Tractography of the parahippocampal gyrus and material specific memory impairment in unilateral temporal lobe epilepsy

Introduction Temporal lobe epilepsy (TLE) is associated with disrupted memory function. The structural changes underlying this memory impairment have not been demonstrated previously with tractography.
Introduction
Temporal lobe epilepsy (TLE) is the most common form of refractory focal epilepsy (Crawford, 2000). Parahippocampal structures, which are critically implicated in the generation and propagation of seizures in TLE (Avoli et al., 2002; Bertram, 2006; Du et al., 1993; Plate et al., 1993; Rutecki et al., 1989; Spencer and Spencer, 1994), are essential for declarative memory (Eichenbaum, 2000). Longitudinal neuropsychological studies have shown that persisting epilepsy is associated with progressive memory impairment (Dodrill, 2002; Helmstaedter et al., 2003). Those who undergo anterior temporal lobe resection (ATLR) are at further risk of memory impairment, the nature of which depends on whether surgery is on the dominant or non-dominant side (Ivnik et al., 1987; Spiers et al., 2001).
Neuropsychological assessment, quantitative MRI, and latterly functional MRI (fMRI) indicate the role of the medial temporal lobe structures (MTL) in sustaining material specific memory functions (Powell et al., 2005), and the reorganisation of memory that occurs with TLE (Powell et al., 2007a). These and lesion deficit studies (Frisk and Milner, 1990; Smith and Milner, 1981) have shown that memory deficit after ATLR is related to the functional integrity of the parahippocampal structures. The strength of connections, or structural connectivity, of the parahippocampal gyrus in TLE has not been evaluated, or related to function.
Tractography is a technique which uses diffusion tensor imaging (DTI) data to delineate white matter tracts, and to quantify their volume, and infer structural characteristics (Johansen-Berg and Behrens, 2006). Using one such tractography technique, Probabilistic Index of Connectivity or PICo (Parker et al., 2003; Parker and Alexander 2003), we evaluated the structural connectivity of the parahippocampal gyrus in TLE, with the hypothesis that this would be impaired ipsilaterally to the seizure focus, and that the degree of any such impairment would correlate with material specific memory function.
Methods
Subjects
We studied 18 patients (median age 33.5 years; range 22–47 years; 11 males) with medically refractory TLE undergoing pre-surgical evaluation at the National Hospital for Neurology and Neurosurgery, London, UK. All patients had undergone structural MRI at 1.5T (Duncan, 1997). Of the eight left TLE patients, seven had hippocampal sclerosis (HS) (one also had a ganglioglioma in the left fusiform gyrus) and one had a MTL dysembryoblastic neuroepithelial tumour (DNET). Of the ten right TLE patients, seven had HS, one had a right MTL glioma, another had a right MTL DNET, and the other had right superior temporal focal cortical dysplasia (FCD). Video-EEG had confirmed seizure onset in the MTL ipsilateral to the clinically defined seizure site, and all patients had a normal, contralateral hippocampus based on qualitative and quantitative MRI criteria (Woermann et al., 1998).
All patients were on anti-epileptic medication, and were fluent English language speakers. Handedness was determined using the Edinburgh handedness inventory (Oldfield, 1971), and language dominance was determined using a range of fMRI tasks which have been described previously and include the use of verbal fluency, and reading tasks (Powell et al., 2006). All patients underwent a standardised pre-surgical neuropsychological assessment (Baxendale et al., 1998). Patient demographics, neurological test results, and surgical outcome data are listed in Table 1. The ILAE classification of post-operative seizure outcome following epilepsy surgery was used (Wieser et al., 2001). We also studied 10 right-handed native English speaking, healthy volunteers (median age 29.5 years; range 23 to 50 years; 7 females). The study was approved by the National Hospital for Neurology and Neurosurgery and the Institute of Neurology Joint Ethics Committee, and informed written consent was obtained from all subjects.
MR data acquisition
MRI studies were performed on a 1.5-T GE Signa Horizon scanner (General Electric, Wakashua, Milwaukee, Wisconsin, USA). Standard imaging gradients with a maximum strength of 22 m Tm- 1 and slew rate 120 Tm- 1s- 1 were used. All data were acquired using a standard quadrature birdcage head coil for both RF transmission and reception. The scanning protocol also included a coronal T1-weighted volumetric acquisition sequence with 1.5-mm-thick slices, and hippocampal volumes were determined according to a previously described method (Moran et al., 1999).
Diffusion tensor imaging
The DTI acquisition sequence was a single-shot spin-echo planar imaging (EPI) sequence, cardiac gated (triggering occurring every QRS complex) (Wheeler-Kingshott et al., 2002), with TE = 95 ms. Sets of 60 contiguous 2.3-mm thickness axial slices were obtained, covering the whole brain, with diffusion sensitizing gradients applied in each of 54 non-colinear directions (maximum b value of 1148 mm2 s- 1 (δ = 34 ms, Δ = 40 ms, using full gradient strength of 22 mTm- 1)) along with 6 non-diffusion weighted (b = 0) scans. The field of view was 24 cm, and the acquisition matrix size was 96 × 96, zero filled to 128 × 128 during reconstruction so that the reconstructed voxel size was 1.8 × 1.8 × 2.3 mm3. The DTI acquisition time for a total of 3600 image slices was approximately 25 min (depending on the heart rate).
We used the method of Parker and Alexander (Parker et al., 2003; Parker and Alexander, 2003) to reduce fibre orientation ambiguities in voxels containing fibre crossings. Voxels in which the single tensor fitted the data poorly were identified using the spherical-harmonic voxel-classification algorithm of Alexander et al. (Alexander et al., 2002). In these voxels a mixture of two Gaussian probability densities was fitted and the principal diffusion directions of the two diffusion tensors provided estimates of the orientations of the crossing fibres (Tuch et al., 2002). In all other voxels a single tensor model was fitted. For all voxels, fractional anisotropy (FA) maps were generated from the single tensor fit (Pierpaoli et al., 1996; Pierpaoli and Basser, 1996).
Tractography
All scans were transferred to a Unix workstation for processing. We used the PICo algorithm extended to cope with crossing fibres (Parker et al., 2003; Parker and Alexander, 2003) to track from anatomically defined regions of interest (ROIs) within the parahippocampal gyrus. This algorithm adapts the commonly used streamline approach to exploit the uncertainty due to noise in one or more fibre orientations defined for each voxel. This uncertainty is defined using probability density functions (PDFs) constructed using simulations of the effect of realistic data noise on fibre directions obtained from the mixture model (Parker and Alexander, 2003). The streamline process is repeated using Monte Carlo methods to generate maps of connection probability or confidence of connection from the chosen start region(s).
The anatomical definition of the ROI was based on a previously published tractography analysis of the parahippocampal gyrus in healthy subjects (Powell et al., 2004). Viewing the FA images in three orthogonal planes using MRIcro (http://www.psychology. nottingham.ac.uk), the centre of the white matter tract just anterior to the brainstem, and posterior to the cerebral peduncles was selected, such that the parahippocampal gyrus was defined at its longest in the corresponding sagittal view (Fig. 1). The corresponding coronal slice was then used to select two adjacent voxels in a left-right direction, such that they both lay within the white matter tract on axial and sagittal views. This process was repeated in one anterior and one posterior coronal slice. This method was chosen as the parahippocampal gyrus runs anterior to posterior, inferior to superior and medial to lateral within the medial temporal lobe. The principal eigenvector of each voxel, when viewed using PICo and projected on the axial plane was orientated anterior-posterior. A threshold of FA ≥ 0.1, and curvature threshold of 180° were set for tractography.
Neuropsychological tests
The list learning and design learning tests were used to assess material specific memory function (Coughlan and Hollows, 1985). In the verbal learning task the subject is read a list of 15 words five times, and on each presentation attempts to recall as many of the words as possible. The overall percentage of correct responses was used as the measure of verbal memory efficiency. For non-verbal memory we employed a design learning task; the subject is presented with a visual design on five occasions with recall being tested after each presentation. The percentage of correct responses over the five trials was used as a second measure of non-verbal memory efficiency. These tests form part of our presurgical memory assessment in TLE cases, and have proven least affected by performance anxiety, have a good test-retest reliability and are sensitive indicators of medial temporal lobe function (Baxendale et al., 1998). Neuropsychological test results are listed in Table 2.
Data analysis
All data were analysed using SPSS (11.0.0). It was first verified whether all parameters were normally distributed using the Kolmogorov–Smirnov test for normal distribution. Group differences for age were determined by a one-way analysis of variance (ANOVA), and gender distribution was assessed using the Pearson's χ2 test. The age of onset of epilepsy, duration of epilepsy, and frequency of complex partial (CPS) and secondarily generalized seizures (SGS) in right and left TLE patients were compared using the Mann–Whitney U test.
Each subject's output tractography connection probability map was spatially normalised by mapping into a standard space using the MNI template provided by SPM2 (Wellcome Department of Imaging Neuroscience, London; http://www.fil.ion.ucl.ac.uk/spm). Binary masks at a threshold connection probability value of 0.05 were then constructed. Our group has previously demonstrated in this same group of patients and control subjects, that the threshold 0.05 strikes a balance between losing non-specific low probability connections, while retaining the main body of the pathways (Powell et al., 2006). Binary masks at this threshold were therefore averaged across each group, to produce variability (or commonality) maps indicating the degree of spatial variability and overlap of the identified connections (Parker et al., 2005). A voxel commonality value C of 1.0 indicates that every individual had a connection identified in this voxel, while a C value of 0 indicates that none of them did (Parker et al., 2005). Tracts were then assessed visually in all three planes for visual symmetry and size/extent using MRIcro.
Normalised tract volumes were calculated for the connecting tracts from the left and right PHG of each control and patient at a threshold of 0.05 (Toosy et al., 2004). An asymmetry index for volume (AIvol) defined as AIvol = [100 × (Right Volume − Left Volume)] / [(Right Volume + Left Volume)/2] was calculated (Jutila et al., 2001), and the mean values between groups compared using a one way ANOVA analysis. Comparisons between the control AI values, and left and right TLE AI values were carried out using post-hoc Dunnett t-tests. Two way mixed ANOVA with one between subjects factor (group –— controls or TLE (both left and right)) and one within subjects factor (hemisphere — left or right) was used to test for the effect of interaction between group and hemisphere on volume, and unpaired t-tests were used to compare the patient and control group tract volumes.
The mean FA of the connected volume was calculated in native space for the left and right tracts in controls and patients. This was carried out by multiplying the native, thresholded, binarised images with that subject's whole brain FA image, in order to calculate the mean intensity value of the voxels. An asymmetry index for FA (AIFA) defined as AIFA = [100 × (Right FA - Left FA)] / [(Right FA + Left FA)/2] (Jutila et al., 2001) was calculated, and the mean values between groups compared using one way ANOVA analysis. Comparisons between the control AI values and left and right TLE AI values were carried out using post-hoc Dunnett t-tests. Two way mixed ANOVA with one between subjects factor (group — controls or TLE (both left and right)) and one within subjects factor (hemisphere — left or right) were used to test for the effect of an interaction between group and hemisphere on FA, and unpaired t-tests were used to compare the patient and control group tract FA values.
Pearson's correlation test was used to evaluate the evidence for a correlation of hippocampal volume with tract volume and FA, and tract volume with tract FA, ipsilateral and contralateral to the seizure focus. Performance on material specific memory measures was investigated for evidence of a correlation with tract volume and FA in right and left TLE groups, omitting patient 14 because of his atypical language dominance.
Results
Demographic analysis
There was no significant difference in the mean age or gender distribution of participants in the three groups (controls, left TLE, right TLE). There was no significant difference in the age of onset, duration of epilepsy or frequency of CPS and SGS between left and right TLE patients.
Qualitative tract analysis
In controls, PHG connections were visually symmetric. Connections between the para-hippocampal gyrus and anterior temporal lobe, orbitofrontal areas and posterior temporal and extrastriate occipital areas were observed as documented previously (Powell et al., 2004). There was a clear decrease in ipsilateral compared with contralateral connections in left TLE (Fig. 2), though an ipsilateral reduction was not evident in the commonality map of the right TLE group (Fig. 3).
Quantitative tract analysis
There was a significant difference in AIvol between groups [F(2,25) = 3.31 p = 0.05]. The AIvol was greater in left TLE patients than in controls (p = 0.05) with a mean 22% reduction in volume on the left (Table 3). There was no significant difference between the AIvol in right TLE patients and controls. There was no significant interaction between group and hemisphere for tract volume, and no significant differences between left and right volumes in left/right TLE patients, and controls.
There was a significant difference in AIFA between groups [F(2, 25) = 4.92 p = 0.02], attributable to reduced FA on the left in left TLE (p = 0.02 against controls). There was no significant difference between the AIFA in right TLE patients and controls. There was a significant interaction between group and hemisphere on tract FA [F(2, 25) = 4.35 p = 0.02], with FA being lower on the left in left TLE patients, compared with controls (p = 0.03). Although a similar trend was present in right TLE patients, with FA being lower on the right side compared with controls, this was not significant (p = 0.06).
Correlation of tract volume and FA with pre-surgical material specific memory
All but one patient were left hemisphere dominant for language. Patient 14 was left handed and right hemisphere dominant on both fMRI and intracarotid amytal testing. He was therefore omitted from the correlation analysis. In the left TLE patients, FA of the left PHG connections were correlated significantly with pre-surgical verbal learning (r = 0.88, p = 0.002), and right FA correlated significantly against pre-surgical design learning (r = 0.63, p = 0.05) (Fig. 4). Left and right tract volumes were not significantly correlated with verbal learning and design learning, respectively. In the right TLE patients, there were no significant correlations between left or right tract FA or volume, with pre-operative verbal learning or design learning respectively.
Correlations of hippocampal volumes with tract volumes and tract FA
There was no significant correlation between ipsilateral hippocampal volume and ipsilateral tract volumes or FA nor between contralateral hippocampal volumes and contralateral tract volume or FA.
Correlation analysis of tract volume with tract FA
There was a significant correlation between tract FA and tract volume (r = 0.61, p = 0.008) ipsilateral to seizure focus (Fig. 5), but not contralateral to seizure focus.
Discussion
Our principal finding was that in TLE the white matter connections of the parahippocampal gyrus ipsilateral to the seizure focus had smaller volumes and decreased FA. This was statistically significant in left but not right TLE patients. Furthermore, in left TLE, decreased FA was associated with poorer performance on both material specific memory measures. These results are consistent with the hypothesis that TLE involves dysfunction and structural changes in a network that includes the parahippocampal gyrus.
The connections of the human parahippocampal gyrus, visualized with tractography, have been described in a control population (Powell et al., 2004). Using a different tractography algorithm (Fast Marching Tractography or FMT) connectivity was found between the parahippocampal gyrus and the anterior temporal lobe, orbitofrontal areas, posterior temporal lobe and extra-striate occipital lobe via the lingual and fusiform gyri. These findings are similar to those in this control group.
This is the first study to use tractography to quantitatively assess the structural changes in the parahippocampal gyrus in TLE. Several volumetric MRI (Bernasconi et al., 2003; Bonilha et al., 2003) and voxel-based morphometry (VBM) studies (Bernasconi et al., 2004; Bonilha et al., 2004; Keller et al., 2002b,a, 2004) have investigated the extra-hippocampal structural changes in unilateral TLE. The para-hippocampal gyrus or its sub-regions including the entorhinal cortex, parahippocampal cortex and perirhinal cortex have been shown to be affected in TLE. Several studies suggested that the degree of atrophy and regional distribution was more extensive in left TLE, though none were specifically designed to evaluate this (Bernasconi et al., 2004; Bonilha et al., 2004; Keller et al., 2002b). It has however been recently demonstrated that the distribution and severity of extra-hippocampal grey matter loss is more extensive in left TLE patients than right TLE patients (Bonilha et al., 2007). Our study is the first to show that the white matter connections of the parahippocampal gyrus are affected by unilateral TLE. This process appears to be more severe in left TLE compared with right TLE. A similar pattern was observed in another tractography study that assessed language pathways in TLE patients (Powell et al., 2007b), although in this case there was also asymmetry of connections in controls, with more extensive connections in the speech dominant hemisphere. Ultimately, longitudinal quantitative MRI studies will be necessary to determine the effects of underlying cause, seizures, medications, and co-morbidity on the white matter structures and connections of the brain.
Parahippocampal connections and memory in temporal lobe epilepsy
The memory deficits associated with TLE, particularly left TLE, can be disabling. Consequently, much attention has been focused on the effects of both chronic TLE and ATLR on memory. While the hippocampus plays a critical role in the initial formation of memories (Eichenbaum, 2000), the parahippocampal region is thought to be involved in the intersection between perception and memory, and the translation of material into a more permanent storage in the cortical association areas (Eichenbaum, 2000; Murray and Bussey, 1999). Animal models have shown that the parahippocampal region is important for recognition memory (Brown and Aggleton, 2001; Eichenbaum, 2000), and that selective lesions to the parahippocampal area can severely impair memory (Suzuki et al., 1993). Furthermore, the functions of the medial temporal lobe are highly lateralized and the classic model of material specific memory predicts that lesions in the left hippocampal system impair verbal memory retrieval (Hermann et al., 1997), while those in the right hippocampal system affect non-verbal memory, though these findings are less consistent (Alessio et al., 2004).
In left TLE patients there was a significant correlation between parahippocampal tract FA and material specific memory measures on both the left and right sides. No significant correlations were seen with respect to tract volume in left TLE, and neither volume nor FA in right TLE. The finding of fewer occipital connections in the left TLE group compared with right TLE patients and controls may be significant in this respect (Figs. 2 and 3). Animal models suggest that potential roles for these connections may involve the priming of mesial temporal lobe structures to facilitate the consolidation of visual memory, or enhancing the visual processing of emotionally significant stimuli (Suzuki et al., 1993; Suzuki and Amaral, 1994). Visual cues may be important not only for non-verbal memory, but may also be useful in verbal memory. This could explain the correlation of FA found with verbal and non-verbal memory in left TLE patients. In the right TLE patients the occipital connections did not seem to be as affected, and hence these patients may have been able to use visual cues to aid both verbal and non-verbal memory.
Several other studies have also observed correlations between MRI findings and memory dysfunction in TLE. Some have shown a correlation between left and right hippocampal MRI volumes, and verbal and non-verbal memory respectively (Baxendale et al., 1998; Kalviainen et al., 1997; Lencz et al., 1992). Others have shown correlations between hippocampal T2-signal, or abnormalities of MR spectroscopy and material specific measures (Gadian et al., 1996; Wendel et al., 2001). Lui et al. (Lui et al., 2005) examined 18 TLE patients and found correlations between apparent diffusion coefficients (ADC) in the left and right hippocampi and parahippocampal gyri and, verbal and non-verbal memory respectively. Others have demonstrated correlations between localized diffusion measures and domains of cognitive functioning in diseases as varied as schizophrenia (Nestor et al., 2004), HIV related dementia (Ragin et al., 2005), age related cognitive decline (Charlton et al., 2006), mild cognitive impairment (Rose et al., 2006), and Alzheimer's disease (Yoshiura et al., 2002). There are also studies that have observed that the relationship between the side of pathology and memory dysfunction appears to be more evident in those with left TLE than right TLE. Alessio et al evaluated the relationship between several medial temporal lobe structures and memory in 39 patients, and found a correlation between the degree of left sided hippocampal atrophy and verbal memory deficits, but not between right sided hippocampal atrophy and visual memory deficits (Alessio et al., 2004).
In this study, it also appears that tractography derived parahippocampal FA is a more sensitive marker than volume of the functional integrity of tracts that form the hardwiring of circuits needed for memory (Charlton et al., 2006). Other tractography studies have shown that FA is a more sensitive and robust measure than volume of pathology in white matter tracts (Heiervang et al., 2006). In biological terms the interpretation of reduced anisotropy is complex (Beaulieu, 2002), and depends on the context or disease in which it is found (Alexander et al., 2007). In epilepsy, it may represent neuronal loss, gliosis, and structural disorganisation (Wieshmann et al., 1999).
The association between hippocampal volume and parahippocampal Volume/FA
No association between hippocampal volume, and tract volume or FA was found in this study. Other studies have reported mixed findings. While Bonhila et al found no correlations between the severity of hippocampal atrophy and grey matter volume in the parahippocampal region (Bonilha et al., 2003), Jutila et al reported positive correlations but only in a subgroup analysis of those patients with the most severe hippocampal atrophy (Jutila et al., 2001). This suggests that despite the fact that the hippocampus and parahippocampal gyrus are significantly interconnected, the structural size relationship between the two structures is not linear. There was also a significant correlation between ipsilateral tract volume and FA in the current study.
Limitations of Study
Only modest numbers of patients and controls were available for this study, as the scanner used was decommissioned and replaced with one of higher field strength, making comparison with more recent data impossible. These small numbers, particularly in the left TLE group, may have contributed to the pattern of observed changes in left but not right TLE patients. Furthermore, for this reason several morphological and functional factors that can influence cognition and memory in chronic epilepsy were not included as co-variates in the statistical analysis. The pathological basis of TLE was not homogenous throughout the group, and the degree of hippocampal atrophy was varied. Secondly the spatial resolution of the tractography was limited and we did not separately evaluate the entorhinal cortex, parahippocampal cortex, and perirhinal cortex (Duvernoy, 1998). The entorhinal cortex is considered to be the route by which data reaches the hippocampus. The perirhinal, and parahippocampal cortex on the other hand, provide the incoming connections to the entorhinal cortex, conveying information from the polymodal and unimodal cortices (Squire, 1991). A sub-regional analysis is an area that should be explored in future tractography studies.
Conclusion
This tractography study has shown disruption of the architecture and atrophy of the connections of the parahippocampal gyrus ipsilateral to the seizure focus in patients with refractory TLE, and these structural changes were associated with memory deficits evident on psychometric testing. This information has both diagnostic and prognostic implications. Larger, longitudinal studies at a higher resolution will enable both sub-regional analysis, and the investigation of other factors that may contribute to neuronal loss and structural changes, and subsequent memory impairment in patients with TLE.

tractography
parahippocampal gyrus
memory
epilepsy


Interactions of Shiga-like toxin with human peripheral blood monocytes

The cytotoxic effect of Shiga-like toxin (Stx; produced by certain Escherichia coli strains) plays a central role in typical hemolytic uremic syndrome (HUS). It damages the renal endothelium by inhibiting the cellular protein synthesis. Also, the monocyte has a specific receptor for Stx but is not sensitive for the cytotoxic effect. In this work, monocytes were studied as a potential transporter for Stx to the renal endothelium. Coincubation of isolated human monocytes loaded with Stx and target cells (vero cells and human umbilical vascular endothelial cells) were performed. Transfer was determined by measuring the protein synthesis of target cells and by flow cytometry. Furthermore, the effect of a temperature shift on loaded monocytes was investigated. Stx-loaded monocytes reduced the protein synthesis of target cells. After adding an antibody against Stx, incomplete recovery occurred. Also, adding only the supernatant of coincubation was followed by protein synthesis inhibition. Stx detached from its receptor on the monocyte after a change in temperature, and no release was detected without this temperature shift. Although the monocyte plays an important role in the pathogenesis of HUS, it has no role in the transfer of Stx.
Introduction
Hemolytic uremic syndrome (HUS) is a clinical syndrome consisting of three characteristic features: hemolytic anemia, thrombocytopenia, and acute renal failure [1]. In the new classification of HUS, infections due to Shiga-like toxin (Stx)-producing bacteria belong to the category “etiologically advanced” [2]. This work focuses on the form in which Stx-producing Escherichia coli is the most common pathogen [1]. It can produce several types of Stx, of which Stx1, Stx2, and Stx2c are most frequently associated with HUS [3, 4]. Stx plays a crucial role in the pathogenesis because of its cytotoxic effect on the renal endothelium. Both renal tubular epithelial cells and glomerular visceral epithelial cells (podocytes) are also sensitive to the toxic effect of Stx [5, 6]. It can inhibit the protein synthesis of these cells after specifically damaging the ribosomal RNA [7]. However, the question of how this toxin is targeted mainly to the kidney remains unsolved. Stx was never detected in the serum of patients, but it was detected in renal biopsy material of patients with HUS [8]. As a specific treatment for HUS is still lacking, more insight into the transport of this toxin might lead to new intervention strategies.
After oral ingestion of the bacteria through contaminated food or water, the noninvasive bacteria adhere to the intestinal epithelial cells of the distal small bowel and colon. This leads to a rearrangement of the morphology of the cells and initiates inflammation [9, 10]. Bacterial flagellin plays an important role in this process [11]. Stx can probably reach the circulation because of active transport in these cells and also passively after damage to the intestinal cells [12]. Subsequently, it has to be transported in the circulation to reach its primary target, the renal endothelium.
It is very tempting to look at the blood cells as a carrier for the toxin. Stx can bind to a specific receptor, which is a globotriaosylceramide (Gb3, Pk Antigen, CD77) [13]. This receptor is present on renal endothelial cells but also on blood cells. Stx binding has been described on red blood cells [14], B lymphocytes [15], and platelets, which also have an additional binding possibility (glycolipid, band 0.03) [16]. Several groups showed the existence of a specific binding of Stx on monocytes [17, 18, 19]. After binding to its receptor, Stx can be internalized. Whereas in epithelial cells the toxin follows the retrograde transport route and becomes cytotoxic, in monocytes it is targeted to the lysosomes and will get degraded [19]. During this transport, the monocyte becomes activated. This will lead to an increase of transcription factors, such as nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1), and an upregulated production of cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, and IL-8 [17, 20]. These events will have a pro-inflammatory effect.
We postulated that, as the monocyte has a specific receptor, it might also function as a carrier to transport Stx to the renal endothelium. To investigate this hypothesis, Stx was loaded to isolated monocytes from healthy donors and coincubated with target cells [vero cells and human umbilical cord venous endothelial cells (HUVEC)]. The level of transfer was determined by measuring the protein synthesis of these target cells and the transfer of fluorescein isothiocyanate (FITC)-labeled B subunit of Stx1 with flow cytometry.
Materials and methods
Materials
Stx2 was kindly provided by Dr. M. Karmali (Public Health Agency of Canada, Ontario, Canada). FITC-labeled Stx1B subunit and 125I-Stx1B subunit were a gift from Dr. L. Johannes (Institut Curie, Paris, France). Stx1B subunit is a useful tool for studying binding in monocytes [19]. It is the binding part of the toxin, whereas the enzymatic A subunit will only stimulate the uptake of the toxin and does not affect binding [21]. Vero cell medium consists of M199 (Gibco; Paisley/UK), fetal calf serum (FCS, Greiner Bio-One; Kremsmunster/Austria), penicillin/streptomycin (Gibco, Paisley/UK), and glutamine (MP Biomedicals; Eschwege/Germany). HUVEC medium is made of M199, human serum (HS; Cambrex; Walkersville/USA), newborn calf serum (NBCS; Gibco, Paisley/UK), penicillin/streptomycin, glutamine, heparine (Leo Pharma BV, Breda/The Netherlands), and endothelial-cell growth factor [22].
Ethylenediamine tetraacetic acid (EDTA) tubes were purchased from BD Vacutainer (Alphen aan de Rijn/The Netherlands). The MACS kit for negative selection of monocytes was provided by Miltenyi Biotec (Bergisch Gladbach/Germany). Hank’s balanced salt solution (HBSS) was ordered from MP Biomedicals (Eschwege/Germany). Human serum albumin (HSA) from Sanquin (Amsterdam/The Netherlands) and porcine gelatin from Fluka (Neu-Ulm, Germany) was used. Trichloroacetic acid and TNF-α was obtained from Sigma-Aldrich Chemie B.V. (Zwijndrecht/The Netherlands). The antibody against Stx2 (TMA-15) is well characterised [23]. It was a kind gift from Dr. Yamagami from the Department of Biomedical Research from the Teijin Institute, Tokyo, Japan. 3H-leucine and Ficoll-paque PLUS was purchased from Amersham Biosciences (Uppsala, Sweden). Culture plates were ordered from Corning Inc. (Corning, USA).
Culture of vero cells and HUVEC
Vero cells (renal epithelial cells of the African green monkey) were grown to confluency on 24-well plates (ordered from ATCC; Middlesex, UK). These cells have a high basal expression of Stx-receptor CD77. HUVEC were isolated, and these cells were grown to confluence on gelatin-coated 24-well plates [24]. Every 2 days, fresh medium was added to the cells. In contrast, HUVEC were preincubated for 24 h with TNF-α (10 ng/ml) to upregulate the expression of CD77.
Isolation of monocytes and loading with Stx2
Fresh venous blood (20 ml) from 40 healthy donors was collected into EDTA tubes. Monocytes were isolated by negative selection using antibody-labeled beads (CD3, CD7, CD16, CD56, and CD123). After centrifugation of blood over Ficoll (20 min 400 g without break at room temperature), the interphase (containing monocytes, lymphocytes, and platelets) was collected. Platelets were removed by centrifugation (200 g 10 min at room temperature) before adding the beads. The purity of monocytes after the magnetic isolation was (as determined by flow cytometry) 80–85%. After the isolation, the monocytes were resuspended in HBSS with 1% HSA and placed on ice. In every experiment, monocytes from one donor were used. To load the monocytes with Stx2, the toxin was added to a concentration of 10 nM during a time period of 3 h [17]. The cells remained on ice. After proper washing to remove all unbound Stx2, the cells were resuspended in vero cell or HUVEC medium (in which HS and NBCS were substituted by FCS). Monocytes stayed viable after this loading, as determined by trypan blue exclusion.
Coincubation Stx2-loaded monocytes and target cells
These experiments were performed in two different experimental settings: with transfer of loaded monocytes from 4° to 37°, and without change of temperature. To start with the first setting, the Stx2-loaded monocytes were added to a monolayer of target cells [HUVEC (n = 10) or vero- cells (n = 9)] in a concentration of 1 × 106 per well. This was performed during 24 h at 37°C. For comparison, also monocytes without Stx were used. To determine the specificity of the effect of Stx2, the loaded monocytes were preincubated with a well-characterized antibody against Stx2 (TMA-15, 1 μg/ml, approximately 150× excess). All experiments were performed in duplicate. To measure the transfer of Stx2 from the monocyte, the protein synthesis of the target cells was determined by adding 3H-Leucine (0.67 μCi/ml). Subsequently, intracellular proteins were precipitated by treatment with trichloroacetic acid (TCA), and the radioactivity was measured in a liquid scintillation counter. In the other experimental setting, monocytes were loaded in a similar way with FITC-labeled Stx1B subunit; 1 × 106-loaded monocytes were coincubated with 1.25 × 105 vero cells in suspension for 3 h at 4°C (n = 5) while being continuously rotated. At this temperature, bias due to internalization of the toxin could be avoided [19, 25]. After 3 h, the presence of Stx1B subunit FITC on the vero cells was determined by flow cytometry. For every experiment, at least 1,000 cells were measured.
Study of supernatant Stx-loaded monocytes
To study the effect of the supernatant of Stx2-loaded monocytes, it was collected after 16 h of coincubation on vero cells (n = 11). The supernatants were centrifuged to remove possible monocytes and added again to fresh vero cells. Protein synthesis was measured by adding 3H-leucine and measuring the incorporation after 24 h of incubation at 37°C. Also, the antibody against Stx2 was used to determine a possible effect of free Stx2. Furthermore, monocytes were loaded with 125I-Stx1B subunit to investigate whether it could be released after a change in temperature from 4° to 37°C (n = 3). For each experiment, between 3 and 4 × 106 monocytes are loaded with 200 nM Stx1B subunit (3,600 cpm/ng protein). After loading, unbound Stx1B subunit was removed by centrifugation. Subsequently, the monocytes were placed at 37°C for 2 h. They were then centrifuged, and cells and supernatant were measured separately for the presence of 125I-Stx1B with a gamma counter. This was compared with the amount of binding to the monocytes before the temperature change. The experiment was performed in duplicate.
Statistics
All data presented are expressed as a range with the median. Significance of increase or decrease of protein synthesis compared with controls was analyzed using the Wilcoxon signed ranks test. The statistical level of significance was defined as P < 0.05.
Results
Coincubation Stx2-loaded monocytes and target cells
To investigate whether monocytes can transfer Stx2 to target cells (vero cells and HUVECs), Stx2-loaded monocytes were added to a monolayer of these cells. After coincubation, protein synthesis of vero cells and HUVEC was measured. If the Stx2 was transferred to the target cells, there was protein synthesis inhibition, as this is the biological effect of Stx2 in both types of target cells. After 24 h of coincubation at 37°C, in both cell types, there was protein synthesis inhibition (Fig. 1). In vero cells, greater protein synthesis reduction was measured than in HUVEC. Directly adding 10 nM Stx2 to vero cells reduced protein synthesis to 5.5% (data not shown). When an antibody against Stx2 was added to the monocytes before coincubation with vero cells, protein synthesis inhibition was partly restored (Fig. 2). Only in one out of five experiments was there complete recovery.
Fig. 1Coincubation of Shiga-like toxin (Stx)2-loaded monocytes with target cells [vero cells and human umbilical cord venous endothelial cells (HUVEC)]. To investigate whether monocytes could transfer Stx2, toxin-loaded monocytes were coincubated with vero cells (n = 9) and HUVEC (n = 10). This led to protein synthesis inhibition, as could be determined with the incorporation of 3H-leucine. The coincubation with unloaded monocytes was used as a control and set at 100%. ** P < 0.01Fig. 2Effect of the addition of an antibody against Shiga-like toxin (Stx)2. The monocytes of five healthy donors were coincubated with vero cells (Stx2-loaded monocytes, white bar; Stx2-loaded monocytes with Stx2 antibody, black bar). Addition of the antibody against Stx2 led to partial protein synthesis recovery in four donors and complete recovery in one donor. Coincubation with unloaded monocytes was used as a control and set at 100%
In equal experiments performed with HUVEC (n = 3), TMA-15 also partly prevented  protein synthesis inhibition (data not shown).
Study of supernatant Stx-loaded monocytes
Because the addition of an antibody against Stx2 was not sufficient for complete protein synthesis recovery in four out of five tested donors, we investigated the possible presence of an additional inhibitor in the supernatant after 16 h of coincubation. This was performed by re-adding the supernatant of coincubated Stx2-loaded monocytes and a monolayer of vero cells to new vero cells. Figure 3 shows that this incubation again led to an inhibitory effect on vero-cell protein synthesis in contrast to the supernatant of unloaded monocytes. But this reduction was less than with direct coincubation. The effect could be partly blocked by adding the antibody against Stx2. This means that there is unbound Stx2 present in the supernatant. Next, we investigated whether the temperature change of the Stx-loaded monocytes from 4° to 37°C could lead to toxin release. For this reason, we loaded the monocytes with 125I-Stx1B subunit. The amount of radioactivity on or inside the monocytes was measured before and after incubation of the cells for 2 h at 37°C (Table 1). Vero cells were used as a control. In vero cells as in monocytes, the toxin was released from its receptor after the incubation.
Fig. 3Addition of supernatants coincubation to vero cells. After coincubation of Shiga-like toxin (Stx)-loaded and unloaded monocytes, supernatants were collected and re-added to fresh vero cells (n = 11). The supernatant from the toxin-loaded monocytes induced, again, protein synthesis inhibition. This inhibition could be partially restored with a Stx2 antibody (AB). The incubation of supernatant with unloaded monocytes was used as a control and set at 100%. ** P < 0.01Table 1Amount of 125I- Shiga-like toxin (Stx)1B subunit on cells after change of temperatureStx-loaded cellsBefore 4→37°C (cpm)After 4→37°C (cpm)Vero cells1,034,383671,060Monocytes donor 11,437427Monocytes donor 21,070374Monocytes donor 31,121677cpm counts per minute
Transfer of Stx to target cells without change in temperature
A change in temperature from 4° to 37°C released Stx from its receptor on the monocyte. To study again the possibility of Stx transfer from monocyte to target cells, we loaded isolated monocytes with Stx1B subunit labeled with FITC. Coincubation of these monocytes with vero cells in suspension without a change in temperature did not result in a transfer of the B subunit to the vero cells (n = 5, Fig. 4).
Fig. 4Transfer of Shiga-like toxin (Stx)-loaded monocytes to vero cells without temperature shift (white: normal monocytes; black: Stx-loaded monocytes; grey: vero cells). The x-axis represents the intensity of the fluorescein isothiocyanate (FITC) signal, the y-axis represents the number of cells. a FITC-labeled Stx1B subunit was bound to monocytes from a healthy donor, showing an increase in intensity. b Coincubation of vero cells and unloaded monocytes; note the high basal signal of vero cells. c After 2 h of coincubation with vero cells in suspension at 4°C, there was no transfer of the FITC signal. The cells remained in position
Discussion
After coincubation of Stx2-loaded monocytes with target-cells (vero cells and HUVEC), protein synthesis inhibition could be detected. As the biological effect of Stx is protein synthesis inhibition, a transfer was expected. This effect could not be due to the presence of monocytes, because it is well described that Stx has no inhibitory effect on peripheral blood monocytes [17]. However, the addition of an antibody against Stx only partly restored the inhibition. We hypothesized that in parallel to transfer of Stx2, a possible additional inhibiting factor was present in the supernatant during the experiment. To further investigate this possibility, the supernatant of this coincubation was re-added to fresh vero cells. Again, there was protein synthesis inhibition, which could partly be decreased by blocking with the Stx2 antibody. The conclusion was made that there another inhibitory factor needed to be present (possibly released by activated monocytes), but unbound Stx2 was also present [26]. Apparently, Stx1B subunit is released from its receptor when there is shift of temperature from 4° to 37°C, as shown in this study and also by Ramegowda and Tesh [18]. This finding stresses the caution that must be taken when drawing conclusions from in vitro experiments performed at 4°C. In Fig. 5, all findings are schematically summarized.
Fig. 5Schematic summary of performed experiments. a Monocytes loaded with Shiga-like toxins (Stx) at 4°C. b After shifting Stx-loaded monoytes to target cells at 37°C, Stx was released from its receptor. This led to protein synthesis inhibition of target cells. However, transport could not be excluded (but this was performed in experiments without a shift in temperature). Probably, some toxin was internalized and monocytes became activated. This could lead to cytokine and proteases production (possible cytotoxic factors). c When the supernatant is re-added to new target cells, there are released Stx and secreted products present. This induced, again, protein synthesis inhibition
As these in vitro experiments were not suitable to investigate a possible transfer, experiments were performed without a change of temperature. No transfer of the binding part of the toxin could be detected in this setting. Experiments could not be performed at 37°C because the toxin is internalized by the monocytes during 3 h of incubation. Because of these in vitro experiments, we believe that the monocyte cannot function as a transporter for Stx in the circulation. However, monocytes still seem to play an important role in pathogenesis. As a component of the innate immune system, they play a central role in immunity and inflammation. Fernandez et al. showed that patients in the acute period of HUS have monocytes with phenotypic (reduced expression of CD14, CD64, and CD11b) and functional [decreases lipopolysaccharide (LPS)-induced TNF-α production and Fcγ-dependent cytotoxicity] differences compared with healthy children and acute uremic children [27].
Some unanswered questions remain. What is the inhibitory factor released by monocytes after loading with Stx? Why is there still unbound Stx2 present in the supernatant after 16 h of coincubation? It is surprising that Stx2 is not completely bound and internalized by the numerous receptors on monocytes and vero cells. Also, how Stx is transported in the circulation remains unsolved. Kimura et al. described that serum amyloid P (SAP) can bind Stx2 and function as a neutralizing factor [28]. However, in humans, there was no correlation between circulating SAP and the development of HUS [29].
As renal endothelial damage is already present after the occurrence of clinical symptoms of HUS, it is very important to develop efficacious early prevention. Understanding the mechanism in which the toxin is specifically targeted to the kidney can lead to novel intervention strategies.

shiga-like toxin
monocyte
hemolytic uremic syndrome
vero cells
acute renal failure
huvec


Fiberoptic endoscopic evaluation of swallowing in intensive care unit patients

Aspiration in critically ill patients frequently causes severe co-morbidity. We evaluated a diagnostic protocol using routine FEES in critically ill patients at risk to develop aspiration following extubation. We instructed intensive care unit physicians on specific risk factors for and clinical signs of aspiration following extubation in critically ill patients and offered bedside FEES for such patients. Over a 45-month period, we were called to perform 913 endoscopic examinations in 553 patients. Silent aspiration or aspiration with acute symptoms (cough or gag reflex as the bolus passed into the trachea) was detected in 69.3% of all patients. Prolonged non-oral feeding via a naso-gastric tube was initiated in 49.7% of all patients. In 13.2% of patients, a percutaneous endoscopic gastrostomy was initiated as a result of FEES findings, and in 6.3% an additional tracheotomy to prevent aspiration had to be initiated. In 59 out of 258 patients (22.9%), tracheotomies were closed, and 30.7% of all 553 patients could be managed with the immediate onset of an oral diet and compensatory treatment procedures. Additional radiological examinations were not required. FEES in critically ill patients allows for a rapid evaluation of deglutition and for the immediate initiation of symptom-related rehabilitation or for an early resumption of oral feeding.
Introduction
Pulmonary aspiration is a serious cause of morbidity and mortality in patients with a depressed sensorium, patients with neuromuscular discoordination, or patients having structural disorders of the upper aerodigestive tract. It is a leading cause of nosocomial infection in the intensive care unit (ICU). The most common manifestations of pulmonary aspiration are pneumonia, pleuropulmonary infection, and acute airway obstruction [1].
Hospital-acquired pneumonia is the second most common nosocomial infection in the critically ill patient and is associated with the greatest mortality and increased morbidity and cost of care. Incidence of HAP varies in different populations of critically ill patients and generally ranges from 9 to 20%. The etiologic agents differ according to the population studied, duration of hospital stay, time after intubation, and prior antimicrobial therapy [2]. Risk factors include nonmodifiable factors like age, chronic obstructive pulmonary disease, severe head trauma, and multiple trauma, and modifiable factors like large-volume gastric aspiration, duration of mechanical ventilation, elevated gastric pH, histamine type two blocker therapy, ventilator circuit change frequency, self-extubation, and silent aspiration following scheduled extubation [2]. When patients are admitted to the ICU, they are at increased risk of more frequent aspiration events because of a variety of factors, including depressed level of consciousness (often caused by excess analgesia and sedation), forced supine position, and the presence of nasogastric or endotracheal tubes [3].
There is a growing body of evidence suggesting that intubation for longer than 48 h may cause at least transient injury to the larynx with a subsequent reduction in the protective mechanism and increased incidence of oropharyngeal secretions once the patient is extubated. The presence of an orotracheal tube has been shown to alter the mechanoreceptors and chemoreceptors of the pharyngeal and laryngeal mucosa, causing dysfunction of the swallowing reflex. The prevalence of swallowing dysfunction post-extubation has been reported to occur in between 20 and 83% of those patients intubated longer than 48 h. This wide range of estimate has partly been attributed to the variation in the diagnostic tools [4]. The impact that diagnosis using invasive diagnostic techniques may have on the epidemiological characteristics of HAP in critically ill patients is unknown, but may potentially improve aspiration-related specific therapy and ultimately clinical outcome.
Based on these considerations, we prospectively evaluated a diagnostic protocol using routine fiberoptic endoscopic evaluation of swallowing (FEES) in critically ill patients at risk to develop aspiration following temporary transnasal intubation or tracheotomy and tried to define the impact of post-extubation FEES on the initiation of aspiration-related treatment.
Material and methods
We conducted a prospective, interventional, clinical study at Klagenfurt General Hospital. The hospital is a 1,400-bed tertiary referral centre that serves a population of approximately 1,000,000 and runs seven ICUs (paediatrics and neonatology, 1; neurology, 1; neurosurgery, 1; cardiology, 1; general internal medicine, 1; and anaesthesiology, 2) with a total of 84 beds.
We offered a standardized endoscopy protocol for critically ill patients considered to be at risk for silent aspiration following transnasal intubation or tracheotomy for ventilation. Patient selection for endoscopic assessment of swallowing was done by ICU physicians using a protocol that we designed to help in the early recognition of risk factors for, or overt signs of, aspiration. This protocol included a taxonomic summary of symptoms and clinical signs potentially related to aspiration. Rating was applied after the stop of mechanical ventilation. Symptoms were defined as:Weight loss,unexplained fever >38.0° centigrade,coughing,bronchitis/pneumonitis,impaired voice,witnessed regurgitation/aspiration event at the bedside accompanied by coughing, choking, and/or expectoration of material,prolonged oral feeding,aversion for oral intake of liquids and solids,disturbance of bolus transport,frequent postural changes during oral intake, andregurgitation.If such conditions were observed in previously intubated patients following extubation, or in patients with tracheotomies, physicians at ICUs were encouraged to request endoscopic assessment of swallowing.
The FEES procedure was consequently provided by staff members of the department of Oto-Rhino-Laryngology, Head and Neck Surgery. The team consisted of a laryngologist and a nurse who had both previously gone through a specialized training in performing FEES procedures in patients with deglutition disorders.
Over the 45-month period from 1 January 2002 to 30 September 2005, we were called to perform 913 endoscopic examinations in 553 patients treated in one of the ICUs. Four hundred and 46 patients underwent one single endoscopy, and 107 had repeated examinations. Two hundred and 95 patients had shortly before been extubated after transnasal tracheal intubation, and 258 patients had indwelling tracheotomies. For further data interpretation, patients were sub-grouped according to their route of ventilation (transoral/transnasal intubation vs. tracheotomy). Findings and recommendations from the initial FEES procedure in all 553 patients were accumulated for this study. The results of 360 control procedures were not further analyzed.
Details of the FEES procedure have previously been described in detail. Therefore, only a short summary of the procedure shall be given here: The FEES procedure is a portable examination, easily taken to bedside in ICUs. Since our patients were usually bedridden, we performed the examination in a in bed with the head of bed elevated to approximately 70° with the bend of the bed is at the patient’s lower back (904/913 patients, i.e., 99%). In four patients (0.4%), the examination was carried out with the patient in reverse Trendelenberg at 30°–45°, and five patients (0.6%) were able to sit on a chair during the procedure. A fiberoptic laryngoscope was passed transnasally to the oropharynx, where the larynx and surrounding structures could then be visualized. Patients were led through various tasks to evaluate the sensory and motor status of the pharyngeal and laryngeal mechanism. Stained liquid and semi-liquid boluses were then given to determine the integrity of pharyngeal deglutition. The interior larynx and airway were examined for evidence of food penetration within the laryngeal vestibule and aspiration of food below the true vocal folds before and after each swallow. In each case, the nasogastric tube was removed prior to the procedure. We assessed structural changes of the larynx and pharynx, timing and direction of movement of the bolus through the pharynx, the ability to protect the airway and to uphold airway protection for a some seconds, the capability to clear the bolus during deglutition, presence of pooling and residue of material in the hypopharynx, and timing of bolus flow and laryngeal closure.
Aspiration was defined as the entry of material into the airway below the levels of the true vocal cords. Silent aspiration was defined as aspiration occurring in the absence of acute symptoms (i.e., lack of cough or gag reflex as the food or liquid bolus passed into the trachea).
Findings were documented in a standardized form to allow for monitoring therapeutic interventions over time, and for later data analysis and evaluation.
Results
Over a 45-month period, we were called to perform 913 endoscopic examinations in 553 patients. Of these, 295 patients had previously been extubated, and 258 patients had indwelling tracheotomies. Silent aspiration or aspiration with acute symptoms (cough or gag reflex as the bolus passed into the trachea) was detected in 383 (69.3%) of all patients during initial FEES. Prolonged non-oral feeding via a naso-gastric tube was initiated in 49.7% of all patients. In 13.2% of patients, a percutaneous endoscopic gastrostomy was initiated as a result of FEES findings, and in 6.3% an additional tracheotomy to prevent aspiration had to be set off. In 59 out of 258 patients (22.9%), tracheotomies were closed, and 30.7% of 553 patients could be managed with the immediate onset of an oral diet ± compensatory treatment procedures without further diagnostic evaluation. Logopedic instruction of the patient and/or rehabilitation using compensatory strategies to reduce the risk of aspiration were an integral part of our rehabilitation program and included one or more of the following:Modifications in volume and tempo of food presentationoral feeding with consistency modificationshead rotationholding the chin down during deglutition to narrow the airway entrancesupraglottic swallow: This technique uses simultaneous swallowing and breath-holding, closing the vocal cords and protecting the airway.One hundred and seven patients were scheduled for repeated FEES, usually to assess the progress of the rehabilitation program.
Signs and symptoms encountered during initial FEES are condensed in Table 1. Table 2 stratifies the route of feeding in the two subgroups of our study population. Table 3 classifies the type of aspiration detected during FEES. Table 4 summarizes the recommendations derived from our endoscopic findings for the further management of patients with aspiration.
Table 1Symptoms of aspiration in 553 patientsPatients after transnasal tracheal intubation (n = 295)Patients with indwelling tracheotomies (n = 258)Unexplained fever17 (5.8%)39 (15.1%)Coughing68 (23.1%)31 (12.0%)Bronchitis/pneumonitis57 (19.32%)39 (15.1%)Impaired voice57 (19.32%)0 (0%)Witnessed regurgitation/aspiration event 74 (25.1%)149 (57.8%)Others16 (5.4%)23 (8.9%)No data69 (23.3%)19 (7.4%)Since more than one symptom or sign could apply per patient, results sum up to more than 100%Table 2Route of feeding for 553 patients on initial FEESPatients after transnasal tracheal intubation (n = 295)Patients with indwelling tracheotomies (n = 258)Nasogastric tube224 (75.9%)186 (72.1%)PEG15 (5.1%)59 (22.9%)Parenteral12 (4.1%)5 (1.9%)Oral diet44 (14.9%)8 (3.1%)PEG percutaneous endoscopic gastrostomyTable 3Classification of aspiration for 553 patients on initial FEESPatients after transoral or transnasal tracheal intubation (n = 295)Patients with indwelling tracheotomies (n = 258)Silent aspiration51 (17.3%)95 (36.8%)Aspiration115 (39.0%)126 (48.8%)No aspiration129 (43.7%)37 (14.4%)Aspiration = inhalation of material into the airway below the level of the true vocal cords, with acute symptoms (cough or gag reflex as the bolus passed into the trachea), silent aspiration = aspiration occurring in the absence of acute symptomsTable 4Recommendation for further treatment in 553 patients after initial FEESPatients after transoral or transnasal tracheal intubation (n = 295)Patients with indwelling tracheotomies (n = 258)Non-oral feeding (naso-gastric tube) ± logopedic (functional) therapy144 (48.8%)131 (50.8%)Indication for PEG27 (9.1%)46 (17.8%)Indication for non-oral feeding plus tracheotomy35 (11.9%)NAOral feeding ± logopedic (functional) therapy89 (30.2)22 (8.5%)Decannulation and oral feeding ± logopedic (functional) therapyNA59 (22.9%)PEG percutaneous endoscopic gastrostomy, NA not applicable
Discussion
Aspiration is a common event. The North American Summit on Aspiration in the Critically Ill Patient, in a consensus statement published in 2002 [5], estimates the frequency of aspiration in various patient populations is as follows:45% in normal individuals during sleep70% in patients with impaired consciousness0–40% in patients on ETF50–75% in patients with endotracheal tubesWhat determines the clinical consequences of an aspiration event are host factors (age, immune status, underlying disease process, and comorbidities) and the nature of the aspirate. The morbidity and mortality associated with aspiration (with or without subsequent pneumonia) is difficult to ascertain and may vary greatly depending on the patient population that is under study [5].
Despite these general considerations, aspiration is generally accepted as a leading cause of nosocomial pulmonary infection in the ICU [6].
Ajemian reported a 56% (27/48) incidence of swallowing dysfunction following prolonged intubation; 12 (25%) of 48 patients were silent aspirators [7]. In a study by Leder et al. [8], FEES demonstrated swallowing dysfunction in 33% of critically ill trauma patients after prolonged intubation, with 20% having silent aspiration. In a recent study by El Solh and co-workers, aspiration was documented in 52% of ICU patients aged over 65 years and in 36% of those up to age 65. No significant difference in the co-morbidity index and the length of mechanical ventilation was found between aspirators and non-aspirators [4].
Non-specific methods to monitor for and detect aspiration are usually subjective, not standardized or validated, and inaccurate, and there are few outcome valid data. Blue dye has been commonly used to color the feeding solutions, but this method is insensitive and non-specific for assessing aspiration and pneumonia. Testing of pharyngeal secretions with glucose oxidase strips also is not standardized and is too insensitive and non-specific to test for aspiration of feeding solution. The best single test for aspiration remains undetermined to date. Therefore, clinical studies are needed to determine the potential role of particular tests suited for specific indications [9].
Video-fluoroscopy has traditionally been accepted as the “gold standard” for evaluation of a swallowing disorder for the comprehensive information it provides. However, it is not very efficient and accessible in certain clinical and practical situations. This is particularly true for critically ill patients treated at ICUs, who are almost invariably bedridden.
FEES has been shown to be safe and effective for assisting in swallowing evaluation, and in therapy as a visual display to help patients learn various swallowing manoeuvres. [4, 10–13]. It can be used in a wide variety of settings, ranging from office evaluation of swallowing, inpatient and outpatient services to critically ill patient treated at the ICU [14–20].
FEES is not just a screening procedure, it can achieve a complete assessment of the pharyngeal stage of swallowing. It includes five components: assessment of structural changes in the larynx and pharynx, assessment of movement and sensation, assessment of secretions management, direct visualization of swallowing function for food and liquid, and response to therapeutic interventions [12–15, 17].
Aviv and co-workers suggested combining FEES with sensory testing of the laryngeal and pharyngeal mucosa using discrete pulses of air delivered to the epithelium innervated by the internal branch of the superior laryngeal nerve (FEESST: Fiberoptic endoscopic evaluation of swallowing with sensory testing). They performed 1,340 consecutive FEESST procedures on 1,076 adult patients with dysphagia, among them 172 examinations in critically ill patients at the ICU (mostly following cardiac surgery). The authors concluded that FEESST for the purpose of swallowing function can be done safely and yields a 96.5% success rate in terms of completed examinations [19, 21].
Our experience suggests that FEES can be performed easily at the bedside of ICU patients. It is associated with minimal complications for those patients recently liberated from mechanical ventilation, and for patients with indwelling tracheotomy treated at the ICU. However, we cannot determine from our data whether adopting FEES prevented aspiration pneumonia. Barquist et al. [22] conducted a randomized, prospective clinical trial of FEES versus routine clinical management in 70 patients after prolonged intubation. The addition of a FEES examination did not change the incidence of aspiration or postextubation pneumonia. Larger prospective randomized trials of FEES versus conventional patient management will be required to determine whether a difference in the rate of post-extubation pneumonia can be demonstrated. However, in our study, the observations recorded during FEES had immediate impact on the further treatment of patients: prolonged non-oral feeding via a naso-gastric tube was initiated in 49.7% of patients, percutaneous endoscopic gastrostomy in 13.2%, and an additional tracheotomy to prevent aspiration in 6.3%. On the other hand, 81 out of 258 pre-existing tracheotomies were immediately closed, and 30.7% of all 553 patients with endoscopy-proven aspiration could be treated with the immediate onset of an oral diet and compensatory treatment procedures.
After assessing the critically ill patient for risk of aspiration, the clinician still must decide if the patient is ready to be fed. The goal is to identify critically ill patients who are likely to tolerate enteral nutrition and attempt to minimize complications [23]. FEES is certainly a versatile tool in achieving timely and appropriate clinical decisions in patients at risk for aspiration-related morbidity.
In contrast to most of the recently published material on FEES in critically ill patients, we report our observation in a large proportion of patients with indwelling tracheotomies (258 out of 553, i.e., 46.7%) at the time of the examination. Tracheotomy is a commonly performed procedure in ventilator dependent patients. Many critical care practitioners believe that performing a tracheostomy early in the postinjury period decreases the length of ventilator dependence as well as having other benefits such as better patient tolerance and lower respiratory dead space [24]. Table 4 demonstrates that silent aspiration as well as clinically overt aspiration was more frequently encountered in patients with tracheotomies as compared to those that had previously been intubated via an oral or transnasal route. However, the clinical impressions that a tracheotomy or tracheotomy tube increases aspiration risk or that decannulation results in improved swallowing function have not been supported by a recent study. Rather, the need for a tracheotomy indicated comorbidities (e.g. respiratory failure, trauma, stroke, advanced age, reduced functional reserve, and medications used to treat the critically ill) that by themselves predispose patients for dysphagia and aspiration [25]. These data confirmed similar findings previously published by same authors: Neither the presence of a tracheotomy tube nor decannulation affected aspiration status in early, postsurgical head and neck cancer patients. In the acute care setting, no causal relationship between tracheotomy and aspiration status was exhibited [26].
Our results allow no conclusion regarding the impact of tracheotomies on deglutition, since the initial decision to perform a tracheotomy had not been randomly assigned to patients in our two subgroups. The decision to perform tracheotomies was not consistent over different ICUs and largely depended on the anticipated length of assisted ventilation. In our study, FEES was used to determine the need for maintaining tracheotomy, and was accepted by ICU physicians as an important criterion in deciding to close tracheotomies following assisted ventilation.
Conclusion
After the introduction of a standardized endoscopy protocol for critically ill patients considered being at risk for silent aspiration, ICU physicians soon requested FEES routinely for their patients.
FEES in critically ill patients allows for a rapid evaluation of deglutition, for targeted further diagnostic procedures if needed, and for the immediate initiation of symptom-related rehabilitation or for an early resumption of oral feeding. FEES is now accepted at our institution as an important tool in achieving timely and appropriate clinical decisions for ICU-patients at risk for aspiration-related morbidity.
Laryngologists should be encouraged to offer FEES procedures to responsible coordinators of ICUs.

swallowing aspiration tracheotomy deglutition rehabilitation endoscopy dysphagia laryngology