Motor-Skill Learning in Alzheimer’s Disease: A Review with an Eye to the Clinical Practice

Since elderly people suffering from dementia want to go on living independently for as long as possible, they need to be able to maintain familiar and learn new practical skills. Although explicit or declarative learning methods are mostly used to train new skills, it is hypothesized that implicit or procedural techniques may be more effective in this population. The present review discusses 23 experimental studies on implicit motor-skill learning in patients with Alzheimer’s disease (AD). All studies found intact implicit motor-learning capacities. Subsequently, it is elaborated how these intact learning abilities can be exploited in the patients’ rehabilitation with respect to the variables ‘practice’ and ‘feedback.’ Recommendations for future research are provided, and it is concluded that if training programs are adjusted to specific needs and abilities, older people with AD are well able to (re)learn practical motor skills, which may enhance their autonomy.
Introduction
The aging population is growing rapidly and by 2050 the number of elderly people aged 85 years old or older in Europe and North America is estimated to be approximately 19 million (Román 2002). Since age is a high risk factor for dementia (Smith and Rush 2006), the expansion of the aging population and thus the number of people suffering from dementia has momentous consequences for national care systems as well as a large economic impact (Schölzer-Dorenbosch 2005). A way to contain these costly effects is by helping older people to stay independent for as long as possible, implying that the elderly must not stop learning. They apply old skills differently or acquire new skills, like learning how to use a walking aid, which gives rise to the following questions: Are demented elderly people able to learn such new skills? And are we, health professionals, able to train them?
As yet, psychopharmacological interventions, such as the use of cholinesterase inhibitors, may have some benefit in maintaining autonomy of elderly patients suffering from Alzheimer’s disease (AD), as demonstrated by delayed nursing home placement (Becker et al. 2006). Recent pharmacological research also shows promising results for cognition: the treatments evaluated produced a moderate positive effect on global cognitive functioning (Grimley Evans et al. 2004; Schölzer-Dorenbosch 2005; Takeda et al. 2006). In their 2002 review on the placebo-controlled effects of rivastigmine on the cognition of AD patients, Birks et al. (2002) reported statistically-significant increases of 0.8 points on the Mini Mental State (MMS) Examination and 2.1 points on the Alzheimer’s Disease Assessment Scale (ADAS-Cog). They also found benefits in the patients’ activities of daily living, although the difference with placebo was not significant. Takeda et al. (2006) also reported reductions in the cognitive impairment of AD patients for donepezil and galantamine, but again, although showing potential, the improvements did not suggest a major difference in the daily lives of the patients.
These findings do not negate the importance of non-pharmacological approaches and it is possible that the interactions between medication and non-pharmacological approaches may be the most beneficial in maintaining patient’s autonomy. In their review, Luijpen et al. (2003) conclude that the effect of non-pharmacological interventions with respect to cognition and affective behavior in dementia is similar to the effect of pharmacological regimens. Non-pharmacological treatments to improve autonomy in this patient population should hence be considered an additional option, especially since rehabilitation is increasingly being advocated as a means to optimize patients’ overall functioning (De Vreese et al. 2001). Clare (2003) also concludes that neuropsychological rehabilitation applied in the context of progressive disorders like dementia do yield beneficial results.
In the present review we will focus on the ability of Alzheimer’s patients to (re)learn practical motor skills. Currently, explicit or declarative learning methods are the starting points in most rehabilitation programs aimed at motor-skill learning in the cognitively unimpaired population (Van Cranenburg 2004). However, Zanetti et al. (2001) and Rösler et al. (2002) claimed that patients with dementia will profit more from implicit or procedural learning methods by showing that their AD cohorts were able to learn to waltz or to use a telephone when an implicit rather than an explicit learning approach was used. In implicit learning, skills are mastered without awareness, often simply by repeated exposure, and can be unconsciously revived from implicit memory (Buchner and Wippich 1998). The abovementioned studies were all focused on finding the best way to help older people with dementia learn or relearn practical (motor) skills, and although the results are encouraging, the patient samples were always small and it remains unclear how much was learned due to a lack of well-defined performance measures.
In the first part of our review we looked for corroborating evidence in experimental research for intact implicit motor-learning capacity in cohorts of elderly patients diagnosed with AD. In the second part we will elaborate on how these intact learning abilities can be utilized for their rehabilitation while taking the principles from theories of motor-learning into account. Two of the theories’ core variables, i.e., practice and feedback (Schmidt and Wrisberg 2000) will be discussed more extensively, also in the light of research exploring these variables in AD. The results will be translated into practical instructions for more targeted rehabilitation training programs for this patient group.
Materials and Methods
Computerized searches of the literature using the databases of PubMed and PsycLIT were conducted spanning a 20-year period, from 1985 up to and including 2005. The search terms (any field) used were procedural learning, sequence learning, motor-skill learning, or motor learning in combination with Alzheimer’s disease. Only reports published in English were considered. For inclusion in this review the studies had to meet the following criteria: (a) a clinical diagnosis of Alzheimer’s disease based on specified and generally accepted criteria; (b) a procedural task with motor responses; and (c) task performance expressed in time or error measures, and not only in fMRI or other imaging data. Ultimately, 23 studies were included in this review. Three studies will only be discussed in the second part of the review since they explicitly examined the role of feedback and type of practice.
Experimental Research of Implicit Motor-Skill Learning in Alzheimer’s Disease
Implicit Learning Ability
The main results of the studies generated by our search of the literature are shown in Table 1. Four studies using a Maze test in which blindfolded participants had to trace a complex pathway found that the AD patients were able to learn new motor-skills implicitly (Kuzis et al. 1999; Sabe et al. 1995; Starkstein et al. 1997; Taylor 1998). The nine studies that applied a Rotor-Pursuit task, in which participants had to maintain contact between a hand-held stylus and a rotating spot, also reported preserved learning abilities in their AD samples (Beatty et al. 1995; Deweer et al. 1994; Dick et al. 1995, 2001; Heindel et al. 1988; Heindel et al. 1989; Jacobs et al. 1999; Libon et al. 1998; Willingham et al. 1997). This is in agreement with the findings of Poe and Seifert (1997) based on a Puzzle-Assembly task and the results of Rouleau et al. (2002) involving a Mirror-Tracing task. Also, a Serial Reaction-Time Task (SRTT) was used in which participants needed to respond as fast as possible when a stimulus appeared in one of four places by pressing a corresponding response key (Grafman et al. 1990; Knopman and Nissen 1987; Knopman 1991; Willingham et al. 1997). Again, the AD patients showed implicit learning as reflected by the difference in reaction times (RTs) between blocks with a fixed sequence of stimuli presentation (decreasing RTs) and a random block (prolonged RTs). However, there are indications that the implicit learning ability in AD patients is affected because they generated inferior outcomes when accuracy was taken into account (Willingham et al. 1997) or when the data were log-transformed because of the unequal variance in RT (Knopman 1991). Ferraro et al. (1993) found preserved implicit SRTT learning only in the “very mildly demented” group, and less in the “mildly demented” group although it is relevant to mention that none of the other studies used such a subtle severity classification.
Table 1Summary of results of experimental studies on motor-skill learning in Alzheimer’s diseaseAuthorYearSample size and typesTask(s)Amount of learningaResults on learning capacitySabe, L., et al.199520 AD with co-morbid depression, 35 AD without co-morbid depression, 14 depressive, non-demented patients, 16 healthy controlsMaze testAD: 19%Co: 22%The AD patients showed significant deficits in declarative learning but only a minor (although statistically significantly) drop in procedural learning. The AD group with comorbid depression showed a similar learning pattern as the non-depressed AD group.GroupxTrial:p < 0.05Starkstein, S.E., et al.199755 AD (13 with mild, 12 with severe and 30 without anosognosia)Maze testAD no: 48%There was no group difference in declarative learning. As to procedural learning, the patients with severe anosognosia showed a significantly poorer performance whereas the patients with mild or no anosognosia showed no deficits.AD mild: 39%AD severe: −16%–Taylor, R.199858 AD, 58 multi-infarct dementiaMaze test–When age and overall neuropsychological functioning were taken into account, Maze performance was better in the AD patients than in the patients with multi-infarct dementia–Kuzis, G., et al.199915 AD, 15 PD, 10 PD and dementia, 24 healthy controlsMaze testAD: 10%The AD group showed deficits on all measures of explicit memory. There were no significant between-group differences in the measures of implicit memory between the AD, control, and PD groups.Co: 39%–Heidel, W.C., et al.198810 AD, 10 HD, 4 amnestic 20 healthy controlsRotor PursuitAD: 147%The AD patients showed preserved motor-skill learning while the patients with HD showed no motor learning.Co: 115%GroupxTrial: n.s.Heindel, W.C. et al.198916 AD, 13 HD, 17 PD, 22 healthy controlsRotor PursuitAD: 101%The AD patients showed preserved motor-skill learning while the patients with HD showed impaired motor learning.Co: 118%GroupxTrial: n.s.Beatty, W.W., et al.19954 AD, 1 corticbasal degenerationRotor Pursuit–The AD patients showed preserved motor skill learning–Deweer, B., et al.199413 AD institutionalized, 10 healthy controls, 17 AD out patients, 9 healthy controlsRotor PursuitAD in.:86%Explicit memory was severely impaired in the AD patients but they showed normal procedural learning.Co: 48%GroupxTrial: n.s.AD out: 161%Co: 139%GroupxTrial: n.s.Dick, M.B., et al.199512 AD, 12 healthy controlsRotor PursuitAD: 47%Performance significantly improved during the first 40 trials but additional practice provided no further beneficial effects. The AD patients showed minimal retention problems across four retention tests.Co: 81%GroupxTrial: n.s.Libon, D.J., et al.199816 AD, 14 vascular dementiaRotor PursuitAD: 60%The AD patients obtained a lower score on a verbal-learning task-recognition index and high scores on the Rotor Pursuit.–Jacobs, D.H., et al.199912 AD, 12 healthy controlsRotor PursuitAD: 124%The AD patients and the controls were able to learn the motor task.Co: 106%GroupxTrial: p = 0.473Dick, M.B., et al.200118 AD, 18 healthy controlsRotor PursuitAD: 27%In normal-vision trials no differences in learning between the AD patients and the controls were found.Co: 36%GroupxTrial: n.s.Dick, M.B., et al.200399 AD, 100 healthy controlsRotor PursuitThe AD patients and controls receiving constant practice outperformed those in the blocked and random conditions. The AD patients only benefited from constant practice.Poe, M.K. et al.19979 AD, 14 healthy controlsPuzzle Assembly–Even when the subjects had no explicit memory of practicing the task, they all demonstrated savings upon relearning. GroupxTrial: n.s.Rouleau, I., et al.200212 AD, 12 healthy controlsMirror TracingAD: 44%Those AD patients that were able to perform the basic mirror-tracing task did not differ from the controls in level of performance, learning over trails, retention over a delay interval and generalization to other tasksCo: 49%GroupxTrial: n.s.Knopman, D.S., et al.198735 AD, 13 healthy controlsSRTTAD: 22%The AD patients showed learning of the repeated sequence, although they responded more slowly.Co: 38%GroupxTrial: n.s.Graftman, J., et al.199042 AD, 7 PSP, 44 healthy controlsSRTTAD: 36%The AD patients and controls showed motor-skill learning while the PSP patients did not.–Knopman, D.199116 AD, 17 healthy controlsSRTTAD: 37%The AD patients showed learning of the sequence but they showed an inferior level of learning when the data were log-transformed.Co: 33%Ferraro, F.R., et al.199327 very mild AD, 15 mild AD, 17 PD, 26 healthy controlsSRTTAD mild: 11%The very mildly AD patients showed preserved learning comparable with the controls. The mildly AD patients and PD patients showed less implicit learning.AD very mild:22%Co: 20%Willingham, D.B., et al.199720 AD, 20 healthy controlsSRTT, Incompatible SRTT, Pursuit Tracking (randomized and repetitive pattern)SRTT:The dementia ratings predicted the ability to perform tasks but not the ability to learn them. AD patients can have a performance deficit but they have no general deficit in motor-skill learning.AD: 52%Co: 60%GroupXTrial: p > 0.2Pursuit:AD: 12%Co: 17%GroupxTrial: p > 0.2Hirono, N., et al.199736 AD, 19 healthy controlsBi-manual coordinated Tracing taskAD: 37%Skill learning in those AD patients that completed the tasks was as good as in the controls. Co: 39%GroupxTrial: p = 0.193Dick, M.B., et al.199623 AD, 22 healthy controlsTossingThe AD patients given constant practice were able to learn and retain the tossing task similarly well as the controls. The AD patients showed less improvement when practicing at various distances.Dick, M.B., et al.200658 AD, 58 healthy controlsTossingThe AD patients showed significant improvements under constant practice only. None of the practice conditions facilitated intermediate transfer in the AD patients whereas constant practice did benefit them on tests assessing near transfer.AD= Alzheimer’s disease; HD= Huntington’s disease; PD= Parkinson’s disease; PSP=  Progressive supranuclear palsy.aexpressed as a percentage of the difference score between the last and first trial with respect to the score on the first trial. The GroupxTrial interaction for the AD and Co group is also reported when available.
Thus, irrespective of the task used, the studies assessing implicit motor-skill learning in AD we reviewed yielded positive outcomes. Indeed, in their 1997 study, Hirono and colleagues found that patients with mild AD were able to acquire motor and perceptual as well as cognitive skills in various procedural learning tasks.
It should be noted that in all studies the patients that could not perform the task were eliminated from the analyses. Yet, a failure to perform the prescribed task need not necessarily be related to learning problems. Willingham et al. (1997) attributed the phenomenon to other causes like the complexity of the instructions given or the type of skill to be performed. These factors may differ across tasks, which might explain their finding that the ability to complete one task did not predict the rate of improvement in another task. They conclude that AD patients have a performance deficit and not a generalized deficit in motor learning.
Performance and Amount of Learning
From the above discussion of results we can presume that at least a subgroup of AD patients show preserved implicit learning abilities, but to what extent? Here, two aspects in motor learning should be differentiated, i.e., overall performance level and amount of learning (e.g., the increment in Total Time on Target in the Rotor-Pursuit task). All the studies found preserved motor-skill learning in AD patients although their overall performance levels in terms of reaction and movement time were always inferior to those of the controls. However, when we take the level of learning into account, the results are less consistent. Some of the results were not reported with enough detail to show unambiguously the amount of learning the AD patients showed compared to the controls (Poe and Seifert 1997). Some comparative studies did not include a healthy control group in addition to the patient groups (Beatty et al. 1995; Grafman et al. 1990; Libon et al. 1998; Starkstein et al. 1997; Taylor 1998), preventing patient-control comparisons from being made. The AD patients in the SRTT studies showed the same amount of learning (decrease in RT during the blocks with the fixed sequence) as the normal controls (Ferraro et al. 1993; Knopman and Nissen 1987; Knopman 1991; Willingham et al. 1997). In the nine studies that used a Rotor-Pursuit or Tracking task there were also no patient-control differences in amount of learning (Deweer et al. 1994; Dick et al. 1995, 2001; Heindel et al. 1988; Heindel et al. 1989; Hirono et al. 1997; Jacobs et al. 1999; Rouleau et al. 2002; Willingham et al. 1997). Two of the studies using a Maze test reported learning abilities in the AD group but less improvement across trials compared to the controls, (Kuzis et al. 1999; Sabe et al. 1995) findings which are perhaps explainable by the use of a task without visual feedback.
Taken together, the reviewed studies all showed preserved implicit motor-skill learning in AD patients regardless of the task used. Their performance levels, however, never reached the levels of the healthy controls, demonstrated by their prolonged reaction and movement times. The AD patients’ level of learning also differed depending on the task to be performed. Visual feedback appears to have a positive effect on their learning pace. They also seem to experience more problems with implicit learning when performing the SRTT, a task that involves two learning processes. The subjects have to master both spatial and motor regularities (Mayr 1996), and it is the learning of spatial regularities that may be compromised in AD patients, a process that is less implicated in the Rotor-Pursuit task in which normal implicit learning for the patients was found.
Training Patients with Alzheimer’s Disease: Variables in Motor Learning
The studies discussed provide evidence that AD patients can learn new motor skills in an implicit way. It is therefore worthwhile to establish what would be the best way to train them. In the next section we will give a brief account of the two variables practice and feedback that play a role in (re)training motor skills. We will subsequently discuss the variables in relation to the findings reported in the relevant AD studies and conclude by making recommendations of how to enhance the acquisition of new motor skills in this population.
We will first, however, briefly address the existing views on the presence or absence of distinguishable learning stages in explicit and implicit learning. Generally, with explicit learning people tend to pass through three stages in the acquisition of motor skills (Fitts and Posner 1967). The first is the cognitive stage in which the focus is on understanding the task and developing strategies to perform it, requiring cognitive activity such as attention and executive functions. The second phase is the associative stage: the learner has selected the best strategy and now begins to refine the skill. Here, cognitive aspects are less important. And finally, there is the autonomous stage in which the skill becomes automatic, requiring a low degree of attention. Variables such as practice and feedback can be structured differently to enhance learning at each stage. Feedback in the cognitive stage, for example, may need to be more specific and applied more frequently to enhance learning, while feedback may be weaned toward the third stage of learning (Tse and Spaulding 1998).
In implicit learning, on the other hand, there is no clear distinction between these three stages. It has been proposed that in implicit learning the three stages might overlap or be ordered differently. There is support for a parallel development of implicit and explicit knowledge in learning (Willingham and Goedert-Eschmann 1999).
Practice: Theory and Outcome Studies with AD Patients
The principle, “The more you practice, the more you learn,” implies that the amount of practice should be maximized in therapy. But does more practice indeed improve the performance in AD patients? Dick et al. (1995) found that on the Rotor Pursuit both the AD and control group had reached their optimal performance after 40 trials because subsequent practice failed to yield any additional augmenting effect. It would be interesting to determine whether this also holds for other tasks like the Maze test in which, relative to the controls, an inferior amount of learning was observed for AD patients (Kuzis et al. 1999; Sabe et al. 1995).
Since fatigue also plays a role in learning, the next question is how to alternate practice with rest to maximize learning in patients. Schmidt and Wrisberg (2000) distinguish two types of practice. In ‘massed practice,’ the greater proportion of the sessions is dedicated to training, while in ‘distributed practice’ the duration of rest equals or is greater than that of practice. To date, the effects of alternating these two training methods in the generally older AD patient group still requires further investigation.
Another factor that merits closer attention in the context of training programs for AD patients is whether the task should be learned as a whole or per constituent component. Training the components of a task separately before combining them into the whole pattern can be effective if the task itself can be naturally divided into components that reflect the inherent goal of the task (Schmidt 1988). For example, learning to drive a car can be easily divided into the components “learning to shift gear” and “learning to steer,” which can be trained individually. Learning to reach and grasp an item, on the other hand, does not lend itself well for phased training since reaching and grasping are integral components of a single, continuous movement.
The amount of variation in the practice session(s) is also a topic for further study. Task variables like the beanbag’s weight and throwing distance in the Tossing task can be practiced in a random design so that the weight and distance can be varied systematically. Alternatively, they can be offered in a blocked design in which only one task variable per block is practiced repetitively. Another option is to use a constant design in which only one combination of task variables is trained. Note that over time, the connotation of the two terms has shifted: random and blocked practice now refer to the rehearsal of several distinct skills whereas varied and constant practice implies the rehearsal of different variations of the same skill (Schmidt and Wrisberg 2000). Nevertheless, in our report we will use the ‘old’ terms (random, blocked and constant) in their original meanings since these were terms and interpretations used in the reviewed literature. Early evidence suggests that random practice might be most effective for the acquisition and generalizability of a motor skill, whereas during the acquisition of a specific motor skill, performance benefits most from blocked practice (Schmidt 1988).
All available studies reviewed on this matter (Dick et al. 1996, 2000, 2003) show that AD patients learn best under constant practice conditions. According to Dick and his 1996 team, humans use their episodic memory of the training trials to accurately perform a task while learning a skill. They suggest that because AD patients experience problems with episodic memory, constant practice is more effective because repeated running of the same motor program does not require an intact episodic memory. The second reason why random practice may be less effective is that other cognitive functions that play a role in random practice, like the ability to switch tasks and divide attention, are affected in AD patients.
Dick et al. (1996, 2003) explained the AD patients’ superior learning performance under constant practice conditions in terms of the schema theory originally developed by Schmidt (1975), and likewise propose a more open-loop account of motor control. Schmidt assumes the existence of generalized motor programs (GMPs) that are acquired through practice and that define the “form” of the action. These GMPs can be altered to meet environmental demands by a closed-loop system using sensory feedback. Schemata, e.g., for varying weight and distances in tossing, are learned and allow the action to be scaled to the environment (Schmidt 2003). When they considered their results in terms of this theory, Dick et al. (1996, 2003) concluded that AD patients can develop and access a GMP in training situations that emphasize movement consistency. However, they do not form the motor schemas needed to successfully achieve a movement when the environmental demands change because they are unable to encode and to store the different types of information about a motor pattern.
There are three other training approaches that can produce the desired learning effect: guidance, observation, and mental practice (Schmidt 1988). Guidance should only be used at the onset of training because experiments have shown that practice under unguided conditions seems to be more effective for retention and transfer (Shumway-Cook and Woollacott 1995). Observation conveys information about how a skill should be performed and seems to be especially beneficial for the acquisition of new movement patterns (Magill 1993). Our automated computer search and an extra search combining the three keywords with Alzheimer dementia both failed to generate any relevant studies that employed one of these training methods. The only study that provided some additional information on the topic is a report by Dick et al. (1988) which showed that AD patients could recall preselected (subject-defined) movements more accurately than constrained (experimenter-defined) movements on a linear positioning apparatus. This was explained by the patients’ ability to profit from mental preparation of the movement prior to its execution. Without further systematic investigation, however, it cannot be inferred that the ability to profit from mental preparation also means AD patients will profit from mental practice. More research into the effects of all three practice types in AD is needed.
Feedback: Theory and Outcome Studies with AD Patients
A second crucial variable that influences motor learning is type of feedback. Intrinsic feedback encompasses the sensory information generated by motion, and extrinsic feedback entails information from an external source like a therapist (Schmidt and Wrisberg 2000). There are various ways to provide extrinsic feedback. It can be delivered during or after the movement, immediately following movement completion or delayed, and in a verbal or a non-verbal fashion. It can contain information on average performance (summary feedback) or it may reflect each movement or performance (constant feedback; Schmidt 1988). It is generally believed that constant feedback enhances only motor performance, not the level of learning (Shumway-Cook and Woollacott 1995). With less frequent feedback, learners have to rely more on other cues, which entails more elaborate encoding (Schmidt 1988). Extrinsic feedback can moreover be divided into ‘knowledge of results,’ in which the movement outcome is given in terms of the goal, and ‘knowledge of performance,’ so that the feedback concerns the movement pattern itself (e.g., in a Tossing task: increase the swing of your arm).
In almost all studies on motor-skill learning in AD, visual feedback was employed. Only the Maze tasks were administered under blindfolded conditions and the amount of learning in the AD patients proved inferior to the amount found for the controls (Sabe et al. 1995; Kuzis et al. 1999). In most Rotor-Pursuit tasks, the velocity of the target was individualized to equate initial performance. Controls generally tracked at a faster rate than the AD patients (Deweer et al. 1994; Dick et al. 1995; Jacobs et al. 1999; Libon et al. 1998). Possibly, AD patients can only perform this task at a slower rate because they rely more on visual feedback than controls.
Only one study using a Rotor-Pursuit task explicitly examined the role of visual feedback on performance in AD patients, showing a drop in performance when the visibility of the moving target was reduced during the learning phase (Dick et al. 2001). In contrast to that of the normal controls, the patients’ performance did not improve across trials in the restricted-vision condition. In the full-vision condition the patients showed normal learning.
It can be tentatively concluded that for AD patients, constant visual feedback is important in learning motor skills, but more research is needed to confirm this hypothesis. We did not find any studies that were concerned with the frequency of external feedback, and whether knowledge of results and knowledge of performance makes a difference in this patient group. Based on the results cited above, it may be hypothesized that both forms of feedback knowledge probably place too much weight on the cognitive abilities in AD patients and therefore contribute little to successful performance.
Conclusions and Recommendations
People with Alzheimer’s disease are able to implicitly (re)learn motor skills to a certain extent and under specific conditions. The experimental research to date shows preserved implicit motor learning irrespective of the task used. Patients are capable of acquiring motor skills without awareness simply by repeated exposure, although their performances will not reach normal levels. This is expressed in their protracted performance relative to that of unimpaired controls. Moreover, extent of learning will differ depending on the task to be mastered.
The preserved implicit learning ability in AD can be of use for physical therapists working with this elderly patient group. Physical therapists can call upon neuropsychologists to provide information on their patients’ learning capacities since they have quantitative measures at their disposal to assess a patient’s level of functioning. However, the memory and learning tests currently available in the clinical practice evaluate explicit or declarative memory (Spaan et al. 2003). In order to get a satisfactory differential picture of the learning capacities in demented patients, implicit (motor) learning tasks need to be added to the neuropsychological assessment.
The evidence of intact implicit learning in AD further prompted the question how these intact learning abilities can best be translated into rehabilitation programs targeting this patient group. Learning is central in rehabilitation and knowledge of the system under treatment, like the motor system, must be combined with knowledge of how learning principles must be applied to achieve a successful training program (Baddeley 1993). With respect to patients with dementia, apart from the subtype of dementia and its specific neuropsychological syndrome, the training programs should apply the principles that emerge from theories of learning.
The studies we reviewed showed that in (re)learning motor skills, constant, or rather frequent and consistent practice is important in AD patients. This way of learning draws less on episodic memory and other cognitive functions compromised in AD patients. These data also suggest that practice under dual-task conditions should also be avoided.
Because AD patients have difficulty in generalizing the motor skills learned during the sessions, training has to take place in an environment that closely resembles the one in which the skill is going to be used and presumably with tools used by the AD patient in his or her daily life. If, for instance, an AD patient is trained in the use of a microwave, the device used during the training should be the same as the one available in the patient’s household. The amount of training a patient needs will depend on the task being trained. The role of fatigue is also important in this respect. The effects of massed and distributed practice in this generally older patient group need to be addressed in future investigations.
Patients with AD appear to remain dependent upon visual feedback throughout training and performance. Screening and subsequent correction of visual problems or the use of visual aids can be effective in the training process in this group for whom vision problems are very common (De Winter et al. 2004). The type and point in time when external feedback needs to be given and its effect on learning in AD also warrants attention in future research.
In the introduction of our review we asked whether patients with Alzheimer’s disease might have intact motor-skill learning abilities. The answer is twofold. Clearly, AD patients show preserved implicit learning abilities that can be utilized in teaching (motor) skills, yet transfer to other skills is minimal. Accordingly, the professionals delivering the training programs should tailor the contents to the particular needs and abilities of this patient group or the individual patient. When the above guidelines are kept in mind and when our knowledge on this topic are widened, non-pharmacological interventions might contribute significantly in helping elderly people suffering from dementia to keep their autonomy. The extent to which pharmacological intervention may enhance these behavioral mechanisms and foster independent living in AD patients has yet to be determined.

motor-skill learning
alzheimer’s disease
rehabilitation
non-pharmacological interventions
procedural learning
implicit learning


C1q deficiency promotes the production of transgenic-derived IgM and IgG3 autoantibodies in anti-DNA knock-in transgenic mice

C1q-deficient mice have been shown to develop a lupus-like disease and to display an impaired clearance of apoptotic cells that are enriched in lupus autoantigens. However, the role of C1q in the regulation of autoreactive B cells remains debatable. To explore this we crossed MRL/Mp C1q-deficient mice with knock-in transgenic (Tg) mice expressing an anti-ssDNA antibody (VH3H9R and VH3H9R/VLκ8R). Analysis of the VH3H9R mice showed that in the absence of C1q higher titres of Tg-derived IgM and IgG3 anti-ssDNA antibodies were detectable. In contrast, in the VH3H9R/VLκ8R C1q-deficient animals no increase in Tg antibody levels was observed. In both models the lack of C1q induced a marked reduction of marginal zone B cells and this was paralleled by a significant increase in the percentage of plasmocytes. Thus, one could postulate that in the absence of C1q the failure to clear efficiently dying cells provides an additional stimulus to the autoreactive Tg B cells resulting in their emigration from the marginal zone B cell compartment with subsequent increase in plasmocytes. However, the lack of C1q led to an increased production of Tg IgM and IgG3 antibodies only in VH3H9R mice indicating that additional genetic susceptibility factors are required to break self-tolerance.
1
Introduction
Hereditary deficiencies of early components of the classical pathway of the complement system are known to predispose to systematic lupus erythematosus (SLE). Among these, C1q deficiency exhibits the strongest association with prevalence greater than 90% suggesting that a physiological activity of the early part of the classical pathway normally protects against the development of SLE (Pickering et al., 2000). Mice with targeted deletion of the C1q gene (C1qa−/−) developed a spontaneous lupus-like disease characterised by the development of anti-nuclear autoimmunity and glomerulonephritis associated with the presence of multiple apoptotic bodies (Botto et al., 1998). Introgression of C1q deficiency onto different genetic backgrounds revealed that in mice C1q operates as a disease modifier. C57BL/6.C1qa−/− mice displayed no increase of IgG autoantibodies (autoAbs) or glomerulonephritis (Mitchell et al., 2002), whilst C1q deficiency backcrossed onto the lupus-prone MRL/Mp background accelerated both the onset and the severity of the autoimmune disease (Mitchell et al., 2002). Consistent with these observations, C1q reconstitution by bone marrow transplant attenuated the autoimmune disease present in MRL/Mp.C1qa−/− mice (Cortes-Hernandez et al., 2004).
Currently, there are two main hypotheses to explain the role of complement in the development of SLE, neither of which is mutually exclusive. The first model, defined as the tolerance hypothesis, proposes a role for complement in determining the activation thresholds of lymphocytes, whereby complement enhances presentation of autoantigens to self-reactive immature B cells resulting in their elimination (Prodeus et al., 1998). The second one, known as the ‘waste disposal’ hypothesis, suggests that in addition to its role in the clearance of immune complexes, complement is involved in the physiological disposal of apoptotic cells (Botto et al., 1998), that have been shown to express lupus autoantigens on their surface (Casciola-Rosen et al., 1994). C1q plays a significant role in the clearance pathway of cellular debris by binding directly or indirectly to apoptotic blebs where it activates complement and mediates phagocytosis by professional and non-professional phagocytes (Korb and Ahearn, 1997; Mevorach et al., 1998; Nauta et al., 2004; Quartier et al., 2005; Taylor et al., 2000). Therefore, improper removal of dying cells in the setting of C1q deficiency could result in the stimulation of autoreactive cells leading to autoimmunity.
Immunoglobulin Tg models have been instrumental in understanding B cell regulation revealing several key mechanisms, including receptor editing, deletion, anergy and ignorance. The hen egg lysozyme (HEL)–anti-HEL (IgHEL) Tg model in particular has been widely used to demonstrate elimination of self-reactive clones by membrane-bound expressed antigen, anergy induction by soluble antigen (sHEL) or ignorance when the amount of antigen is so low that it does not reach the threshold to induce anergy. More recently, it has been shown that intracellular membrane-bound HEL failed to induce tolerance and was instead autoimmunogenic positively selecting IgHEL B1 cells and inducing large numbers of IgM autoantibody-secreting plasma cells (reviewed in Ferry et al., 2006). Taken together, these findings suggest that in this model the fate of self-reactive B cells is determined not only by the abundance, the avidity of the target self-antigen and the affinity of the B cell receptor but also by the location of the “auto”-antigen. The role of complement has been tested in the IgHEL Tg (MD4)–sHEL (ML5) model by crossing the double Tg mice with mice deficient in C1q, C4, C3 or CD21/CD35 (Cutler et al., 2001; Prodeus et al., 1998). IgG anti-HEL Abs remained undetectable in all complement deficient mice, but C4 and CD21/CD35 deficient B cells displayed reduced surface IgM modulation, indicating a lower degree of anergy induction in these mice (Prodeus et al., 1998). However, this was not observed in the C1q deficient mice (Cutler et al., 2001). This discrepancy could either indicate that C4 operates independently from C1q or reflect differences in the genetic background of the mice used (Cutler et al., 2001). Nevertheless sHEL is not the ideal model to study the maintenance of tolerance in SLE as it is neither a natural autoantigen, nor are soluble plasma proteins typically targeted by the SLE autoAbs.
More recently, a model targeting an SLE antigen, DNA, was generated by Weigert and co-workers (Chen et al., 1995). In this model the rearranged variable heavy chain (VH) gene derived from a double stranded DNA-binding hybridoma developed in the autoimmune strain MRL/Mp.lpr/lpr, was inserted at the Igh locus and was referred to as VH3H9R. In contrast to conventional Tg mice, this knock-in model allows the Tg locus to undergo normal editing, isotype switching and somatic mutation. A variety of light chains can combine with the VH3H9 to yield anti-DNA Abs (Radic et al., 1991) but only few light chains are able to “silence” VH3H9R so that it no longer binds to DNA. By virtue of this characteristic the mice expressing only the VH3H9 chain (VH3H9R mice) can generate anti-DNA specificities. However, when the VH3H9R mice were crossed with a Tg knock-in light chain VLκ8 (Prak and Weigert, 1995) to generate monospecific Tg mice (VH3H9R/VLκ8R mice) (Chen et al., 1997a), this combination of heavy and light chain V regions (VH3H9/VLVκ8) bound only ssDNA and not dsDNA (Prak and Weigert, 1995). Previous studies with the VH3H9R mice have shown that the autoreactive Tg B cells accumulated in the splenic marginal zone and were regulated by anergy on non-autoimmune backgrounds such as BALB/c (Chen et al., 1995; Erikson et al., 1991) and C57BL/6 (Fukuyama et al., 2005; Sekiguchi et al., 2002). However, tolerance could be broken in this model if T cell help was provided in the form of a chronic graft versus host disease (Sekiguchi et al., 2002). Consistent with these observations, the double VH3H9R/VLκ8R knock-in Tg B cells were regulated by anergy in non-lupus prone mice (BALB/c), whilst in autoimmune prone MRL/Mp.lpr/lpr animals Tg B cells escaped tolerance induction and underwent class-switching and affinity maturation (Brard et al., 1999). These experiments suggested that the lpr mutation in the MRL background allowed the Tg autoreactive B cells to receive T cell help during a germinal center reaction.
To determine whether C1q is involved in selection of self-reactive B cells, we bred the C1q-deficient mice with the VH3H9R and the VH3H9R/VLκ8R mice and monitored the regulation and activation of anti-DNA Tg B cells over a period of 10 months. The mice in this study were on the autoimmune prone background MRL/Mp expressing the CD95 (Fas) gene. The analysis of these mice revealed that the lack of C1q can influence the levels of IgM and IgG3 Tg-derived antibodies only in the VH3H9R model.
2
Materials and methods
2.1
Mice
MRL/Mp mice were obtained from Harlan Olac, Bichester, UK. MRL/Mp.C1qa−/− deficient mice were generated as previously described (Mitchell et al., 2002). VH3H9R.MRL/Mp (Chen et al., 1995), VLκ8R.MRL/Mp (Prak and Weigert, 1995) and VH3H9R/VLκ8R.MRL/Mp mice were kindly provided by Prof. M. Weigert (Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL). MRL/Mp.C1qa−/− mice were crossed with the VH3H9R/Vκ8R.MRL/Mp mice and the resulting VH3H9R/VLκ8R.MRL/Mp.C1qa+/− were then crossed either with MRL/Mp or MRL/Mp.C1qa−/− in order to generate littermate animals. Mice were bled every 3 months starting from 2 months of age and at 10 months they were sacrificed. The mice were genotyped by PCR using specific primers. PCR primers were as follow: mC1qA/5′ (5′-GGGGCCTGTGATCCAGACAGG-3′), mC1qA/In2− (5′-TAACCATTGCCTCCAGGATGG-3′) and neo (5′-GGGGATCGGCAATAAAAAGAC-3′) for the C1q genotyping; MW114 (5′-CTGTCAGGAACTGCAGGTAAGG-3′) and MW162 (5′-CATAACATAGGAATATTTACTCCTCGC-3′) for the VH3H9R genotyping (Erikson et al., 1991); MW133 (5′-GGTACCTGTGGGGACATTGTG-3′) and MW157 (5′-AGCACCGAACGTGAGAGG-3′) for the VLκ8R genotyping (Carmack et al., 1991). Animals were kept under specific pathogen-free conditions. All animal care and procedures were conducted according to institutional guidelines and approved by the local ethical committee.
2.2
Flow cytometry
Flow cytometry was performed using a four-color staining of cells and analyzed with a FACSCalibur™ (Becton Dickinson, Mountain View, CA). The following Abs were used: anti-B220 (RA3-6B2), anti-CD5 (53-7.3), anti-CD11b (M1-70), anti-CD19 (1D3), anti-CD23 (B3B4), anti-CD21/CD35 (7G6), anti-CD90.2 (53-2.1), anti-CD138 (281-2), anti-IgM (II/41). All Abs were purchased from BD Biosciences Pharmingen (San Diego, CA) with the exception of the anti-VH3H9 idiotype (1.209), a kind gift from Prof. M. Weigert (Gay et al., 1993). Biotinylated Abs were detected using an allophycocyanin-conjugated streptavidin Ab (BD Biosciences Pharmingen). Staining was performed in the presence of saturating concentration of 24G2 mAb (anti-FcRII/III). Data were analyzed using WinMDI software (Version 2.8; Scripps Institute).
2.3
Serological analyses
Serum levels of IgM and IgG were quantified by ELISA, as described previously (Cortes-Hernandez et al., 2004). Anti-ssDNA Abs, anti-chromatin Abs, anti-histone Abs and anti-dsDNA Abs were measured by ELISA as described previously (Burlingame and Rubin, 1990; Emlen et al., 1990). Microtiter plates were coated with ssDNA prepared from calf thymus DNA (Sigma), chromatin (Lorne Laboratories Ltd., Reading, UK) or histone (Calbiochem, Merck Biosciences, Darmstadt, Germany). For detecting anti-dsDNA Abs, plates were coated with streptavidin (Sigma). ΦX174 double-stranded plasmid DNA (Promega, Southampton, UK) was biotinylated with Photoprobe biotin (Vector Laboratories, Peterborough, UK) and added to the streptavidin. Serum samples were diluted appropriately in PBS 2%BSA, 0.05%Tween-20, 0.02% NaN3. Bound Abs were detected with alkaline phosphatase conjugated goat anti-mouse IgG (γ-chain specific) (Sigma–Aldrich, Dorset, UK) and anti-mouse IgM (Southern Biotechnology Associates, Inc., Birmingham, AL). All autoAb results are expressed in arbitrary ELISA units (AEU) in reference to a standard curve derived from serum pools containing high titers of autoAbs. The idiotype+ Abs were captured with the anti-VH3H9 idiotype (1.209) (Gay et al., 1993) and detected with alkaline phosphatase conjugated goat anti-mouse IgM or IgG subclasses specific Abs (Southern Biotechnology Associates). The standard curve was derived from an IgM VH3H9R/Vκ8R mAb provided by Prof. Weigert or sera containing high IgG subclasses titers of the Tg.
2.4
Renal assessment
Proteinuria was assessed using Haema-combistix (Bayer Diagnostics, Newbury, UK). Kidneys were fixed in Bouin's solution and paraffin embedded, and sections were stained with periodic acid–Schiff reagent. Glomerular histology was graded in a blind fashion scored on a scale of 0–4, as described before (Carlucci et al., 2007).
2.5
Statistics
The data are presented as median, with range of values in brackets, unless otherwise stated. The non-parametric Mann–Whitney U-test was applied throughout with differences being considered significant for p-values < 0.05. Statistics were calculated using GraphPad Prism Version 3.0 (GraphPad Software, San Diego, CA).
3
Results
3.1
Expression of Tg autoantibodies
C1q deficiency has been shown to accelerate the onset and progression of SLE in MRL/Mp mice (Mitchell et al., 2002), but the mechanisms underlying these effects are still uncertain. The VH3H9R/VLκ8R Tg alleles were transferred onto C1q-deficient mice by crossing VH3H9R/VLκ8R.MRL/Mp with MRL/Mp.C1qa−/− and experimental cohorts of littermate mice were generated. Whilst in previous studies the expression of the Tg alleles was monitored by measuring the Tg-specific immunoglobulin allotype (Steeves and Marion, 2004), this analysis could not be applied in these mice as the Tg alleles and the endogenous immunoglobulin allotype were indistinguishable. Nevertheless, we were able to detect the combination of the VH3H9 with the VLVκ8 using an idiotype specific monoclonal Ab named 1.209 (Gay et al., 1993). As the 1.209 mAb can also recognize the VH3H9 paired with other endogenous light chains such as VLκ4 (Gay et al., 1993), this anti-idiotype Ab allowed us not only to assess the VH3H9R/VLκ8R Tg B cells but also some of the VH3H9R Tg B cells. C1q deficiency did not affect the proportion of peripheral blood B cells expressing the Tg in neither of the two models (VH3H9R/VLκ8R.MRL/Mp.C1qa−/−: 89.1 ± 1.1% versus VH3H9R/VLκ8R.MRL/Mp: 90.7 ± 1.5%, p = 0.4118; VH3H9R.MRL/Mp.C1qa−/−: 25.3 ± 1.6% versus VH3H9R.MRL/Mp: 22.3 ± 2.3% p = 0.1669). The low proportion of idiotype+ B cells in the VH3H9R.MRL/Mp mice indicated that most of the B cells in these mice had either paired the VH3H9R Tg allele with a light chain that was not recognised by the anti-idiotype Ab, or edited the VH3H9R allele, or used the endogenous immunoglobulin allele. In the VH3H9R/VLκ8R.MRL/Mp.C1qa−/− mice the B cell receptor was down-modulated to the same extent as in the VH3H9R/VLκ8R.MRL/Mp mice (data not shown).
Serological analyses were performed in order to establish whether the Tg autoAbs were expressed. The different cohorts of mice were bled at different time points, and the data presented here are those obtained at 10 month of age when the mice were sacrificed. Substantial levels of idiotype+ IgM, IgG3, IgG2a and IgG2b Abs were detected in the VH3H9R/VLκ8R.MRL/Mp and in VH3H9R.MRL/Mp mice indicating that the anti-DNA knock-in Tg alleles on this background were not regulated by anergy unlike when on the C57BL/6 background (Fig. 1). In the VH3H9R/VLκ8R.MRL/Mp.C1qa−/− mice the levels of idiotype+ Abs were similar to those detected in the strain-matched C1q-sufficient animals. In contrast, the VH3H9R.MRL/Mp.C1qa−/− mice had significantly increased levels of idiotype+ IgM and IgG3 Abs compared to the VH3H9R.MRL/Mp mice (Fig. 1A and B), but similar levels of idiotype+ IgG2a and IgG2b Abs (Fig. 1C and D).
We then measured the levels of IgM and IgG autoAbs. A significant increase in the titre of IgM anti-ssDNA Abs was observed in the VH3H9R/VLκ8R.MRL/Mp.C1qa−/− mice compared to the VH3H9R/VLκ8R.MRL/Mp mice (Fig. 2A). Of note, the VH3H9R/VLκ8R.MRL/Mp.C1qa−/− mice displayed only markedly increased levels of IgM autoAbs directed against ssDNA (the specificity encoded by the Tg alleles) but not against other lupus autoantigens such as dsDNA, histone or chromatin (Fig. 2B–D). In the VH3H9R.MRL/Mp mice the pairing of the VH3H9 with endogenous L chains could yield to more specificities. Consistent with this, VH3H9R.MRL/Mp.C1q−/− mice had significantly increased levels of IgM anti-ssDNA, anti-dsDNA, anti-histone and anti-chromatin Abs (Fig. 2A–D). We then analysed the levels of IgG autoAbs and found that these were similar between the C1q-sufficient and -deficient cohorts (Supporting information Table S1).
3.2
Renal assessment
At 10 month of age all the animals were sacrificed. VH3H9R/VLκ8R.MRL/Mp and VH3H9R.MRL/Mp animals showed histological evidence of a mild glomerulonephritis (median score 1.0, range: 0.0–3.0 and 1.5, range: 1.0–3.0, respectively) reminiscent of the disease observed in MRL/Mp non-Tg mice (Mitchell et al., 2002). C1q-deficiency in MRL/Mp mice has previously been shown to worsen the kidney pathology (Mitchell et al., 2002). However, in the Tg mice the absence of C1q did not exacerbate the renal disease compared to the strain-matched C1q-sufficient mice (VH3H9R/VLκ8R.MRL/Mp.C1qa−/− mice: median score 1.0, range: 0.0–3.0, p = 0.4287; VH3H9R.MRL/Mp.C1qa−/− mice: median range 2.0, range: 1.0–2.0, p = 0.4564).
3.3
Flow cytometric analysis of splenic and peritoneal B cells
In order to determine if the serological data were accompanied by phenotypic changes in T and B lymphocytes, we performed a comprehensive analysis of the various splenic and peritoneal subpopulations. Cells of at least 7 mice from each cohort were analysed by FACS at the time of the sacrifice using the combinations of markers shown in Table 1. In agreement with the findings in other genetic backgrounds (Li et al., 2002), the VH3H9R.MRL/Mp mice had a larger marginal zone (MZ) B cell population compared to VH3H9R/VLκ8R.MRL/Mp (Fig. 3A) and an increased proportion of idiotype+ B cells in the MZ compartment compared to follicular (FO) compartment (5.9 ± 1.0 versus 4.2 ± 0.6, paired t-test p = 0.0157) (Table 1). More interestingly, in the VH3H9R.MRL/Mp.C1qa−/− mice, which produced higher levels of IgM autoAbs, the MZ B cell population was significantly decreased compared to the VH3H9R.MRL/Mp animals (Fig. 3A) and less idiotype+ B cells were found in the MZ compared to the FO compartment (3.4 ± 0.7 versus 4.8 ± 1.1, paired t-test p = 0.0042) (Table 1). In the VH3H9R/VLκ8R.MRL/Mp mice there was no preferential localization of idiotype+ B cells in the MZ zone (paired t-test p = 0.056) (Table 1). C1q deficiency was associated with a significant reduction in the percentage of MZ B cells (Fig. 3A) and a decrease of idiotype+ B cells in the MZ compared to the FO area (4.2 ± 0.9 versus 17.5 ± 3.2, paired t-test p = 0.0013). These findings indicated that MZ B cells might have been activated in the absence of C1q. Activated MZ B cells have been shown to migrate into the T cell zone and differentiate into plasma cells. Indeed, the percentage of plasma cells was increased in the two C1q-deficient cohorts compared to the respective MRL/Mp controls (Fig. 3B).
The peritoneal cavity in mice has been shown to be a site where some self-reactive B cells can escape tolerance (Fagarasan et al., 2000) and B-1 cells, one of the major sources of circulating IgM, accumulate. More cells were recovered from the peritoneum of VH3H9R.MRL/Mp.C1qa-/-mice compared to VH3H9R.MRL.Mp mice (p = 0.0051) but not in the VH3H9R/VLκ8R.MRL/Mp.C1qa−/− versus VH3H9R/VLκ8R.MRL/Mp animals (p = 0.1923) (Table 1). When the peritoneal B cell subpopulations were analyzed in more detail, all subsets were increased in the VH3H9R.MRL/Mp.C1qa-/mice compared to the VH3H9R.MRL.Mp mice (Table 1). On the contrary, in the VH3H9R/VLκ8R C1q-deficient animals only B-1a cells were slightly increased compared to their wild type strain-matched controls (Table 1), but this did not reach statistical significance (p = 0.0518). We then analysed the VH3H9/VLκ8 idiotype expression on the different peritoneal B cells. Surprisingly in the peritoneum of the VH3H9R/VLκ8R.MRL.Mp and VH3H9R.MRL.Mp mice the majority of the B cells were not expressing the VH3H9/VLκ8 idiotype (Table 1). Of note the C1q-deficient mice had similar percentage of idiotype+ B cells in the different peritoneal B cell subpopulations compared to C1q-sufficient mice. The reduced proportion of idiotype+ B cells in the peritoneum compared to the peripheral blood and the spleen (data not shown) suggests that in this organ the B cells either preferentially used the endogenous alleles or had been activated and undergone editing.
4
Discussion
C1q deficiency in humans and in mice has been associated with the development of a lupus-like illness. However, the role of C1q in the regulation of autoreactive B cells remains debatable. Here, we explored the regulation of autoreactive B cells by C1q using anti-DNA knock-in Tg models (VH3H9R/VLκ8R and VH3H9R) on the lupus prone MRL/Mp genetic background. The analysis of these mice revealed that the MRL/Mp background was in itself sufficient to allow the expression of anti-DNA Tg autoAbs and that the lack of C1q modified this effect only in the single Tg model (VH3H9R). However, in the absence of C1q the MZ B cell compartment was significantly reduced in both models and this was accompanied by an increase in plasmocytes.
Recently the VH3H9R/VLκ8R anti-DNA knock-in Tg model has been widely used for investigating the mechanisms of regulation of autoAb production in murine models of SLE. There is an increasing evidence that the VH3H9R/VLκ8R and VH3H9R anti-DNA Tg B cells, which have a relatively weak affinity to ssDNA, are regulated by anergy on normal genetic backgrounds such as BALB/c and C57BL/6, but could be induced to lose tolerance when transferred onto lupus models such as (NZB × NZW)F1 or MRL/Mp.lpr/lpr (Brard et al., 1999; Chen et al., 1995; Erikson et al., 1991; Fukuyama et al., 2005; Sekiguchi et al., 2002). In light of these observations it was important for our study to establish whether the autoimmune prone MRL/Mp background was in itself capable of breaking tolerance. MRL/Mp mice are known to develop a mild autoimmune disease which can be accelerated with different disease-modifying genes such as Yaa (Merino et al., 1989), lpr and gld (Cohen and Eisenberg, 1991). The analysis of the idiotype+ (VH3H9R/VLκ8R) Abs revealed that the Tg MRL/Mp mice indeed had in circulation these idiotype+ Abs (IgM, IgG2a, IgG2b, IgG3) indicating that a break of tolerance had spontaneously occurred in these mice. One explanation for this is an intrinsic defect in MRL/Mp B cells and there is some evidence in support of this. MRL/Mp mice have been reported to exhibit a defect in maintaining the developmental arrest of VH3H9/VLλ anti-dsDNA conventional Tg B cells (Mandik-Nayak et al., 1999, 2000) and to have a spontaneous B cell hyperactivity in the absence of Ag in the IgHEL experimental model (Nijnik et al., 2006). However, the VH3H9/VLλ anti-dsDNA autoreactive B cells, despite not being any longer developmentally arrested as in a BALB/c mice, exhibited follicular exclusion and failed to differentiate into plasma cells (Mandik-Nayak et al., 1999). Furthermore Tg MRL/Mp mice expressing IgHEL have been shown to be able to down-regulate their B cell receptor and to be unable to secrete detectable levels of anti-HEL Abs in the presence of sufficient amount of sHEL (Nijnik et al., 2006). Similarly anti-laminin Tg MRL/Mp mice were found to be tolerant (Rudolph et al., 2002). Another potential explanation for the break of the B cell tolerance in the VH3H9R/VLκ8R anti-DNA Tg mice, is that in this model the MRL/Mp background was able to provide sufficient T cell help and the presence of idiotype+ IgG subclasses favour this hypothesis.
We next examined whether C1q could modulate the phenotype of the anti-ssDNA Tg B cells. In the VH3H9R/VLκ8R.MRL/Mp mice the absence of C1q increased significantly the circulating levels of IgM against ssDNA but not against other autoantigens including dsDNA. As the pairing of VH3H9 with VLκ8 prevents the binding of VH3H9 to dsDNA, the elevated amount of IgM anti-ssDNA observed could have been the result of an increased Tg expression. However, the idiotype analysis failed to demonstrate a difference in the levels of IgM idiotype+ Abs between VH3H9R/VLκ8R.MRL/Mp.C1qa−/− and VH3H9R/VLκ8R.MRL/Mp mice, questioning whether the source of the increased levels of IgM anti-ssDNA was indeed the Tg B cells. On the other hand, the VH3H9R.MRL/Mp.C1qa−/− mice displayed significantly higher levels of IgM and IgG3 idiotype+ Abs. As the VH3H9R heavy chain can pair with different endogenous light chains generating a wider range of autoAbs, other specificities were tested. Indeed the absence of C1q increased significantly IgM levels against all the lupus autoantigens analysed.
To gain insight into the mechanisms regulating the autoAb production in the C1q-deficient mice we then carried a detailed analysis of the different B cell populations. Several studies have proposed a possible role for MZ B cells in the development of lupus in mouse models. Although studies in immunoglobulin Tg mice have shown that autoreactive B cells can accumulate in the marginal zone under various experimental situations (Li et al., 2002; Mandik-Nayak et al., 1997, 1999, 2006; Qian et al., 2004; Wen et al., 2005), it remains to be established whether MZ B cells secrete pathogenic autoAbs in any model of lupus. B cells producing potentially pathogenic autoAbs are thought to home to the MZ (Chen et al., 1997b; Li et al., 2002) and sequestration to this site is believed to prevent them from entering into the germinal centres and developing the properties of pathogenic B cells. However, recent studies in lupus-prone mice have reported both enlargements (Grimaldi et al., 2001; Mackay et al., 1999; Wither et al., 2000) and impaired development of the MZ B cell compartment (Amano et al., 2003; Samardzic et al., 2002). Consistent with previous observations in the VH3H9R/VLκ8R model (Wen et al., 2005), the MZ was found to be enlarged in the VH3H9R.MRL/Mp mice but no accumulation of MZ B cells was observed in VH3H9R/VLκ8R.MRL/Mp mice. Importantly C1q deficiency decreased dramatically the proportion of MZ B cells in these mice and this was accompanied by an increase in the percentage of plasma cells. More interestingly, in the C1q-deficient Tg mice we observed a significant disappearance of idiotype+ B cells from the MZ suggesting that these cells had been activated. Marginal zone B cells bearing low affinity self-reactive BCR can react to repetitive Ag and produce natural autoAbs of the IgM isotype upon contact with blood-borne pathogens or self Ag (Oliver et al., 1999). Moreover, MZ B cells can undergo T cell independent switching to IgG, IgA and IgE in response to pathogen associated molecular patterns (PAMP). As C1q has been shown to be involved in the clearance of apoptotic cells, which are enriched in the typical lupus autoantigens, one could postulate that the increased IgM Tg secretion and the reduction of the MZ B cells in the VH3H9R.MRL/Mp.C1qa−/− mice might be related to a failure to clear antigens associated with dying cells. In the absence of C1q the ineffectively cleared autoAgs could stimulate the autoreactive MZ Tg B cells to differentiate into plasmocyte resulting into the decrease of MZ B cells and increase of circulating IgM autoAbs. Similarly, in the spleens of VH3H9R/VLκ8R.MRL/Mp.C1qa−/− mice significantly less idiotype+ B cells were present in the MZ compartment and more plasmocytes were found. However, these cellular changes were not paralleled by an increase of idiotype+ IgM Abs in circulation indicating in this model they were not sufficient to induce autoantibody production.
Another potential source of IgM autoAbs are the B1 cells. B1 cells have long been associated with the secretion of natural Abs against self and foreign pathogens, which can occur without obvious inflammatory response. The importance of B1 cells in the pathogenesis of lupus continues to be debated. A major argument for a role of B1a cells in mouse lupus relates to the expansion of this subset in NZB and (NZB × NZW)F1 mice. Reduction of B1 cells for instance via intraperitoneal injection of H2O delayed disease onset and reduced disease severity in (NZB × NZW)F1 mice (Murakami et al., 1995). However, the expansion of B1a cells was shown not to be critical for the production of IgM or IgG autoAbs in this murine model of SLE (Atencio et al., 2004). The Abs secreted by B1 cells tend to be polyreactive with low-affinity cross-reactivity to a variety of self Ags and these characteristics are very different from the pathogenic IgG Abs produced by lupus mice. In our experimental model, we found an increase in the total number of peritoneal and B1 cells in the VH3H9R.MRL/Mp.C1qa−/− mice compared to their wild type counterparts (Table 1). A similar trend was observed for the B1a cells in the VH3H9R/VLκ8R.MRL/Mp.C1qa−/− mice but this did not reach statistical significance. However, in both models the percentages of idiotype+ B1 cells between C1q-deficient and -sufficient mice were similar. These findings would indicate the lack of C1q may favour the expansion of peritoneal B1 cells and that these cells might have contributed to the increase of serum IgM autoAb levels observed in the VH3H9R.MRL/Mp C1q-deficient mice. Supporting this hypothesis, a recent study showed that C1q deficiency increases the positive selection of B-1 cells and IgM autoAb production by a membrane-bound intracellular auto-Ag (Ferry et al., 2007).
In conclusion, using mice expressing site-directed transgenes for anti-DNA autoAbs we have shown that: (i) the MRL/Mp background was in itself capable of inducing the expression of anti-DNA Tg autoAbs, and (ii) VH3H9R.MRL/Mp.C1qa−/− could influence the production of Tg-derived IgM and IgG3 autoAbs possibly as a result of an impaired disposal of cellular debris. However, we found no evidence of a direct role of C1q in the regulation of self-reactive conventional B cells. Further studies on non-lupus prone genetic backgrounds such as C57BL/6 will be necessary to determine if C1q deficiency can play a more substantial role in shaping the repertoire of the autoreactive B cells.

transgenic
b cells
complement
autoimmunity
ab, antibody
aeu, arbitrary elisa units
fo, follicular
hel, hen egg lysozyme
mz, marginal zone
tg, transgenic
sle, systematic lupus erythematosus
rodent


The use of mechanical bowel preparation in elective colorectal surgery

Background Mechanical bowel preparation (MBP) prior to elective colorectal surgery has been in use for many years. It is considered important in preventing post-operative infectious complications after colorectal surgery. The evidence to support these claims is lacking within the medical literature and yet this still remains standard practice in many hospitals. A literature search was undertaken to ascertain the evidence available regarding the use of MBP in elective colorectal surgery.
BACKGROUND
Mechanical bowel preparation (MBP) prior to elective colorectal surgery has been in use for many years. Early observational studies and long-standing clinical experience have shown that removal of faecal matter from the bowel lumen prior to surgery has been associated with decreased patient morbidity and mortality1. It is still commonly used in routine practice today2. In fact, in a recent survey of members of the American Society of Colon & Rectum Surgeons, 99% of respondents routinely use MBP although 10% question its use3. This is in keeping with common belief that clinical practice often is not evidence based but is based on tradition, previous teaching and anecdote.
MBP is considered important in preventing post-operative infectious complications after colorectal surgery2,4–10. Important infectious complications include wound infection, intra-abdominal abscess formation and anastomotic leakage. There are a number of ways in which MBP is thought to act. It may decrease intraoperative contamination with faecal material thereby reducing the incidence of post-operative wound infection and residual intra-abdominal infection6,7,9,10. It may prevent mechanical disruption of the anastomosis by the passage of hard faeces9 and improves the handling of the bowel intra-operatively2,7. It may reduce the bacterial count within the colon7,10. Conversely, it may also be associated with bacterial translocation through the bowel wall hence possibly contributing to post-operative infectious complications5,11. The evidence to support these claims is lacking within the medical literature and yet this still remains standard practice in many hospitals4,12.
Primary colonic anastomosis is considered unsafe in unprepared bowel but there is little data to suggest that infectious complications are decreased by MBP10. Bowel preparation is unpleasant for patients and can be associated with complications such as dehydration, nausea, vomiting, mucosal lesions, hypokalaemia and other electrolyte disturbances1,9,11. The omission of this practice from pre-operative preparation would be welcomed by nursing staff and patients alike.?
METHODS
A literature search was undertaken to ascertain the evidence available regarding the use of MBP in elective colorectal surgery. This included a search of PubMed, Medline and Embase using the keywords “mechanical bowel preparation”, “bowel cleansing” and “elective colorectal surgery”, a search of recent relevant journals including Diseases of the Colon and Rectum and British Journal of Surgery and backward chaining from articles obtained. The search was restricted to English language articles and a timescale of 10 years was chosen to give a balanced view of this topic.
In this review, mechanical bowel preparation will be defined as an oral preparation given prior to surgery to clear faecal material from the bowel lumen. There are a number of different preparations available including polyethylene glycol, mannitol and sodium picosulphate. Rectal enemas may also be administered before low anterior resections to ensure that the rectum is empty.
Elective colorectal surgery is defined as any surgery undertaken on a planned basis for any condition of the colon or rectum requiring bowel resection and primary anastomosis. This will include colorectal carcinoma and inflammatory bowel disease.
There are a number of recent randomised controlled trials (RCTs) to evaluate the use of MBP prior to elective colorectal surgery,10,11,13,14 and specifically for left-sided resections5. Many of these studies are underpowered therefore introducing the possibility of a Type II error and limiting the use of these results in clinical decision-making (Table I).
Table I
RCTs examining MBP
Zmora 2003
Fa-Si-Oen 2005
Ram 2005
Bucher 2005
Miettinen 2000
No. of patients included
415
250
329
153
267
No. of patients excluded
35
0
Not given
0
12
No. of pt (MBP/no MBP)
187/193
125/125
164/165
78/75
138/129
Mean age (MBP/no MBP)
68/68
68/70 (median)
68/68
63/63
61/64
Cancer % (MBP/no MBP)
78/78
90/92
75/88
32/28
46/55
L colon surgery % (MBP/no MBP)
68/72
48/58
89/85
100/100
45/47
Type of prep
Polyethylene glycol
Polyethylene glycol
Sodium phosphate
Polyethylene glycol
Polyethylene glycol
Antibiotic
Yes
Yes
Yes
Yes
Yes
Same length of prophylaxis
No
Yes
Yes
No
Yes
Rectal enema
Yes
No
No
Yes
No
Anastomosis % (stapled / handsewn)
Not given
7/93 (MBP) 8/92 (no MBP)
94/6 (MBP) 98/2 (no MBP)
Not given
60/30 (MBP) 62/28 (no MBP)
Surgeon/trainee %
Not given
42/59 (MBP) 50/50 (no MBP)
37/63 (MBP) 32/68 (no MBP)
Not given
Not given
Anastomotic leak % (MBP / no MBP)
3.7/2.1 (NS)
5.6/4.8 (NS)
0.6/1.2 (NS)
6/1 (NS)
4/2 (NS)
Wound infection % (MBP / no MBP)
6.4/5.7 (NS)
7.2/5.6 (NS)
9.8/6.1 (NS)
13/4 (NS)
4/2 (NS)
Intra-abdominal abscess % (MBP / no MBP)
1.1/1 (NS)
Not given
0.6/0.6 (NS)
1/3 (NS)
2/3 (NS)
NS = not significant
This lack of power in studies is somewhat overcome by the use of meta-analyses and systematic reviews of the literature but the reader must be aware that these methods also have their limitations. A number of meta-analyses and systematic reviews were used in this review1,2,7–9,15.
DISCUSSION
Six systematic reviews were identified in the literature assessing the role of MBP in preventing infectious complications following colorectal surgery1,2,7–9,15.
The meta-analysis carried out by Platell & Hall7 found a statistically significant increase in the incidence of wound infection in those patients receiving MBP when considering the three included RCTs. This may have been influenced by the rate of wound infection seen in one trial that used a five day regime of MBP. The anastomotic leakage rate was also higher in the MBP group but not significantly so. Each RCT used a different type of MBP and this lack of standardisation affects the validity of the results. The included studies were also underpowered thereby introducing a high possibility that they failed to detect a significant difference in the results (type II error). Evidence from the prospective and retrospective studies was in favour of no MBP in pre-operative period.
A subsequent review of the literature by Zmora et al9 appraised four RCTs. One of the studies found an increased risk of anastomotic leakage and intra-abdominal infection but no increased risk of wound infection in the group of patients receiving MBP. The remaining RCTs found no significant difference in intra-abdominal infection rate but a slight increase in wound infection rate in the MBP group.
There were conflicting results in the non-randomised studies with some showing an increased rate of infection and others reporting no difference in infection rates between the groups.
The remaining four meta-analyses only included RCTs1,2,8,15. All authors agreed that MBP was of no benefit in preparation for colorectal surgery and it may be detrimental to the patients' outcome1,2,8. Wille –Jorgensen et al15found that although initial analysis showed a significantly higher rate of anastomotic leakage in the MBP group, this significance disappears when sensitivity analyses are applied thereby weakening the conclusion that MBP leads to an increased rate of anastomotic leakage.
Slim et al8 found there was significantly more anastomotic leakage in the group of patients receiving MBP and a tendency to a higher rate of wound infection but this was not statistically significant. This group repeated the analysis excluding the poor quality trials and the results still favoured a no MBP regime although this was not statistically significant.
Only two meta-analyses looked at MBP in rectal surgery specifically1,15. Willie-Jorgensen et al15 found that when results were stratified for colonic and rectal surgery there was no trend in either direction. Guenaga et al1 found that the results of stratification favoured no MBP but this was not statistically significant. This is of more clinical importance as it may be difficult to perform a low anterior resection and anastomosis with a loaded rectum15. Both authors suggest that further trials evaluating the use of rectal preparation with enemas may be useful. Guenaga et al1also mention that the use of pre-operative radiotherapy would be an important consideration in assessment of bowel preparation for rectal surgery as many patients with rectal cancer undergo pre-operative radiotherapy.
All but one of the five RCTs examining MBP5,10,11,13,14 found no significant difference in the rate of anastomotic leakage and wound infection between patients receiving MBP or not10,11,13,14. The largest trial was undertaken by Zmora et al10 with 415 patients recruited. There are several flaws in the methodology of this trial introducing bias and compromising the validity of the results.
There was no difference found in the rate of post-operative infectious complications between the two groups. The rate of diarrhoea post-operatively was significantly more common in the group receiving MBP but this is of little clinical significance, as many patients will experience an increased stool frequency once the bowels become active. The authors acknowledge that separating the role of MBP in post-operative infection rate is difficult and ideally all other measures should be constant. They also note that the study is underpowered to detect a 5% difference in infection rate.
The RCT conducted by Bucher et al5 comparing MBP with no MBP in patients undergoing elective left-sided colorectal surgery found an increase in the total incidence of infectious abdominal complications in the group receiving MBP (22% v 8%; p=0.028). This led the authors to conclude that there was good evidence to suggest that the practice of MBP should be re-evaluated. They gave an enema pre-operatively to all patients undergoing an anterior resection regardless of whether they had been randomised to MBP or not, decreasing the internal validity of the results. If anastomotic leak rate (a more clinically important outcome than wound infection) were to be used as the primary end-point then the study would need 514 patients in each group.
The trial conducted by Ram et al14 was not properly randomised, introducing methodological bias and limiting the value of the results of this study. There was no definition of sample size and patients with low rectal anastomosis were excluded. Again, the assessor of outcome was not blinded to the intervention, introducing another source of bias. No statistically significant difference in the frequency of infectious complications was observed between the groups yet the authors concluded that “mechanical bowel preparation is unnecessary for safe elective colonic and colorectal surgery". But they recommend MBP in selected cases including the resection of small tumours when palpation of the colon may be necessary or when intra-operative colonoscopy may be performed.
Fa-Si-Oen et al13 conducted a well-designed multi-centre RCT, reported in 2005. Approximately half the resections carried out in this study were left-sided. This is important as it is now generally accepted that right-sided anastomosis is safe without MBP. This study excluded patients undergoing rectal surgery. There was no significant difference in wound infection or anastomotic leak rate but the bacterial swab results used to define wound infection in this study were only correctly obtained in 185 out of 250 patients therefore this may not be an accurate reflection of the true rate of wound infections. This study could not demonstrate an additional protective effect for MBP but it was an interim analysis and was underpowered. As a result, conclusions for clinical practice cannot be drawn from these results.
Miettinen et al11 reported the results of a prospective, randomised study including patients undergoing rectal surgery. There was no significant difference in infectious complications found between the two groups but it is difficult to conclude on the influence on anastomotic leakage from these results as the study included patients who did not undergo an anastomosis.
A number of these reported trials are underpowered thereby limiting their ability to detect a clinically significant difference in outcome between the two study groups10,11,13. One way of overcoming the problem of small sample sizes is to carry out a multi-centre trial where a larger number of patients are easier to recruit. A limitation is that they introduce heterogeneity in operative and peri-operative techniques. This is important, as surgical technique may be the single most important factor in influencing the surgical outcome9. All these studies agree that elective colorectal surgery may be safely performed without MBP and that there is no evidence to continue this invasive practice with potentially negative side effects.
Memon et al16 carried out a retrospective non-randomised trial based on operating surgeon preferences using a questionnaire. The validity of this questionnaire is unclear as no pilot study was carried out prior to the collection of definitive data. Follow-up of the patients was obtained using the hospital records therefore relying on accurate clinical notes, which are not always available.
One hundred and thirty six patients who underwent elective left-sided colorectal procedures for non-obstructive large bowel pathologies were identified using the hospital computer system. Coding errors may mean that some eligible patients were excluded from the analysis. This, along with the lack of randomisation, would introduce significant bias.
No statistical difference was found between the two groups for all infectious complications and mortality. The authors recognise the limitations of their results and do not recommend any changes in practice but do suggest that a prospective randomised trial should be performed to demonstrate the impact of MBP on morbidity and mortality in patients undergoing elective colorectal surgery.
A prospective, observational trial performed by van Geldere et al17 assessed the outcome of 250 consecutive patients who underwent resection and primary anastomosis of the colon and upper rectum under the care of a single surgeon. None of these patients received MBP pre-operatively. Both emergency and elective procedures were included in analysis. Results were favourable with an overall wound infection rate of 3.3% and an anastomotic failure rate for left-sided resections of 1.2%. The authors recommend that more powerful randomised trials are needed but in the hands of a single surgeon, primary anastomosis of unprepared bowel is safe with relatively few complications.
A small observational study conducted by Ahmad et al4 found an anastomotic leak rate of 4.2% and a wound infection rate of 8.5%. The average age of the sample was lower than that of the typical population undergoing colorectal surgery. This fact, plus the small sample size, compromises the external validity or the extent to which the results can be generalised to other samples or situations.
CONCLUSION
There are a number of meta-analyses, systematic reviews and RCTs looking at the efficacy of MBP in preventing post-operative infectious complications following elective colorectal surgery. Unfortunately many of these trials are underpowered and have a high chance of a type II error10,11,13. Most authors recommend that colorectal surgery is safe without pre-operative MBP but that there may some situations in which it may be beneficial (e.g. if there is a small tumour or the possible need for intra-operative colonoscopy)14.
The implication for clinical practice in this situation is that there is not enough strength of evidence at present to recommend a change in practice. There is a need for further higher powered trials to try to answer this question definitively. The only way that this may be achieved is by multi-centre trials where it is easier to recruit a large number of patients but it must be taken into consideration that this will introduce heterogeneity in the operative and peri-operative techniques which may have an influence on overall outcome9. There is a need for larger clinical trials in this area to address whether MBP, with its potential side effects, is truly necessary prior to elective colorectal surgery.
Further studies are required to assess the use of rectal preparation alone prior to rectal surgery1,15 and also to include patients who have undergone pre-operative radiotherapy1 as this is a common occurrence in patients who have rectal carcinoma and these patients may subsequently undergo resection with primary anastomosis. It is clear that further research is needed to clarify the role of MBP in elective colorectal surgery to ensure that the patients are receiving the most appropriate treatment with the least adverse effects.

mechanical bowel preparation
colorectal surgery