation.

Comparison of connexin 43, 40 and 45 expression patterns in the developing human and mouse hearts.

The mouse is currently widely used as a model organism in the analysis of gene function but how developmentally regulated patterns of connexin gene expression in the mouse compare with those in the human is unclear. Here we compare the patterns of connexin expression in the heart during the development of the mouse (from embryonic day 12.5 to 6 weeks postpartum) and the human (at 9 weeks gestation and adult stage). The extent of connexin43 expression in the ventricles progressively increased during development of the mouse heart. The developmental pattern of expression for connexins 40 and 45 in the mouse heart was similar, but not identical, and in the ventricles showed a progressive and preferential expression in the conduction system. In general, these dynamic changes of connexins 43, 40 and 45 during mouse cardiac development appear to be mirrored in the human.

Changes in concentration, localization and activity of catalase within the human placenta during early gestation.

Using villous tissue from accurately dated gestational age placentae, this study identified significant changes in the protein concentration, enzyme activity and localization of catalase, an enzyme responsible for the intracellular metabolism of hydrogen peroxide, during the first and early second trimester of pregnancy. Enzyme activity was found to increase approximately threefold between weeks 6 and 17, with the greatest increase between 12 and 17 weeks. Immunostaining of tissue sections was supportive of these findings, demonstrating a progressively stronger signal between weeks 6 and 17. Immunostaining also demonstrated that the main cell types expressing catalase were the cytotrophoblast cells as well as a subset of the stromal cells. Between 13-17 weeks gestation, however, it was possible to detect catalase within the syncytiotrophoblast also, although with a much reduced intensity of staining. At the ultrastructural level, immunogold labelling of catalase clearly showed that staining was predominately compartmentalized within peroxisomes, although non-peroxisomal staining was also seen. Immunoreactivity also demonstrated, via morphological identification, that the stromal cells containing detectable levels of catalase were placental macrophages (Hofbauer cells). These results are in agreement with the proposal that the placenta exists in a physiologically low oxygen environment during the early part of gestation. In this environment oxidative activity of the sort resulting in the generation of hydrogen peroxide would presumably be suppressed, thereby limiting the requirement for catalase until oxygen tension begins to rise.

A reappraisal of the contrasting morphological appearances of villous cytotrophoblast cells during early human pregnancy; evidence for both apoptosis and primary necrosis.

Villous cytotrophoblast cells display a range of morphological appearances that are assumed to reflect different stages of differentiation. Here we demonstrate that apoptosis and primary necrosis can also occur in these cells during normal early pregnancy, and should be included in the list of possible phenotypes. Samples from 30 placentae of 6-15 weeks gestational age were examined. Cytotrophoblast cells displaying highly condensed chromatin, but no karyorhexis, were observed detached from the basement membrane, and represented 0.49% (s.d.+/-0.36) of the total population. Their cytoplasm was heavily vacuolated, and their mitochondria swollen, indicating secondary necrosis. By contrast, extremely pale-staining cells with large rounded nuclei (volume-weighted mean volume 471.6 microm(3) compared to 250.1 microm(3) for euchromatic cells) were frequently observed (5.97% of total, s.d.+/-4.31). These cells displayed loss of euchromatin, a paucity of cytoplasmic organelles, and swelling of the mitochondrial intracristal space and endoplasmic reticulum. Nuclei of these cells displayed a significantly higher level of gold labelling using the TUNEL technique compared to euchromatic nuclei [1.0 particles/microm(2) (s.d.+/-0.13) vs 0.12 particles/microm(2) (s.d.+/-0.03),P< 0.05], confirming increased DNA fragmentation. We conclude that these cells are undergoing primary necrosis. The stimulus for both forms of cell death remains unknown, but may be associated with syncytiotrophoblastic stress.

Comparison of in vivo dissolution processes in hydroxyapatite and silicon-substituted hydroxyapatite bioceramics.

The incorporation of silicate into hydroxyapatite (HA) has been shown to significantly increase the rate of bone apposition to HA bioceramic implants. However, uncertainty remains about the mechanism by which silicate increases the in vivo bioactivity of HA. In this study, high-resolution transmission electron microscopy was used to observe dissolution from HA, 0.8 wt% Si-HA and 1.5 wt% Si-HA implants after 6 and 12 weeks in vivo. Our observations confirmed that defects, in particular those involving grain boundaries, were the starting point of dissolution in vivo. Dissolution was observed to follow the order 1.5 wt% Si-HA>0.8 wt% Si-HA>pure HA and it was found to be particularly prevalent at grain boundaries and triple-junctions. These observations may help to explain the mechanism by which silicate ions increase the in vivo bioactivity of pure HA, and highlight the enhanced potential of these ceramics for biomedical applications.

Ultrastructural and cytochemical study of neurones in the rat dorsal motor nucleus of the vagus after axon crush.

The cell populations in the dorsal motor nucleus of the vagus (DMNV) of the rat were studied by light microscopy and transmission electron microscopy, including retrograde labeling with horseradish peroxidase and histochemical demonstration of the distribution of the activity of the enzymes acetylcholinesterase (AcChE) and butyrylcholinesterase (BuChE). Two types of neurones were observed: 1) Larger Type A cells, which stain for both AcChE and BuChE and which project into the vagus nerve trunk, and 2) smaller Type B cells, which stain lightly for AcChE but not for BuChE and which do not project into the vagus nerve. Standardised vagal crush at the mid-cervical level causes loss of cholinesterase activity in Type A neurones within a few days but has no effect on Type B neurones. Changes in nuclear morphology of Type A neurones are pronounced at 10 weeks postinjury, indicating that degeneration is irreversible even by this stage. The number of Type A cells projecting to the vagus nerve reduces as a function of time, presumably as these cells die. Only a small number of Type A neurones persist at 2 years postinjury.