Brief Report: Eye Movements During Visual Search Tasks Indicate Enhanced Stimulus Discriminability in Subjects with PDD

Subjects with PDD excel on certain visuo-spatial tasks, amongst which visual search tasks, and this has been attributed to enhanced perceptual discrimination. However, an alternative explanation is that subjects with PDD show a different, more effective search strategy. The present study aimed to test both hypotheses, by measuring eye movements during visual search tasks in high functioning adult men with PDD and a control group. Subjects with PDD were significantly faster than controls in these tasks, replicating earlier findings in children. Eye movement data showed that subjects with PDD made fewer eye movements than controls. No evidence was found for a different search strategy between the groups. The data indicate an enhanced ability to discriminate between stimulus elements in PDD.
Introduction
While most diagnostic criteria of Pervasive Developmental Disorder (PDD) are characterised by impairments, mostly with respect to social and communicational abilities, subjects with PDD excel on certain visuo-spatial tasks that supposedly reflect superior processing of fine detail (see Dakin & Frith, 2005, for a review). An especially robust finding seems to be the behavior of subjects with PDD on visual search tasks (Plaisted, O’Riordan, & Baron-Cohen, 1998; Plaisted, Saksida, Alcantara, & Weisblatt, 2003; O’Riordan, Plaisted, Driver, & Baron-Cohen, 2001). In such tasks, subjects are required to detect a target in a display containing a variable number of distracters. If the task difficulty is increased by adding distracters, usually RTs increase. In studies on PDD, two search task versions that differed in difficulty have been used. In both tasks children with PDD showed shorter RTs, as compared with normally developing controls (Plaisted et al., 1998; Plaisted et al., 2003; O’Riordan et al., 2001). The reason for this superior performance is not clear, but has been related to enhanced ability to discriminate between stimulus elements in subjects with PDD (O’Riordan & Plaisted, 2001), an explanation that has also been proposed for the superior performance of subjects with autism on other visuo-spatial tasks (Plaisted et al., 2003, see also Bertone, Mottron, Jelenic, & Faubert, 2001).
So far, however, there have been no studies that have validated the claim for enhanced stimulus discrimination as an explanation for superior performance in search tasks in PDD more directly. It is well-known that stimulus discriminability affects eye movement parameters, especially number of fixations and fixation duration (Hooge & Erkelens, 1999). Therefore, studying eye movements during search tasks in subjects with PDD is a first step to gain more insight into the neurocognitive mechanisms of atypical visuo-spatial processing in this group. If increased stimulus discriminability is indeed the underlying factor for superior performance of subjects with PDD, it is expected that they need fewer and/or shorter fixations to identify the target. Also, eye movement parameters can be used to test alternative hypotheses for shorter reaction times in search tasks in PDD. More specifically, it has been argued that (healthy) subjects in a visual search task have a tendency to move their eyes even though in some situations it would be a better strategy not to do so, since longer fixations allow better extraction of (peripheral) information (Hooge & Erkelens, 1999; see also Rayner, 1998). It is well possible that individuals with PDD use a different search strategy in which they show longer, but less, fixations than controls.
The first aim of the present study was to use eye movement parameters to test the two hypotheses described above for superior visual search in subjects with PDD. A second aim was to replicate the findings of Plaisted et al. (1998) and O’Riordan and Plaisted (2001) in high functioning adults with PDD. For these reasons we used the same search tasks as described in O’Riordan et al. (2001, second experiment) in a group of high functioning adults with PDD, matched on gender, IQ, and age to a control group.
Methods
Participants
Two groups of young male adults participated, a group of eight high functioning individuals with PDD and a group of eight normal control individuals. The clinical subjects were recruited via the Department of Child and Adolescent Psychiatry at the Utrecht University Hospital. The study was described to the subjects and written informed consent was obtained. All subjects were administered the Wechsler Intelligence Scale, revised Dutch edition (WAIS). Diagnoses of either Autistic Disorder or Asperger Syndrome were made by a child psychiatrist and based on DSM-IV criteria. The parents of all subjects with PDD were administered the Autism Diagnostic Interview Revised (ADI-R) (Lord, Pickles, McLennan & Rutter, 1997; Lord, Rutter & LeCouteur, 1994). Due to technical problems, eye-movement data of one subject with PDD and one control subject were lost. The individual ADI scores of the remaining seven PDD subjects can be found in Table 1. Unpaired t-tests revealed that there were no significant differences between the remaining seven PDD and seven control subjects with respect to either age, TIQ, VIQ, or PIQ (see Table 2). None of the participants showed any visual or oculomotor pathology other than refraction anomalies. Subjects were allowed to wear their contact lenses or glasses.
Table 1ADI scores on the four domains for individual PDD subjectsADI scores individual subjectsSocial interaction (cutoff 10)Communication (cutoff 8)Stereotypy (cutoff 3)Age of onset (cutoff 1)12218202181390324163542616855242410362120437281752Table 2Mean age and IQ characteristics of the control and PDD groupControlPDDAge21.222.1TIQ115.3121.9VIQ119.1124.0PIQ106.7114.0
Set up and Data Analysis
Subjects sat in front of a LaCie Blue Electron lll 22′ Screen (0.394 m × 0.295 m, 1240 × 1024 pixels at 85 Hz,) at a distance of 0.64 m. Stimuli were generated by a Apple PowerMac G4/450 DP using a Matlab program based on routines taken from the Psychophysics Toolbox (Brainard, 1997; Pelli, 1997) and the EyeLink Toolbox (Cornelissen, Peters & Palmer, 2002).
Movements of the left eye were measured at 250 Hz with the EyeLink 1 eye monitor. Head movements were prevented by the use of a chinrest. Data were stored on disk and were analysed off-line by a self-written Matlab program. The velocity signal of eye movements was searched for peak velocities above 20°/s. Each peak (in the velocity signal) was considered a potential indicator of the presence of a saccade. The exact onset of the saccade was determined by going backward in time to the point where the velocity signal dropped below the average velocity plus two standard deviations during the stable fixation period before the saccade. The exact offset of the saccade was determined by going forward in time to the point where the velocity signal dropped below the average velocity plus two standard deviations during the stable fixation period after the saccade. This method was adopted from Van der Steen and Bruno (1995). This procedure was followed by rejection/acceptation based on minimum saccade duration of 12 ms and minimum amplitude of 1°. When a saccade was removed, fixation time before and after this saccade and the duration of the saccade were added together.
Stimulus and Task
Two tasks were tested in separate sessions for each participant, with the order counterbalanced within participant groups. Both tasks contained tilted as well as vertical line elements. The tasks only differed in which of the two elements was target or distracter. Since an earlier study has shown a difference in task difficulty depending on target orientation (O’Riordan et al., 2001), the condition including a vertical target among tilted distracters will be referred to as the easy condition, and the condition with a tilted target (among vertical distracters) as the hard condition. Each task contained two crossed factors; set size (4, 16, or 25 items) and probe, target present or target absent. There were 30 trials at each unique combination of factors, yielding a session of 180 trials per task. The sequence of different tasks was randomised within each session. In each session, the participant was informed about the nature of the target (either vertical or tilted), but did not know when a target would be present or absent, or what the display size would be on any trial. The participants performed a binary choice RT task indicating whether a target was present or absent by a button press on each trial.
Procedure
The order in which the two conditions were presented was counterbalanced. Prior to each task participants were given 10 practice trials. Participants were instructed to respond as quickly and accurate as possible by pressing the five key on the numeric pad when the target was present and the four key when the target was absent. Thereafter, the eye link camera was attached to the head and calibrated. On each trial the sequence of events was as follows: a fixation cross was presented. The participant had to fixate at the cross and press the space bar subsequently. Fixation of the fixation cross was used for on-line drift correction of the eye tracker. Then, the search display appeared and remained visible until the participant responded. After the response the next trial started.
Results
To assure that a possible superior task performance of the individuals with PDD was not due to a difference in detection criteria (e.g., if speeded reaction time is accompanied by a decrease in number of correct answers), we performed an independent t-test on the error rates of both groups. No significant difference in error rate between the groups was found. One of the subjects in the PDD group showed a high number of errors and a deviant pattern in reaction time data, and was excluded from further analysis. The results described below are therefore based on six subjects with PDD, and seven controls.
The mean RT, first fixation times, remaining fixation times, and number of fixations data were analysed using a repeated measures ANOVA, with one between subjects factor of group (control or autistic) and three within subject factors, condition (condition 1: tilted target or condition 2: vertical target), probe (target present or target absent) and set size (4, 16, or 25 items). Analyses of the first and remaining fixations were done separately, because there is evidence that they belong to different distributions (Hooge & Erkelens, 1996). Unless otherwise stated, a significance level of p < .05 was adopted for all statistical comparisons. Only main effects or interactions with Group will be considered.
RT Analysis
The analysis of variance revealed a main effect of Group, F(1,11) = 6.6, p < 0.05, reflecting the fact that individuals with PDD were significantly faster overall than the control individuals (737 vs. 1016 ms). A significant interaction Group × Display was found (F(2, 22) = 3.8, p < .05), as well as an interaction of Group × Display × Probe (F(2, 22) = 4.7, p < .05). Partial analyses showed that the Groups difference was significant at all set sizes, but that the effects were largest for set size 25 (all p < .05) (resp., F(1,11) 5.7, 5.4, 6.8). The interaction Group × Probe was marginally significant for set size 25 only (F(1,11) = 4.0, p = 0.72), indicating that at this set size, the Groups difference was largest if the target was absent (See Fig. 1).
Fig. 1Means and standard errors of the reaction times for each group in trials with (left panel) and without a target (right panel), for the easy and hard condition
Eye Movement Analysis
The analysis of number of fixations revealed a significant effect of group (F(1,11) = 10.3, p < .01). The individuals with PDD made significantly less fixations than the control group (means, respectively, 1.3 and 2.1). In addition, there were significant interactions between Group × Probe, Group × Display and Group × Probe × Display. When tested per level of Display, subjects with PDD showed significantly fewer fixations for all display sizes (F(1,11) = 8.2, p = .015/9.4, p = .011/10.8, p = .007), but the effects were largest for the 25 set size. Interactions of Group × Probe effects were marginally significant for all set sizes (F(1,11) = 4.7, p = .052/4.0, p = .072/4.4, p = .06), indicating that group differences in number of fixations were largest in the target absent condition (see Fig. 2).
Fig. 2Means and standard errors of the number of fixations for each group in trials with (left panel) and without a target (right panel), for the easy and hard condition
No significant group differences were found for fixation times at either the first fixation or the remaining fixations.
Discussion
The present study was aimed to test two hypotheses on superior performance in visual search tasks in subjects with PDD. It has been suggested earlier that this superior performance reflects an enhanced ability to discriminate between stimulus elements (O’Riordan et al., 2001). We tested this hypothesis by measuring eye movements during search tasks in high functioning adult men with PDD and an age- and IQ-matched control group. In case of enhanced stimulus discriminability, shorter fixation times and/or fewer fixations were expected in the PDD group, as compared to the control group. The alternative explanation, that subjects with PDD use more efficient search strategies, was also tested. A more efficient search strategy would be reflected in a longer fixation time.
The reaction time data showed that subjects with PDD were better than the control group for both easy and hard search tasks, especially in the largest set size and in trials in which the target was absent. These findings are in accordance with earlier studies on visual search in children with autism, indicating faster RT in the same type of tasks (O’Riordan et al., 2001; Plaisted et al., 1998). This indicates that superior search performance in subjects with PDD is a robust finding that can be demonstrated in both children and adults.
Analyses of fixations during the search tasks showed that subjects with PDD made fewer fixations than controls. This difference in number of fixations was especially clear for the largest set size and for the condition in which the target was absent, analogous to the RT effects. In addition, it was noted that many subjects with PDD showed an absence of saccadic eye movements in target present trials, indicating that they were able to localize the target in a single glance. No differences between groups were found in fixation time. The absence of group differences in fixation time indicates that subjects with PDD followed the same search strategy as normal controls. However, the decreased number of fixations during the task in the PDD group is in agreement with the hypothesis of enhanced stimulus discriminability in subjects with PDD, as suggested by O’Riordan and Plaisted (2001). The finding that the pattern of effects for fixation times was strongly similar to the RT effect provides additional evidence that superior performance in this task is indeed related to enhanced discriminatory abilities in PDD.
The question what the locus of this enhanced discriminability could be has been addressed by Cohen (1994), who tested a neural network model of processing in subjects with PDD. This model showed that an increase in processing units resulted in a strong ability to discriminate and learn stimulus patterns, along with a weak ability for generalization. Indeed, there is anatomical evidence for abnormal development of the cyto-architecture of the cerebral cortex of subjects with PDD, resulting in an increase in processing units. Recent post-mortem studies in subjects with PDD show an increased number of micro columns, albeit of reduced size (Casanova, Buxhoeveden, Switala, & Roy, 2002; Casanova et al., 2006).
Concluding, the measurement of eye movements during search tasks shows that the superior behavior of subjects with PDD in these tasks cannot be attributed to strategy difference, but point indeed to increased stimulus discriminability in this group.

eye movement
search task
pdd
perception
eye fixation


The role of metacaspase 1 in Plasmodium berghei development and apoptosis

The malaria parasite encodes a wide range of proteases necessary to facilitate its many developmental transitions in vertebrate and insect hosts. Amongst these is a predicted cysteine protease structurally related to caspases, named Plasmodium metacaspase 1 (PxMC1). We have generated Plasmodium berghei parasites in which the PbMC1coding sequence is removed and replaced with a green fluorescent reporter gene to investigate the expression of PbMC1, its contribution to parasite development, and its involvement in previously reported apoptosis-like cell death of P. berghei ookinetes. Our results show that the pbmc1 gene is expressed in female gametocytes and all downstream mosquito stages including sporozoites, but not in asexual blood stages. We failed to detect an apparent loss-of-function phenotype, suggesting that PbMC1 constitutes a functionally redundant gene. We discuss these findings in the context of two other putative Plasmodium metacaspases that we describe here.
1
Introduction
Malaria remains the most important parasitic human disease responsible for millions of deaths each year. The life cycle of malaria parasites is complex involving multiple life stages and requiring two hosts: mosquito and human. The uptake of gametocytes by the mosquito leads to rapid gametogenesis and fertilisation. Ookinetes are formed in the mosquito midgut lumen, which cross the midgut wall and transform into oocysts. Each oocyst can produce thousands of sporozoites, which after release traverse the mosquito hemolymph and infect the salivary gland tissues before they can enter the human host via the mosquito bite. Following inoculation, sporozoites infect hepatocytes and develop into liver schizonts. Liver schizont produce merozoites, which upon release into the blood stream initiate the erythrocytic cycle, the prime cause of clinical symptoms in malaria patients.
Existing malaria control strategies are hampered by increasing parasite resistance in the field to established antimalarial drugs, as well as by widespread insecticide resistance in the mosquito vectors. Effective antimalarial vaccines are only at the preliminary stages of development. There is thus a great need to develop novel strategies for malaria control. Proteases are attractive antimalarial targets because of their indispensable roles in parasite development and infectivity. A wide variety of proteases of malaria parasites have been described [1]. Amongst these are cysteine proteases belonging to the Peptidase_C14 family (clan CD) which include caspases. Caspases belong to a distinct class of cysteine proteases with a so called Caspase Hemoglobinase Fold (CHF) [2]. Recently, two new families of predicted CHF proteases closely related to the caspases were identified, named paracaspases and metacaspases [3].
In metazoa, caspases are centrally involved with the molecular machinery of programmed cell death (apoptosis), and are responsible for many of the biochemical and morphological changes that accompany it. They cleave a variety of proteins ultimately leading to the disintegration of the cell [4]. Metacaspases have thus far only been identified in organisms that do not possess classic caspase genes: plants, fungi and the parasitic protists Trypanosoma, Leishmania and Plasmodium, and there is some evidence linking metacaspases to apoptosis in these organisms [5–10]. In Plasmodium falciparum a metacaspase gene (GenBank accession CAD52669, here named PfMC1) has been described possessing consensus histidine and cysteine residues that typically form the catalytic dyad in this family of proteases [1,3], suggesting that this parasite species may possess a mechanism of programmed cell death. Indeed, apoptosis-like DNA fragmentation has been reported to occur in blood stage parasites of P. falciparum in response to chloroquine treatment [11]. In addition, ookinetes of Plasmodium berghei were reported to undergo an apoptosis-like mechanism of cell death resulting in very substantial parasite losses [12]. In this study we investigate the role of PbMC1 in the reported programmed cell death in Plasmodium by constructing PbMC1-deficient parasites. We discuss our findings in the context of two additional Plasmodium metacaspase-like proteins that we describe here.
2
Materials and methods
2.1
Parasite maintenance, culture and purification
P. berghei ANKA (clone 2.34) parasites were maintained in female TO mice by mechanical blood passage and regular transmission in Anopheles stephensi, as described [13]. Ookinete cultures were set-up as described [12,13] both with and without prior removal of white blood cells by CF11 columns. In general, 21–24 h ookinete cultures were centrifuged at 1500 × g for 5 min and the cell pellet incubated on ice in 20 volumes of 0.17 M NH4Cl to lyse the red blood cells. In some experiments ookinetes were further purified on Nycodenz cushens. Finally, parasites were concentrated by centrifugation at 1500 × g for 5 min, resuspended in RPMI 1640 or phosphate buffered saline, and immediately analysed.
2.2
Generation of PbMC1-KO parasites
A 720 bp fragment was amplified by PCR from P. berghei genomic DNA with primers IF-MC3′-F (TAAACCATTGGTCATACCAAAAAATAATCAAAAAAATAACCAA) and IF-MC3′-R (CGGGCCGCTCTAGCATGACCAGGCTCAATAATTGAACA) and introduced into NdeI-digested pLP-DHFR2 via In-Fusion PCR cloning (BD Biosciences). The resulting plasmid, pLP-DHFR/MC, contains a loxP-prokaryotic promotor cassette (BD Biosciences), followed by a modified Toxoplasma gondii dihydrofolate reductase (dhfr) gene cassette [14], followed by the 3′UTR of pbmc1. A 680 bp fragment was amplified by PCR from P. berghei genomic DNA with primers pDNR-ΔMC-F (ACGAAGTTATCAGTCGACGGTACCCCATCATAAAGCAAAAAGC) and pDNR-ΔMC-R (ATGAGGGCCCCTAAGCTTATTTAACATAAATTTTGTCCATTT) and introduced into SalI/HindIII-digested pDNR-EGFP via In-Fusion PCR cloning. The resulting plasmid, pDNR-ΔMC/EGFP, contains two loxP sites flanking the 5′UTR plus first 11 codons of pbmc1 fused in-frame to the enhanced green fluorescent protein (egfp) gene, followed by the 3′UTR of P. berghei dhfr. The pbmc1-specific insert contained within pDNR-ΔMC/EGFP was introduced into pLP-DHFR/MC via Cre-loxP site-specific recombination (BD Biosciences) to give the transfection vector pLP-ΔMC/EGFP. Prior to performing transfections this plasmid was digested with KpnI and NotI to remove the vector backbone. Parasite transfection, pyrimethamine selection and dilution cloning were performed as previously described [15]. Genomic DNA extraction, and Southern blot were performed as previously described [14].
2.3
Apoptosis assays
Phosphatidylserine (PS) translocation was studied with fluorescein isothiocyanate (FITC)-conjugated Annexin V in conjunction with propidium iodide, by using the Annexin-FITC Apoptosis Detection Kit (Sigma–Aldrich) according to manufacturer's instructions. DNA condensation in the nucleus was assessed using acridine orange staining as described [12]. DNA fragmentation was assessed by in situ terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labelling (TUNEL), by using the ApopTag® Fluorescein In Situ Apoptosis Detection Kit (Chemicon International) according to manufacturer's instructions. Caspase activation was assessed with sulphorhodamine-VAD-FMK inhibitor of caspases, by using the CaspaTag™ Pan-Caspase In Situ Assay Kit (Chemicon International) according to manufacturer's instructions.
3
Results
3.1
Description of Plasmodium metacaspases
Using BLAST searches of available Plasmodium genome sequences with the amino acid sequence of PfMC1 we identified orthologues in P. berghei and other Plasmodium species including P. chabaudi, P. yoelii, P. knowlesi, P. vivax and P. gallinaceum. Multiple alignment of the predicted PxMC1 proteins revealed conserved amino- and carboxy-terminal domains, separated by a region of variable length and sequence. These amino- and carboxy-terminal domains had, in Pfam and SMART searches, significant homologies with a C2 domain (a Ca2+-dependent membrane targeting module, Pfam identifier PF00168) and with the Peptidase_C14/Caspase domain (Pfam identifier PF00656), respectively (Fig. 1A). We also identified two paralogues of PxMC1 (named PxMC2 and PxMC3) in all Plasmodium species, each containing a region of significant homology to the Peptidase_C14/Caspase domain in Pfam and SMART searches. In contrast to the PxMC1 proteins however, sequence conservation in the much larger PxMC2 and PxMC3 proteins appeared to be limited to their C-terminal regions containing the putative caspase domains (Fig. 1A).
Multiple alignment of the predicted caspase domains of PxMC1, PxMC2 and PxMC3 from P. berghei, P. falciparum and P. vivax, along with a metacaspase from yeast (YCA1) [6] shed further light on their structures (Fig. 1B). Like PfMC1, all PxMC1 orthologues possess histidine and cysteine residues predicted to form the catalytic dyad in this family of proteases. It is interesting to note that PbMC1 has the predicted catalytic cysteine located one residue upstream of the consensus position for this amino acid, with a proline in the consensus position (Fig. 1B). The same is true for PcMC1 and PyMC1 (data not shown). In contrast, PvMC1 has two adjacent cysteines, at the consensus position and the preceding residue (Fig. 1B). The same is found in PkMC1 and PgMC1 (data not shown). Catalytic dyad histidine and cysteine residues are absent in the consensus positions in the PxMC2 and PxMC3 proteins (Fig. 1B). However, PfMC3 and PvMC3 have a cysteine at the preceding amino acid position, similar to the situation in PxMC1 of rodent malaria species.
3.2
Generation and molecular analyses of PbMC1 knockout parasites
Because PxMC1 proteins display the greatest sequence similarity with the consensus Peptidase_C14 domain and, of the three Plasmodium metacaspases described here, are therefore most likely to be active enzymes, we decided to investigate the expression of PbMC1 and its contribution to P. berghei parasite development. To this purpose we generated genetically modified parasites in which the PbMC1 coding sequence was removed and replaced with a reporter gene, enhanced green fluorescent protein (EGFP). In the strategy used, the 5′UTR and 3′UTR of the pbmc1 gene allow for double homologous recombination [15,16], introducing the EGFP coding sequence downstream of the first 11 codons of PbMC1 (coding for MSLQMDKIYVK), as well as inserting a modified T. gondii dihydrofolate reductase/thymidylate synthase (tgdhfr/ts) gene cassette conferring resistance to the antimalarial drug pyrimethamine into the pbmc1 locus (Fig. 2A).
After transfection of purified P. berghei schizonts [15] we readily obtained pyrimethamine-resistant parasites. Diagnostic PCR amplification across the predicted integration sites showed that correct integration of the DHFR/TS cassette into the PbMC1 locus had occurred (data not shown). This was confirmed by assessing the integrity of a clonal population of the genetically modified parasite (named PbMC1-KO) by Southern blot analysis of HincII-digested genomic DNA. Accordingly, a probe corresponding to the 5′UTR plus PbMC1 coding region gave rise to expected bands of 4.5 and 2.1 kb in the parental wild-type (WT) parasites (Fig. 2B). In contrast, in the PbMC1-KO parasites expected bands of 4.5 and 0.9 kb were observed. The 0.9 kb fragment corresponds to 360 bp of the 5′UTR of pbmc1, its first 11 codons, plus 490 bp of the egfp gene (Fig. 2B). The 4.5 and 0.9 kb bands are more weakly labelled than the 2.1 kb band, because a smaller portion of the probe anneals to these fragments (Fig. 2A). A probe corresponding to the tgdhfr/ts gene gave rise to bands of 2.6 and 0.9 kb in the PbMC1-KO parasites, but no signal in the WT parasite sample as expected (Fig. 2B). These combined results are fully consistent with the structural removal of the PbMC1 coding sequence, and the integration of the reporter and selectable marker genes into the pbmc1 locus.
3.3
PbMC1 expression and loss-of-function phenotype
PbMC1-KO parasites developed normally in mice and were morphologically indistinguishable from WT parasites in Giemsa-stained blood smears. Using UV microscopy EGFP fluorescence was readily observed in a subpopulation of parasitized erythrocytes. Upon gametocyte activation (by placing the infected blood in ookinete medium) the large majority (>90%) of fluorescent parasites emerged from the host cell (clearly visible by the rounding up of the parasite) by 15 min, typical of gametogenesis. The number of remaining intracellular fluorescent parasites was considerably lower than the percentage of trophozoites present in the sample as assessed by Giemsa staining. These are therefore likely to constitute immature gametocytes, or gametocytes that had failed to emerge. We were further able to distinguish between male and female gametocytes by observing exflagellation (i.e. the release of male gametes); this involved gametocytes that were not green. From these combined observations we conclude that the pbmc1 gene is expressed only in female gametocytes (Fig. 3A). As far as we could ascertain all females were positive and all males negative. Normal differentiation of gametocytes into ookinetes both in vitro and in vivo was observed. These ookinetes again displayed bright EGFP-based fluorescence under UV light (Fig. 3B). WT and PbMC1-KO parasite-infected A. stephensi mosquitoes developed comparable numbers of oocysts, indicating that the PbIMC1a-KO ookinetes are capable of normal midgut invasion and subsequent ookinete-to-oocyst transition on the hemocoel side of the midgut wall. Examination of oocysts on the midgut wall by UV microscopy displayed EGFP fluorescence in oocysts and the sporozoites contained within (Fig. 3C). Fluorescent sporozoites were observed in the mosquito salivary glands (Fig. 3D), which were infectious to mice upon infected mosquito bite. These combined results show that the pbmc1 gene is expressed in female gametocytes and all downstream mosquito stages including sporozoites, but not in the asexual blood stages. The apparently normal development of the PbMC1-KO parasite both in the vertebrate and insect hosts shows that pbmc1 is not an essential gene and may be functionally redundant.
3.4
Apoptosis in ookinetes
It has been reported that, in P. berghei, a large proportion of ookinetes undergo cell death, displaying typical apoptosis-like features such as PS translocation, nuclear condensation and DNA fragmentation [12]. As caspases are well known to play a central role in apoptosis, we wanted to assess the effect of PbMC1 knockout on this process. Initially, we assessed the level of apoptosis-like cell death in WT ookinetes. Annexin V conjugated to FITC (staining green), which specifically binds to PS, was used to assess the proportion of ookinetes displaying PS translocation to the outer leaflet of the cell membrane. Propidium iodide (staining red) was used simultaneously to assess plasma membrane integrity (cell viability). Across 13 experiments conducted we observed an average 77% viable ookinetes (no staining), while an average 21% appeared dead (red only staining) (Fig. 4). The average proportion of ookinetes displaying PS translocation (green staining) was less than 3%. Only 0.5% of ookinetes examined showed PS translocation and were propidium iodide negative (green only staining) (Fig. 4). In addition to this assay, we used acridine orange staining to look for nuclear condensation, and TUNEL to assess DNA fragmentation in P. berghei ookinetes, as described [12]. We found no evidence for either of these processes occurring.
Given the low numbers of positive cells in the above assays, we decided to look for the activation of caspases, a key event in caspase-dependent apoptosis, by using a fluorochrome-labelled inhibitor of caspases (CapaTag). In mammalian cells, these labelled inhibitors are permeant and covalently bind to the active centres of caspases with a 1:1 stoichiometry. In this assay, WT ookinetes displayed an average 3.8 ± 0.5% of positive cells (two experiments) after 21 h of culture, which, at 24 h had gone up to an average 14 ± 9% (four experiments). Under the same conditions, PbMC1-KO ookinetes showed an average 2.6 ± 0.3% positive cells (two experiments) at 21 h, and 13 ± 6% (five experiments) at 24 h of culture. We also used a green fluorescent P. berghei parasite with an intact pbmc1 gene (PbGFPCON [17]), that displayed an average 3.8% and 9.0% of positive ookinetes at 21 and 24 h, respectively. Thus, knockout of PbMC1 does not appear to significantly affect the percentage of ookinetes that bind CaspaTag.
4
Discussion
In this paper we describe the generation of P. berghei parasites in which the caspase-like protein PbMC1 is replaced by the fluorescent reporter EGFP, allowing us to study both its expression profile and its loss-of-function phenotype using a single genetically modified parasite line. Our EGFP reporter data show the pbmc1 gene to be transcribed, and probably translated, in female gametocytes and all downstream mosquito stages including sporozoites, but not in asexual blood stages. However, we cannot rule out that the pbmc1 gene may be subject to translational repression. There are many examples of genes that are transcribed in the female gametocyte, but which have a translational block and are not translated until later in development, as was recently published [18]. Interestingly, pbmc1 is not one of the genes identified by these authors, supporting a scenario of direct translation. Knockout of PbMC1 expression does not seem to adversely affect any stage of parasite development, at least in vivo in the mouse and mosquito, or in the culture systems used in this study. This was surprising because pxmc1 is a highly conserved single copy Plasmodium gene, and because PxMC1 is predicted to possess consensus catalytic dyad residues and thus is likely to possess caspase activity. Although the two paralogues, PxMC2 and PxMC3, do not share the typical consensus catalytic dyad residues of the Peptidase_C14/Caspase family, it is possible that they constitute active zymogens by using alternate, functionally equivalent catalytic residues. Indeed, functional replacement between cysteine, serine or threonine residues has been reported for other cysteine and serine proteases [19–21]. If this is the case it is conceivable that one or both of the paralogues could carry out the role of PbMC1, hence leading to functional redundancy and resulting in an apparently normal mutant phenotype as observed. Interestingly, five metacaspase genes have been identified in Trypanosoma brucei, TbMCA1-TbMCA5, and two of these also lack a conserved histidine residue (replaced by a tyrosine, TbMCA1) and/or cysteine residue (replaced by a serine, TbMCA1 and TbMCA4) (reviewed in [22]). Whether any of these molecules are active enzymes remains to be determined.
It has been previously shown in plants and fungi [6,9] that metacaspases can be involved in the regulation or the execution of programmed cell death. Because an apoptosis-like mechanism of cell death was reported to take place in ookinetes of P. berghei
[12] we hypothesized that PbMC1 could be involved in this phenomenon. Of three typical apoptotic markers: PS translocation, nuclear condensation and DNA fragmentation, only PS translocation gave positive results in our hands. PS translocation does also occur when cells die from accidental cell death (oncosis), in which case membrane integrity is lost concomitantly. Accordingly, we cannot truly determine whether ookinetes that in our assay stained doubly positive for both Annexin V and propidium iodide were in the later stages of apoptosis, or whether they died from an unrelated death mechanism. The fact that a subset of ookinetes stained positive for Annexin V suggests that the low numbers of positive cells observed are not a result of a reduced PS content in the parasite plasma membrane. Overall, the results based on these three apoptotic markers indicate that apoptosis-like death in ookinetes, in our hands, is low.
When we monitored for potential apoptotic ookinetes using CaspaTag, we obtained up to 14% positive cells. Surprisingly, no reduction of CaspaTag labelling was observed in PbMC1-KO parasites. Although PbMC1 is the most likely target of the CaspaTag inhibitor, it is possible that other targets of CaspaTag are present in ookinetes, which could mask any reduction in CaspaTag binding in the PbMC1-KO parasite. Clearly, the other two putative metacaspases are prime candidates. Indeed, P. falciparum transcriptome and proteome analyses indicate that PfMC2 is expressed in gametocytes and sporozoites [23–25]. Hence, its expression in other mosquito stages including ookinetes is likely. This scenario could also explain the normal development of the PbMC1 null mutant parasites by assuming a level of functional redundancy between the three Plasmodium metacaspases. Expression and knock out experiments with the other metacaspases are underway to test this hypothesis.
Caspase activation is one of the earliest events in apoptotic mammalian cells. Hence, early apoptotic cells have activated caspases, but display no changes in plasma membrane integrity [26]. It was therefore notable that no EGFP-based fluorescence was observed in CaspaTag positive ookinetes neither in the PbMC1-KO nor PbGFPCON parasite lines, indicating that these cells had lost plasma membrane integrity before CaspaTag labelling occurred. This is consistent with results from Al-Olayan et al. [12] who reported that their caspase inhibitor-positive ookinetes were propidium iodide permeable. Moreover, we found that pre-treatment with the unlabelled inhibitor failed to reduce subsequent binding of CaspaTag (data not shown). In mammalian cells this has been reported to reduce CaspaTag binding by over 90% [26]. We should thus consider the possibility that these caspase inhibitors may have different specificities in Plasmodium than they do in mammalian cells.
It is difficult to explain why in our hands the number of potentially apoptotic ookinetes detected was far below the numbers previously reported [12]. Despite numerous attempts we were unable to find conditions that led to increased numbers of ookinetes displaying apoptotic markers. We cannot rule out that small differences in the experimental set-ups might explain the conflicting findings. Alternately, the level of apoptosis detected in P. berghei ookinetes may depend on multiple factors that are still to be defined, for instance the exact conditions under which the gametocytes are generated in the mouse host. A not dissimilar discrepancy also exists for the reported apoptosis-like DNA fragmentation that occurs in blood stage P. falciparum parasites in response to chloroquine treatment [11], an observation that was not corroborated in a more recent study [27].

metacaspase
plasmodium berghei
apoptosis


Early changes in arterial structure and function following statin initiation: Quantification by magnetic resonance imaging

Effective LDL-cholesterol (LDL-C) reduction improves vascular function and can bring about regression of atherosclerosis. Alterations in endothelial function can occur rapidly, but changes in atherosclerosis are generally considered to occur more slowly. Vascular magnetic resonance imaging (MRI) is a powerful technique for accurate non-invasive assessment of central and peripheral arteries at multiple anatomical sites. We report the changes in atherosclerosis burden and arterial function in response to open label statin treatment, in 24 statin-naïve newly diagnosed stable coronary artery disease patients. Patients underwent MRI before, and 3 and 12 months after commencing treatment. Mean LDL-C fell by 37% to 70.8 mg/dL (P < 0.01). The plaque index (normalised vessel wall area) showed reductions in the aorta (2.3%, P < 0.05) and carotid (3.1%, P < 0.05) arteries at 3 months. Early reductions in atherosclerosis of aorta and carotid observed at 3 months were significantly correlated with later change at 12 months (R2 = 0.50, P < 0.001; R2 = 0.22, P < 0.05, respectively). Improvements in aortic distensibility and brachial endothelial function that were apparent after 3 months treatment were sustained at the 12-month time point.
1
Introduction
HMG CoA reductase inhibitors or ‘statins’ reduce cardiovascular events by approximately 25–30% in patients with stable atherosclerotic disease [1,2]. Previous studies of ultrasound carotid intima media thickness (CIMT) have suggested relatively slow regression of atherosclerosis after 1–2 years of high dose statin treatment [3,4]. More recently, intravascular ultrasound (IVUS) of the coronary arteries has demonstrated coronary plaque regression following intensive LDL-C reduction in over 300 patients treated with 40 mg rosuvastatin for 2 years [5]. Corti et al. [6] were among the first to use serial magnetic resonance imaging (MRI), to demonstrate reduction in carotid and aortic atherosclerosis in 18 patients in response to 12 months of statin treatment. Subsequently, the same group demonstrated that more effective lipid lowering, to LDL-C < 100 mg/dL, was associated with a larger decrease in plaque size at 12 months [7]. Similarly, Saam et al. [8] have recently identified statin treatment as an independent predictor of reduced annual rate of carotid atheroma progression, measured using MRI.
In the setting of acute coronary syndromes, early initiation of ‘intensive’ statin treatment can rapidly reduce cardiovascular risk within 4–16 weeks [9,10]. However, given the apparently slow changes in atheroma burden observed in previous studies [6], it has been hypothesised that the early clinical benefits of statins cannot reflect structural changes within arteries, but are due to ‘pleiotropic’ effects of statins such as improvement in endothelial function and reduction in inflammation and thrombosis [11]. Indeed, numerous studies have demonstrated rapid improvement in endothelial function in response to statin treatment [12–14]. However, there is also evidence that rapid changes in plaque size and composition might also be possible. Animal models of atherosclerosis indicate that potent correction of dyslipidaemia can result in prompt regression and favourable remodelling of plaques after only 9 weeks [15]. Furthermore, in humans, a recent study of treatment with intravenous apoAI-Milano induced modest but significant regression of coronary atherosclerosis after only 6 weeks [16].
We have previously shown that MRI can assess both central and peripheral vascular function, including measurements of arterial stiffness and endothelial function [17,18]. These parameters are of additional interest because they have been shown prospectively to predict cardiovascular risk [19,20]. By assessing both atherosclerosis and vascular function in the same patients we hoped to examine the extent to which changes in structural parameters might be anticipated by early functional changes. In this study, we used magnetic resonance imaging to evaluate, in vivo, the changes in structure and function in human aorta, carotid and brachial arteries at 0, 3 and 12 months in response to statin therapy. Notably, the mean post-treatment LDL-C achieved in this study reached the contemporary target of 70 mg/dL [21].
2
Methods
2.1
Study population
Newly diagnosed patients (n = 32) with coronary artery disease were recruited from the Cardiology Department of a single tertiary centre. Diagnosis was based on a history of typical symptoms of angina together with an exercise ECG that showed ischaemic-type ST segment changes or a stenosis of ≥50% in at least one coronary artery at angiography. No patients had taken statins prior to study enrolment. No other cholesterol modifying agents were permitted, but no further restrictions were placed on concomitant cardiovascular medications. Patients with acute coronary syndromes or contra-indication to MRI were excluded. MRI was performed at baseline, 3 and 12 months. At each time point, venous blood samples were obtained after a 12-h fast. Statin treatment was withheld until immediately after the first MRI scan, when a statin was started according to the preference of the managing clinician. Decisions regarding cholesterol treatment and management of cardiac risk factors were taken by the responsible clinicians. Statin dose titration, whilst allowed, was not protocol-driven. The study protocol was approved by the local Research Ethics Committee and all subjects gave informed written consent. Of the 32 subjects initially recruited, 8 did not complete all three study time points: 1 patient died from an out of hospital cardiac arrest, 5 were unwilling to return for follow-up scans due to claustrophobia and 2 were lost to follow-up. The 8 patients who did not complete the study were not significantly different from the 24 who did in terms of either their risk factors (age, cholesterol, diabetes, hypertension and smoking) or MRI measures of atheroma or vascular function at baseline. The data reported below refer to the 24 patients, who completed the study protocol.
2.2
Vascular MRI protocol
Imaging was performed on a 1.5 T magnetic resonance scanner (Siemens Sonata, Erlangen, Germany) as previously described [17,18]. In brief, aortic imaging was performed using a combination of a two-element array surface coil placed on the chest and spine coil array. For carotid artery imaging, a two-element array surface coil was used (Machnet BV, Eelde, Netherlands) and for brachial artery imaging a surface coil was attached at the right elbow. Brachial artery blood pressure was monitored using a blood pressure cuff on the left arm. For quantification of aortic wall area, ECG-gated double-inversion recovery (black-blood) fast spin echo images were acquired during breath-hold (Figs. 1b and 2b). Typical parameters were FOV 200 mm, TR 750 ms, TE 11 ms, in plane resolution 0.8 mm × 0.8 mm, slice thickness 5 mm. Using an oblique sagittal image of the aorta as a pilot, 11 serial axial images were acquired with 5 mm interslice gap to cover the entire descending thoracic aorta. The midpoint of the right pulmonary artery in cross section was used as the anatomical reference for the first slice in baseline and follow-up scans. For the carotid arteries axial ECG-gated T2 weighted, black blood images of the neck were acquired during free breathing (Fig. 2b). Sequence parameters: FOV 150 mm, TR 2 R–R intervals, TE 81 ms, resolution 0.5 mm × 0.5 mm in plane, slice thickness 3 mm, no interslice gap. Nine images of the common carotid artery were acquired using the common carotid bifurcation as the anatomical reference position for baseline and follow-up scans. Care was taken to place aortic and carotid image slices perpendicular to the long axis of the vessel on the pilot images in order to limit partial volume effects. For analysis of aorta and carotid plaque images all identifying data were removed apart from a code number so that observers were blinded to both patient identity and study time point. The external vessel boundary and vessel lumen were contoured manually by one of two experienced observers (J.L., C.S.) using CMRtools software (Imperial College, London, UK). Vessel wall area was calculated from the difference between these two contours, and then normalised to external vessel area to yield a plaque index (PI), as previously described [22,23]. Plaque index for each patient was then expressed as the mean of all aortic or carotid slices. In keeping with previous studies of carotid atherosclerosis, a mean value was obtained for left and right carotid arteries combined [3,4]. A subset of images from four randomly selected patients (>100 images) were analysed by both observers to assess inter-observer variability.
Steady state free precession (SSFP) acquisitions were used to determine aortic distensibility and brachial artery reactivity, as previously described [17,18]. Post processing of aortic and brachial images was performed using semi-automated edge detection methods developed with Matlab software (Mathworks Inc.) [24]. Maximum and minimum aortic cross-sectional areas over the cardiac cycle were measured, from which distensibility was calculated as the relative change in area divided by the pulse pressure. To assess brachial artery flow mediated dilatation (FMD), cross sectional images of the brachial artery were acquired at baseline and following release of a cuff inflated to 50 mmHg above systolic blood pressure on the forearm for 5 min. After 10 min, further brachial artery images were acquired following administration of 400 μg of sublingual glyceryl trinitrate to elicit maximal (endothelial-independent) dilatation. Maximum percentage change in cross sectional area was used to determine the response to each stimulus.
2.3
Serum and plasma assays
Cholesterol and lipoprotein assays were performed using a Cobas-Mira Analyser (ABX Diagnostics, Shefford, UK). Total cholesterol was assayed using the enzymatic CHOD-PAP method and triglycerides were assayed using the enzymatic GPO-PAP method. HDL-cholesterol was assayed using a homogenous second generation PEGME method (Roche Diagnostics, Burgess Hill, UK). Apolipoprotein AI (apoAI) and apolipoprotein B (apoB) were assayed using immunoturbidimetric methods, using reagents supplied by ABX Diagnostics. C-reactive protein was analysed using ELISA (MP Biomedicals, UK) according to the manufacturer's instructions.
2.4
Statistical analyses
Statistical analysis was performed using SPSS 12.0 (SPSS Inc., Chicago IL). The Kolmogorov–Smirnov test was used to assess whether data were normally distributed. Measurements at each time point were compared using repeated measures ANOVA for normally distributed data and Friedman analysis of variance by ranks for non-normally distributed data. Post-hoc analysis of paired time points was performed using a Bonferroni correction. Categorical data were analysed by the χ2 test. Data are presented as mean ± standard deviation or median and interquartile range as appropriate. Statistical significance was assigned at P < 0.05.
3
Results
3.1
Clinical and biochemical measures
Baseline characteristics of the 24 patients who completed all three study time points are shown in Table 1. The most common statin dose used was simvastatin 40 mg daily (63% patients); other statin prescriptions are detailed in Table 1. Mean baseline total cholesterol was 187.9 mg/dL (LDL-C 112.7 mg/dL), however 3 months after commencing statins mean LDL-C was reduced by 37% to 70.8 mg/dL (P < 0.01) with a corresponding 24% (P < 0.01) reduction in apoB. Mean LDL-C at 12 months (79.3 g/dL) appeared slightly greater than at 3 months, but this was not statistically significant. Triglycerides and CRP did not change significantly at 3 or 12 months, though HDL cholesterol did show significant increase by 12 months – lipid and apolipoprotein data are shown in Table 2.
Compared to baseline, at 3 months there were no significant changes in the proportion of patients taking either angiotensin converting enzyme inhibitors (12/24 versus 8/24: χ2 = 0.77; P = 0.38) or beta adrenoceptor blockers (22/24 versus 18/24: χ2 = 1.35; P = 0.25). By 12 months, the number of patients taking ACE inhibitors had increased significantly compared to baseline (17/24 versus 8/24: χ2 = 5.3; P < 0.05), though this would not have influenced the study findings at the early time point of 3 months. Beta blocker usage was not changed at 12 months (19/24 versus 18/24: χ2 = 0.11; P = 0.74). There was no significant change in either blood pressure or heart rate over the course of the study (Table 3).
3.2
Atherosclerosis regression
All 24 patients had aortic plaque images of sufficient quality for analysis. Four patients had carotid plaque images at one or more time points of insufficient quality for analysis; therefore they were excluded from statistical analysis. Images (total > 100) from four randomly selected patients were analysed by both observers yielding inter-observer coefficients of variation for plaque index of 4.8% in the aorta, and 2.9% in the carotid. In both the aorta (Fig. 1a) and the carotid arteries (Fig. 2a), there were statistically significant reductions in plaque index after 3 months of statin treatment. Mean aortic plaque index decreased from 0.303 ± 0.024 at baseline to 0.296 ± 0.022 at 3 months (P < 0.05 versus baseline) and 0.288 ± 0.024 at 12 months (P < 0.01 versus baseline). Plaque regression in the aorta between 3 and 12 months was also significant (P < 0.05). In the carotid artery, plaque index fell from 0.446 ± 0.053 at baseline to 0.432 ± 0.046 at 3 months (P < 0.05 versus baseline) and 0.416 ± 0.032 at 12 months (P < 0.01 versus baseline). Plaque index for the carotid artery between 3 and 12 months did not show significant reduction (P = 0.09). The number of patients with early regression in the aorta at 3 months compared to baseline was 18/24, similar to that at 12 months where 20/24 showed regression (χ2 = 0.126, P = 0.72) (Fig. 1c). The number of patients with measurable early regression at 3 months in the carotid was 14/20, which was not significantly different to the 18/20 observed at 12 months (χ2 = 1.4, P = 0.24); see Fig. 2c. Furthermore within patients, the early (3 months) change in plaque index of aorta and carotid arteries showed significant correlation with the final change observed at 12 months (Figs. 1c and 2c).
Mean aortic lumen area was 431 ± 77 mm2 at baseline, 434 ± 77 mm2 at 3 months (P = 1.0 versus baseline) and 442 ± 85 mm2 at 12 months (P = 0.09 versus baseline). Mean lumen area in the carotid arteries was 44 ± 9 mm2 at baseline, 44 ± 8 mm2 at 3 months (P = 1.0 versus baseline) and 43 ± 8 mm2 at 12 months (P = 0.55 versus baseline).
3.3
Physiological measures
After 3 months of statin treatment, aortic distensibility increased by >20% at each of the three locations along its length. This effect was sustained but did not increase further at 12 months (Table 3). Flow-mediated dilatation of the brachial artery, a measure of endothelial function, also improved after 3 months by >30% (Table 3). Endothelial independent relaxation induced by GTN was not significantly changed after 3 months, but did show a significant increase by 12 months.
3.4
Relationship between variables
Within individual patients there was no correlation between MRI quantification of atheroma burden in the aorta and carotid at baseline. Although at a group level, endothelial function and aortic compliance improved and atheroma burden diminished, there was no correlation of these changes within individual patients. Furthermore, there were no significant associations between measures of vascular function or atheroma burden and any of: attained LDL-C; change in LDL-C, HDL-C, apoB, apoA-I, or CRP.
4
Discussion
In this study, we have observed that regression of atherosclerosis in response to statin treatment can occur earlier than previously appreciated in both the aorta and carotid arteries. The robustness of this observation is enhanced by the finding that, within individual patients, regression at the early time point of 3 months was closely related to the magnitude and direction of change at 12 months. Patients also showed early and sustained improvement in aortic distensibility and in flow mediated vasodilatation of the brachial artery.
The magnitude of atheroma regression observed in our study after 3 months is consistent with previous longer-term studies of regression in response to statin treatment [6,7,25]. A lesser LDL-C reduction than that achieved in our study was not associated with early plaque regression after 6 months statin treatment [6]. Intensive LDL-C reduction has been reported to achieve greater regression, but this study did not include an early time point [7]. Thus, the early regression observed here is a new finding that accords with the analysis of Nissen et al. [5] in which atheroma regression, assessed by intravascular ultrasound in the coronary arteries, was predicted by attained mean LDL-C < 70 mg/dL. A recent observational study reported overall carotid wall area progression by 2.2% per year [8], although individual cases of regression of up to 7.9% were reported. Importantly, the population in that study differed by the inclusion of older patients with a higher prevalence of hypertension and selected only patients with carotid plaques of ≥50% stenosis on a prior duplex scan.
Mean lumen size of the aorta and carotid appeared to show slight increase by 12 months, this did not reach statistical significance implying that regression was occurring predominantly by reversal of ‘positive’ vessel remodelling as suggested by Corti et al. [6]. There was significant change in plaque index between 3 and 12 months in the aorta, whilst the carotid artery showed a trend that did not reach significance (P = 0.09) over the same time period. This could reflect the smaller size of the carotid artery compared to the carotid, making a small change harder to detect. Alternatively the plaques in the aorta and carotid may differ in composition, with certain elements of the plaque likely to be more susceptible to removal than others. For instance, lowering plasma LDL-C could slow the rate of lipoprotein deposition in the arterial wall, allowing reverse cholesterol transport mechanisms to predominate, culminating in net regression [26]. Different effects on individual plaque components is suggested by MRI studies of advanced carotid atherosclerosis in which patients treated with aggressive lipid lowering therapy showed a reduction in size of lipid rich areas [27,28]. This study was not designed to examine plaque composition, though initiation of study to address this question has recently been reported [29]. New developments such as lipid selective contrast agents or higher field strength (3 T) imaging with improved resolution might further establish the degree to which plaque regression involves lipid removal [30,31].
Early clinical benefits of statins have also been ascribed to anti-inflammatory effects [32]. The degree of change in coronary atheroma volume measured using intravascular ultrasound has been related to the magnitude of change in both LDL and CRP [33]. However, in the present study, the early changes in plaque size occurred in the absence of significant change in CRP. Lack of measurable change in CRP probably reflects both the relatively small sample size, and the low baseline CRP level in this stable CAD population, comparable to the post treatment CRP levels in other studies [34]. We also found no relationship between the LDL-C attained and change in plaque size, which may again reflect the sample size and a clustering of LDL-C levels in the lower range precluding observation of a quantitative effect. However, the absence of changes in biochemical parameters despite plaque regression highlights the complementary role of MRI as an imaging biomarker.
Rapid improvements in endothelial vasomotor function have previously been demonstrated in patients with atherosclerosis within weeks [12,14] or even days [13] of starting statin treatment. Thus our finding of increased flow mediated dilatation at 3 months was not surprising. By contrast, timing of statin effects on central arterial stiffness is less established. Improvements in large artery stiffness have been demonstrated after a year of statin treatment [35,36], although a shorter study of 8 weeks pravastatin treatment in patients with familial hypercholesterolemia did not show any improvement [37]. Hypertensive patients without coronary artery disease treated with high dose atorvastatin (mean LDL of <70 mg/dL) did show improvements in large artery stiffness after 3 months [38]. Our finding of rapid improvement in aortic stiffness was of similar magnitude and extends this potentially beneficial effect of statin treatment to a coronary artery disease population. We also observed that GTN mediated (endothelium independent) dilatation of the brachial artery was increased after 12 months statin treatment, as has been previously reported [39,40] and which might reflect downstream statin effects e.g. on smooth muscle cell sensitivity to nitric oxide.
We initially hypothesised that changes in arterial function might predict changes in structural parameters, but in this study we found no correlation on an individual patient basis between changes is structure and function. Endothelial function, aortic stiffness and atheroma burden all represent different aspects and stages of disease. Although at a population level all these parameters may change in a favourable direction in response to treatment, individual patients may show variable response in each according to their stage of disease. As a result, it is likely that measures of vascular structure and function will provide complementary insights into vascular disease [18].
The capability of MRI to perform non-invasive assessment of changes in the arterial wall using relatively small numbers of patients is highlighted by this and other studies [6,22]. Accurate and reproducible assessment of the vessel wall is key to detection of atherosclerosis progression or regression [41]. The rationale for this approach has been illustrated by previous carotid intima-media thickness or coronary IVUS studies [42–44]. An early appreciation of effects on both structure and function, as provided by MRI, could be used to guide selection of novel agents prior to investment in major Phase III trials [45]. MRI is an emerging technique and unlike ultrasound measures of IMT and FMD, has not yet been validated as a means to predict future events in large studies. However, recent data shows correlation of aortic plaque burden by MRI with clinical risk scores [46]. As our findings suggest MRI potentially offers additional value through the assessment of arterial structure in multiple locations and complementary measures of vascular function. Therefore, it seems increasingly likely MRI will play a key role in evaluation of new and existing therapies and may even become part of individual patient risk assessment.
4.1
Study limitations
A potential limitation of this study is the absence of a control group. However, given strong evidence of the benefits of early and intensive treatment with statins, a placebo controlled arm or even low intensity statin-treatment arm was not considered ethical. In common with several recent studies [5,6], we therefore conducted a longitudinal study of changes compared to baseline. As a result, it is not possible to exclude the possibility that the changes observed were due to an unknown factor, though in context, this is improbable. These patients were newly diagnosed with coronary artery disease, some of whom were taking vasoactive drugs such as ACE inhibitors and beta adrenoceptor blockers for hypertension prior to study enrolment. However, the increase in use of such medications between study baseline and the 3-month time point was small and not statistically significant and so the vascular changes observed over that period are not likely to be confounded by changes in these other medications. The 2–3% reductions in plaque index observed in this study at 3 months appear relatively modest when compared to decreases in lesion size of greater than 30% observed over a similar time frame in some animal studies. However, the interventions in animal models of atherosclerosis usually involve much more extreme changes in lipid levels than those achievable by statin treatment in humans. Thus, whilst statin treatment to achieve effective LDL-C reduction appears important for plaque regression, additional HDL-C based interventions in order to enhance reverse cholesterol transport may prove even more effective [26]. The patient population studied was overwhelmingly male and Caucasian therefore the findings may not be applicable across all population groups.
5
Conclusions
This study shows that in a population of statin naïve, clinically stable but otherwise unselected coronary artery disease patients, cholesterol reduction using statins to mean LDL-C of approximately 70 mg/dL was associated with rapid regression of atheroma at 3 months. Early changes were highly correlated with changes after 12 months. These rapid structural changes were accompanied by early improvements in arterial stiffness and endothelial function that were sustained to 12 months. Use of multi-modal vascular MRI to detect early changes in atheroma and vascular function in small numbers of patients could prove to be an efficient strategy to screen novel anti-atherosclerotic agents.

statin
magnetic resonance imaging
atherosclerosis
aorta
carotid


The nervous system and chronic kidney disease in children

This paper provides a review of the literature on the nervous system involvement incurred by children and adolescents with chronic kidney disease (CKD), with a particular focus on neuropsychological functioning. In addition to an historical overview of earlier literature, published studies from the past 14 years that address both central and peripheral nervous system function in children with CKD are reviewed (1990–2003). These studies span work in neuroimaging, electrophysiology, and neuropsychology. A key focus for this review is on variables that might affect neurodevelopmental status in these children. The paper concludes with suggestions for achieving progress in the understanding of this complication of kidney disease in children.
Introduction
Associations between end-stage renal disease (ESRD) and neurocognitive dysfunction have been reported for over 25 years. Between 1977 and 1986, approximately 15 publications described dialysis in infants. In these early studies, approximately 65% of infants were reported to have developmental delay and 49% encephalopathy [1]. Between 1976 and 1984, the neurocognitive effects of aluminum-induced neurotoxicity secondary to then current treatments for chronic kidney disease (CKD) were reported [2, 3, 4]. These neurocognitive effects included seizures, speech disorders, dementia, and a slow electroencephalogram (EEG) pattern [2, 3]. These findings were important for the field in that they encouraged modifications to the treatment regimen for dialysis patients and highlighted a confounding factor in the relationship between CKD and neurocognitive integrity. By 1990 the use of aluminum was generally eliminated with improved dialysis water purification techniques and avoidance of aluminum-containing medications. In the early 1990s, erythropoietin was introduced into standard practice, resulting in improved anemia management for children and adults with ESRD and a diminution of anemia-related EEG and cognitive deficits reported in the adult ESRD population [5, 6, 7].
The purpose of this paper is to review the contemporary published literature that focuses on the neurological impact of CKD in children. Papers selected for this review have been published from 1990 to the present, and were selected to coincide with the improvements in treatment, including the diminution of aluminum exposure and availability of erythropoietin that occurred circa 1990. These studies span work in neuroimaging, electrophysiology, and neuropsychology to address central nervous system (CNS) function. We also present available studies addressing the integrity of the peripheral nervous system.
Central nervous system studies
Neuroimaging
Imaging of the brain with computed tomography and magnetic resonance imaging (MRI) has been used to document the structural impact of ESRD in children. Contemporary estimates of the prevalence of cerebral atrophy are obtained from a report of 13 children with chronic renal failure from infancy. Brain atrophy was reported in 3 (23%) of these patients [8]. Publications with a focus on certain disease groups with a high risk for CNS disease, such as cystinosis, Lowe syndrome, and congenital nephrotic syndrome, estimate the prevalence of cerebral atrophy to range between 15% and 100% [9, 10, 11]. A recent study by Qvist et al. [11] described a cohort of 33 children, largely composed of children with ESRD from congenital nephrotic syndrome, tested an average of 6 years after renal transplant. MRI documented CNS infarcts or ischemic changes from both silent and clinically significant cerebrovascular events in 19 (58%). Although relevant for children with CKD, the frequency of brain structure abnormalities in these specific disorders may be greater than in a general pediatric CKD population with a variety of renal diseases.
Electrophysiology findings
Three reports of EEG findings in a cohort of children with CKD have been published since 1990. In one study focal and paroxysmal EEG abnormalities were observed in 12 (36%) of 33 children after renal transplant, with 5 (15%) receiving anticonvulsant therapy [11]. The second report of 14 patients documented unspecified EEG abnormalities in 6 (42%) [8]. This study also documented a normal auditory brain stem evoked response in 12 of 12 children tested [8]. Hurkx et al. [12] evaluated 22 children with chronic renal insufficiency (CRI) and dialysis dependence using somatosensory evoked potential of the right median nerve. They reported an increased interpeak latency (N13–N20) suggesting a delayed thalamocortical conduction. No differences in CRI versus dialysis subgroups were found. Investigations of adults with ESRD demonstrate that these tools are sensitive to kidney disease-related effects and may prove useful in future pediatric studies [5, 6, 13].
Neuropsychology findings
Table 1 provides a brief description of 11 published studies that have been conducted over the past 15 years, with follow-up discussion on each of the studies within the broad domains of cognition listed below. These studies have employed samples ranging from 9 to 62 infants, children, and adolescents with CKD. The subjects experienced different types of treatment for their CKD (e.g., hemodialysis, peritoneal dialysis, kidney transplant, conservative). The research design varied (e.g., longitudinal, pre-post transplant, control groups) and different kinds of assessment methodology were used (e.g., infant development batteries, IQ batteries, specific measures of cognition, experimental tasks). In this regard, drawing specific conclusions for neuropsychological findings will be tentative, at best, but should provide guidance to clinicians and researchers as to how the field should move forward within this assessment domain.Table 1 Cognitive functioning in children and adolescents with end-stage renal disease (ESRD), 1990–2003 (CKD chronic kidney disease, SD standard deviation, CAPD continuous ambulatory peritoneal dialysis)Author/dateSample featuresComparison groupFindingsDavis et al. 1990 [28]n=37Pre-post transplant designMental development20 Dialysis at pre-transplant evaluation Improved from pre- (mean=77.0, range 50–116) to post-transplant (mean=91.4, range 50–117)17 Conservative at pre-transplant evaluationMean age at transplant=17.6 monthsMotor development Improved from pre- (mean=68.7, range 50–86) to post-transplant (mean=85.6, range 65–109)Cognition No change from pre- (mean=92.0) to post-transplant (mean=90.4)Overall development worse with early onset of ESRDFennell et al. 1990 [22]n=56n=56 healthy childrenCognitionMean age=13.6 yearsTesting at 6-month intervals Decreased verbal ability in CKDMean age of CKD onset=6.05 yearsVisual motor skills Decreased in CKDModality:Memory and learning Hemodialysis (7) Decreased in CKD and loss of function over time Peritoneal dialysis (12)Attention Kidney transplant (10) No differences between CKD and controls Conservative (27)Lawry et al. 1994 [18]n=24Transplant compared with dialysis plus conservativeCognitionModality: IQ: no differences between dialysis plus conservative (mean=92.91, SD=16.86) vs. transplant (mean=103.00, SD=11.97) Dialysis (9) Conservative (2) Kidney transplant (13) Correlation between age at onset of illness and IQ (r=0.49)Achievement Dialysis plus conservative less than transplant in maths (P=0.020), reading (P=0.028), and written language (P=0.028)Elzouki et al. 1994 [8]N=15NoneDevelopmental screeningModality: 3 of 15 with developmental delay Dialysis (6) Transplant (3) Conservative (6)Hulstijn-Dirkmaat et al. 1995 [14]n=31NoneDevelopmental indexMean age=2.5 yearsTesting every 6 months Conservative (mean=90.3, SD=14.3) greater than ESRD (mean=67.6, SD=17.3)Modality: Dialysis (16) Conservative (15)Verbal, perceptual performance, and quantitative scales No change over timeMendley and Zelko 1999 [19]n=9Pre-post transplant designCognition (baseline only) Full-scale IQ mean=91.6Mean age at pre-transplant testing=14.2 years Verbal IQ mean=91.4 Performance IQ mean=95.1Mean age at post-transplant testing=15.8 yearsAttention Improvement in sustained attention 1-year post-transplant (P=0.039)Pre-transplant modality:Executive functioning Peritoneal dialysis (5) Improvement in mental processing speed 1 year post transplant (P=0.008) Hemodialysis (3) Conservative (1)Mean age of onset of ESRD=11.9 yearsMemoryMean duration of ESRD prior to transplant=2.5 years Improvement in working memory 1 year post transplant (P=0.016)Warady et al. 1999 [16]n=28 infantsLongitudinal designDevelopment, generalModality: 6 of 28 children below the average range at 1 year of age CAPD at ≤3 months of ageCognitionTransplant at mean age=2.1±0.8 years Verbal IQ: 5 of 18 children below the average range at ≥4 years of ageMean age at follow-up=7.8±2.8 years (range 2.5–12.0 years) Non-verbal IQ: 8 of 18 children below the average range at ≥4 years of age 1 child within impaired range on both verbal and non-verbal IQLedermann et al. 2000 [15], Madden et al. 2003 [17]n=16 infants with ESRDLongitudinal designDevelopment, generalMean age at start of dialysis=0.38 years (range 0.02–1 year) 2 of 8 school-aged children had general delaysDuration of dialysis=17.3 months (range=1–59 months) 2 of 8 children <5 years of age had general delaysMean age at assessment=5.84 (range 1.58–12.00 years)Cognition IQ: 67% in average range, and 20% in low-average range (mean=86.5, range 50–102) Lower IQ scores for children with co-morbid diagnoses (mean=67.0) than for those with ESRD alone (mean=94.2)Attention 7 of 14 children with hyperactivity problemsSocial-behavioral 6 of 14 children displaying conduct problemsBrouhard et al. 2000 [21]n=62n=62 siblingsCognitionMean age: 13.8±0.4 years IQ: ESRD less than siblingsModality: 26 dialysis, 36 transplant No difference in dialysis vs. transplantAcademic achievement ESRD less than controls for all measures of spelling, reading, and mathematics No difference dialysis vs. transplantCorrelation between age of diagnosis and academic achievementCorrelation between parental education and academic achievementQvist et al. 2002 [11]n=33 transplant recipientsNoneCognitionMean age at assessment=8 years (range=7–12 years) Verbal IQ: mean=87.5 Non-verbal IQ: mean=87.5IQ range Low: 3/33 Low-average: 14/33 Average: 14/33 Above average: 2/33Neuropsychological battery No overall group deficits with attention, language, memory, or visuospatial abilities when compared with normative populationNeuropsychological deficits Attention: 8 of 33 children Language: 2 of 33 children Memory: 6 of 33 children Visuospatial: 8 of 33 childrenMotor function Hemiplegia: 3 of 33 children Bilateral infarction: 1 of 33 Cerebral palsy: 1 of 33Auditory function 2 of 33 children with moderate sensorineural hearing loss
General neurocognitive function
In infants and toddlers neurodevelopmental testing is conducted and analyzed in broad categories of overall development, mental development, and motor development. Hulstijn-Dirkmaat et al. [14] compared the general development in 15 toddlers receiving conservative therapy (CRI) with 16 dialysis-dependent children. The children with CRI had a better developmental index compared with the dialysis-dependent children (mean±standard deviation 90.3±14.3 vs. 67.6±17.3) [14]. Ledermann et al. [15] evaluated the long-term outcome of infants requiring peritoneal dialysis. In this study, 2 of 8 (25%) young children demonstrated developmental delay. Warady et al. [16] evaluated 28 infants at 1 year of age, all of whom were dialysis dependent. Of these 28 infants, 6 (21%) scored in the low-average or impaired range of general development. Based on these studies, approximately 20%–25% of children less than 5 years of age with CKD might be expected to show general developmental delays [8, 15, 16].
For older children, IQ is the typical measure of general cognitive function. In the sample of children studied with CKD, the distribution of IQ scores is shifted downward compared with the normal population, with low-average (IQ 80–89) and average (IQ 90–109) range scores predominating [11, 14, 15, 16, 17, 18, 19, 20]. In a study of 62 children with ESRD, dialysis and transplant combined, Brouhard et al. [21] described a significantly lower IQ in the children with kidney disease compared with their sibling controls. In 19 children with a mean age of 6.6±1.3 years who had ESRD from infancy, Warady et al. [16] reported a relatively intact IQ, with 15 of 19 (79%) in the average range. In this group, 13 of 18 (72%) achieved average verbal IQ scores, while only 10 (56%) scored in the average range in the nonverbal subtest [16]. When comparing transplanted patients with dialysis-dependent patients, variable results have been reported. Lawry et al. [18] compared 13 transplanted children with 11 dialysis-dependent children in a cross-sectional study and found a higher mean IQ in the transplant group (103.0±11.97 vs. 92.9±16.86). Conversely, Brouhard et al. [21] compared 36 transplant and 26 dialysis-dependent children in their cross-sectional study and found no significant difference between these patient groups. In aggregate it appears that general cognitive function is impaired in children with CKD, but the published literature is inadequate to fully characterize this effect with respect to CKD modality and differences between verbal and non-verbal IQ [11, 16, 19, 22].
Attention and executive function
The findings in the pediatric CKD literature are mixed with respect to attention and executive function [11, 19, 22]. Fennell et al. [22] reported no differences in measures of sustained attention between children with all treatment modalities of CKD combined and matched controls. Conversely, Mendley and Zelko [19] documented longitudinal improvements in sustained attention (pre-transplant 2.19±1.29 vs. post-transplant 2.95±1.33, P=0.04) and mental processing speed (pre-transplant 2.28±0.72 vs. post-transplant 1.64±0.31 s, P=0.008) 1 year after transplant in 9 children. After renal transplant, Qvist et al. [11] reported no overall group deficits of attention in 33 children compared with the normative population [standard deviation score (SDS)=−0.2±0.4], although 24% of the sample showed generalized attention deficits.
Language
The prevalence of hearing loss among children with CKD is approximately 18%, and unrecognized or delayed diagnosis of hearing impairment may impede language development [11, 23]. Children with known hearing loss tend to be excluded from studies of cognitive function, and so their challenges are not likely to be fully reflected in the published literature [17]. Fennell et al. [22] documented deficits with verbal abstracting abilities in their matched control design for children with CKD. Qvist et al. [11] documented no overall group deficits of language compared with the normative population (SDS=−0.2±0.4), with only 6% of children showing evidence of generalized language deficits. More research needs to be conducted before determining whether language abilities will prove to be an area of concern for children with CKD, particularly with respect to potential delays secondary to hearing impairment.
Visuospatial abilities
Compared with a matched control population, early studies by Fennell et al. [22] documented deficits in visual-motor abilities in a cohort of 56 children with CKD. Qvist et al. [11] documented no overall group deficits with visuospatial abilities in 33 transplant recipients when compared with the normative population (SDS=−0.5±0.5); however, 24% of the cases did show generalized visuospatial deficits.
Memory
In a heterogeneous sample of children with CKD, Fennell et al. [22] reported lower memory scores for children with CKD compared with controls. In addition, a general loss of memory function over time was observed in 26 children with all treatment modalities of CKD over a 12-month testing period [24]. After kidney transplantation, Qvist et al. [11] reported no overall group deficits with memory compared with the normative population (SDS=−0.4±0.5), although 20% of the cases displayed generalized memory deficits. Conversely, Mendley and Zelko [19] documented longitudinal improvements in working memory in 9 children 1 year after transplant compared with the pre-transplant evaluation.
Academic achievement
Academic achievement in children with CKD is vulnerable to deficits in each cognitive domain noted above as well as an increased frequency of school absences. In a study by Lawry et al. [18], the combined dialysis and CRI group (n=11) with a mean chronological age of 14.9±3.3 years achieved at an age equivalent of 13.8±5.8 in mathematics, 15.6±7.7 in reading, and 11.2±7.2 in language. In comparison, the transplant group (n=13) with a mean chronological age of 13.8±3.2 years, achieved at an age equivalent of 16.2±7.8 in mathematics, 16.4±7.7 in reading, and 16.2±9.1 in language. This paper suggested that transplant recipients may have superior academic achievement compared with dialysis-dependent children [18]. Conversely, Brouhard et al. [21] compared the academic achievement of dialysis and transplant patients and normal sibling controls. They reported no difference in academic achievement between dialysis and transplant groups. However, the combined ESRD group of dialysis and transplant achieved below their sibling controls in spelling, arithmetic, and reading [21]. Observational studies of academic placement further indicate that regular education (with or without remedial tutoring) is used for 79%–94% of children with CKD, while 13%–15% receive special education services not related to hearing or visual impairments [11, 16]. This compares with national data in which approximately 10%–15% of students receive special education services [25]. Given the observed frequency of abnormalities across multiple areas of cognitive function, the frequency of special education services used by children with CKD appears low.
Peripheral nervous system
Peripheral neuropathy including diabetic neuropathy and dialysis-related amyloidosis has been reported in adults with ESRD [26, 27]. The frequency of peripheral neuropathy in children with CKD can only be grossly estimated based on the following study. Elzouki et al. [8] assessed nerve conduction velocity in 11 children with CKD and found 1 (9%) had diminished motor nerve conduction. Electromyograms performed in 9 of these patients revealed increased polyphagia of motor unit potentials in 4 (44%) [8]. At present, we are unable to find additional published studies examining peripheral neuropathies in children with CKD.
Key variables affecting neurodevelopmental outcomes
CKD is a complex disorder and, similar to other pediatric disorders, there are undoubtedly a variety of variables that contribute to the neurodevelopmental status of this population. Key variables presented here are postulates for the most part as the studies conducted since 1990 have not been adequately powered to assess multiple influential factors.
The duration of CKD and the age at onset of CKD may affect cognition. Two separate studies have reported a poorer developmental prognosis for children with earlier-onset ESRD [18, 28]. It has been postulated that this may be the result of insults to the CNS at particularly vulnerable periods in development or the result of continuous insults over the majority of postnatal life among some children with long-standing kidney disease. The congenital disorders presenting with kidney disease early in life may influence the perceived relationship between duration of CKD and cognition, as these disorders may have other associated CNS abnormalities, such as pulmonary hypoplasia and hypoxia associated with obstructive uropathies, or structural anomalies with Joubert syndrome and Smith-Lemli-Opitz syndrome [29]. Hulstijn-Dirkmaat et al. [14] identified the presence of co-morbid conditions as a risk factor for cognitive impairment based on mean IQ scored (CKD alone 86.7 vs. CKD with co-morbidity 61.4, P=0.001). Conversely, Fennell et al. [24] found no correlation between age at onset of ESRD and cognition. The available data suggest that the duration of CKD and the age at onset of CKD negatively impact on cognitive functioning, but it remains unclear how these variables contribute to the type and severity of cognitive dysfunction.
Having reached ESRD, the modality of renal replacement therapy may have an impact on neurodevelopment. Several studies have examined small groups of children either before and after renal transplant or as a cross-sectional study comparing renal replacement therapy modality groups [30, 31]. Fennell et al. [24] compared 10 transplant, 7 hemodialysis, and 12 peritoneal dialysis patients. Patients in both the transplant and peritoneal dialysis groups tended to have better attention and memory skills when compared with the performance of hemodialysis patients. Additional investigation is warranted to determine the differential effect of hemodialysis and peritoneal dialysis among children with ESRD with current dialysis adequacy standards [32, 33, 34]. From previous studies, a hypothesis can be formulated that motor skills, memory, attention, and mental processing may improve after renal transplantation [11, 16, 21]. Although existing literature suggests that a successful renal transplant may diminish the adverse developmental effects of ESRD, these same studies document the type and frequency of residual deficits in pediatric renal transplant recipients to be greater than in the normative American population [11, 16, 21].
Complications of CKD such as anemia, hypertension, and malnutrition likely affect neurodevelopment. Anemia has been shown to slow the cognitive event related potential in adults with CKD (mean hematocrit=23.7%) and impair cognitive function among otherwise healthy children aged 6–11 years with hemoglobin levels less than 11.8 g/dl [6, 35]. The optimal hemoglobin for cognitive function in children with CKD remains to be determined. The effects of hypertension on cognition may be related to the degree of blood pressure elevation, brain injury consequent to hypertensive or hypotensive episodes, and side effects of antihypertensive therapy. A recent study by Lande et al. [36] analyzed National Health and Nutrition Examination Survey III (NHANES III) data comparing 217 children with systolic blood pressure greater than or equal to the 90th percentile for age and gender with 4,860 normotensive children. The NHANES III study used subtests of the Wechsler Intelligence Scale for Children and reading and arithmetic subtests of a standardized academic achievement test. In this study, results suggested that hypertension was associated with lower scores in subtests representing memory, attention, and arithmetic. Furthermore, malnutrition in young infants without kidney or other chronic disease has been linked to impaired brain growth and developmental delay [37, 38, 39]. Significant effort has been applied to the nutritional support of children with CKD. However, in national registries of children with CKD, the mean weight z-score is 1.0–1.4 standard deviations below the American age-matched mean, and mean height z-scores are approximately 1.6 standard deviations below the American age-matched mean [40]. Anemia, hypertension, and malnutrition are likely key factors contributing to the cognitive deficits of children with CKD.
Children with CKD are at risk for additional disorders that may accompany congenital kidney disease. Prematurity occurs with a greater frequency in children with congenital kidney disease and is associated with a greater frequency of CNS injuries and later developmental deficits [11, 41]. Socioeconomic status and parental education have a known impact on academic achievement among all children [42]. Although not unique to children with CKD, the identification of poor socioeconomic status or limited parental education may trigger additional educational evaluation and or services for children with CKD in these families. School absences are increased in children with CKD due to the need for outpatient medical visits, hospitalizations, hemodialysis, and acute illness [20]. Finally, the CKD population is at excess risk for sensory deficits, including congenital and acquired hearing impairment (9%-18%) and visual impairment, although the latter has not been examined thoroughly [11, 43]. All of these factors may contribute to the neurodevelopmental dysfunction of children with kidney disease.
Conclusions
Emergent findings appear to apply to three broad groups of children and adolescents: mild-to-moderate CKD, dialysis-dependent children, and transplant-dependent children. What is known about children with mild-to-moderate CKD is severely limited, with no focused studies addressing their neurodevelopmental needs. More is known about the pediatric dialysis population, with deficits in the areas of attention, language, visual-spatial abilities, and memory. However, the differentiation of problems that may relate to disease-specific variables, such as age at onset of kidney failure, anemia, and hypertension, remain relatively unknown and modestly examined at best. For the transplant-dependent group, cognitive deficits appear to persist, supporting the conclusion that the transplant does not result in complete neurocognitive “recovery.”
The low frequency of peripheral neuropathy in children with CKD observed clinically and reported in a single study may be attributed to the low prevalence of diseases with microvascular complications such as diabetes mellitus, advanced atherosclerosis, and dialysis-related amyloidosis [8]. This pediatric advantage may be lost as patients survive into young adulthood. It is unlikely that purely pediatric follow-up studies will be able to characterize this potential late-term complication.
Given the expected long-term patient survival of children with CKD, established guidelines for the provision of renal replacement therapy for children, and the data provided by studies among children with CKD, we believe that an organized, adequately powered approach toward the characterization of the neurodevelopmental impact of CKD is warranted [32, 33, 34, 44]. Ideally, this study or set of studies will include measures of neuroimaging, electrophysiology, and neuropsychology. Future research in this area should characterize potential risk factors, such as age at onset of disease, anemia management, and hypertension to advance our understanding of the magnitude of cognitive dysfunction and aid the identification of modifiable mediators of CNS deficits in children with CKD.

neurodevelopment
cognition
renal failure