Dopamine efflux in the nucleus accumbens during within-session extinction, outcome-dependent, and habit-based instrumental responding for food reward

Rationale Dopamine (DA) activity in the nucleus accumbens (NAc) is related to the general motivational effects of rewarding stimuli. Dickinson and colleagues have shown that initial acquisition of instrumental responding reflects action–outcome relationships based on instrumental incentive learning, which establishes the value of an outcome. Given that the sensitivity of responding to outcome devaluation is not affected by NAc lesions, it is unlikely that incentive learning during the action–outcome phase is mediated by DA activity in the NAc.
Introduction
Despite more than 25 years of intensive research there is still a vigorous and ongoing debate concerning the specific aspects of complex and adaptive behaviors that are mediated by brain dopamine (DA) function (Ikemoto and Panksepp 1999; Redgrave et al. 1999; Salamone and Correa 2002; Schultz 2002; Berridge and Robinson 2003; Joseph et al. 2003; Wise 2004; Kelley et al. 2005; Salamone et al. 2005; Young et al. 2005). Much of the attention is focused on the nucleus accumbens (NAc) and a consensus has formed around the theory that DA innervation of this structure plays a key role in incentive motivation, a Pavlovian conditioned appetitive state that can influence the vigor of approach behavior (Fibiger and Phillips 1986; Robbins et al. 1989; Berridge and Robinson 1998; Cardinal et al. 2002; Parkinson et al. 2002). Consistent with the importance of conditional stimuli (CS+) in the initiation of approach behavior, visual and olfactory stimuli associated with natural rewards such as food or sexually receptive conspecifics can evoke significant increases in DA efflux in the NAc that precede similar changes observed during consummatory behavior (Fiorino et al. 1997; Ahn and Phillips 1999, 2002). Depletion or antagonism of DA function in the NAc eliminates or diminishes the influence of Pavlovian CSs on the level of instrumental responding, i.e., Pavlovian to instrumental transfer (Cardinal et al. 2002; Parkinson et al. 2002), and additionally, shifts response choice from that with higher work demand and a food reward of greater value to that with lower work cost and lower reward value (Salamone et al. 1994; Salamone and Correa 2002).
Contemporary views of instrumental conditioning propose that performance of an instrumental task is controlled by two distinct processes. Based on carefully designed experiments, Dickinson et al. (1995) have demonstrated that during initial learning trials (i.e., after exposure to 120 outcomes), an animal’s performance is based on the knowledge (or expectation) that an instrumental action will lead to a specific biologically significant outcome. After many more trials (i.e., after exposure to 360 outcomes), responses gradually shift from being outcome-dependent to habit-based. It is only in the early action–outcome controlled stage that instrumental performance is sensitive to manipulations that alter the incentive value of the outcome, underscoring the remarkable ability of rats to acquire and encode information relating current incentive value to an action–outcome contingency in as little as 120 outcome trials. Using outcome devaluation and Pavlovian to instrumental transfer tests, lesions of the basolateral amygdala have been reported to impair the capacity of rats to encode the relation between a specific action and the value of an outcome (Corbit and Balleine 2005).
Given the generally accepted role of DA in the NAc in Pavlovian-based incentive motivation, the question arises as to whether DA activity in this region is involved in action–outcome and/or habit-based based stages of instrumental responding. A role for either the core or shell region of the NAc in the formation of action–outcome associations has not been confirmed (Balleine and Killcross 1994; Corbit et al. 2001; de Borchgrave et al. 2002), but deficits in the acquisition of instrumental responding have been reported after blockade of the NMDA class of glutamate receptors in the NAc core (Kelley et al. 1997). In a recent study, Yin et al. (2006) examined DA function during early action–outcome as distinct from later habit-based stages of instrumental responding. Mice, with a knockdown of the DA transporter and chronically elevated levels of DA, showed no deficits in acquisition of an instrumental task and responding early in training was still sensitive to tests of outcome devaluation (Yin et al. 2006). However, in rats with bilateral 6-OHDA lesions of the nigrostriatal DA system, responding on instrumental tasks remained sensitive to reward devaluation despite extensive training sessions (Faure et al. 2005). Thus, the nigrostriatal DA system, and more specifically, its innervation of the posterior lateral striatum, appears to be necessary for transition of instrumental conditioning from an action–outcome stage to a habit-based stage.
Important insights into the role of DA transmission at different stages of instrumental behavior may be gained by examining in vivo changes in DA levels in major areas of DA innervation. Therefore, we conducted microdialysis experiments in the NAc and mediodorsal (MD) striatum at early (5th day) and later (16th day) stages of instrumental learning employing a protocol used successfully to demonstrate that responding after 120 but not 360 reinforced responses is sensitive to outcome devaluation and is therefore action–outcome as distinct from habit-based (Dickinson et al. 1995). Given the finding that quinolinic acid or NMDA-induced cytotoxic lesions of the NAc failed to alter suppression of instrumental responding after outcome devaluation (de Borchgrave et al. 2002), it is unlikely that activity in the NAc is involved in instrumental incentive learning. Rather, the finding that lesions restricted only to the NAc completely abolished Pavlovian to instrumental transfer is consistent with the involvement of DA function in this nucleus in conditioned appetitive states. Therefore, we hypothesize that DA efflux in the NAc may be comparable during both early action–outcome and later habit-based stages of instrumental responding. We also examined changes in DA levels in the MD striatum, as a control site for generalized activity; hence, we predicted no significant changes in DA efflux associated with instrumental responding. The design of this study also incorporated a “within-trial” extinction phase to test the hypothesis that DA efflux in the NAc but not MD striatum would be increased significantly by presentation of a Pavlovian CS+ paired previously with food pellets during instrumental responding in extinction. The relationship between response rate and magnitude of DA efflux in either NAc or MD striatum was also of interest.
Materials and methods
Surgery
Long–Evans male rats (Charles River, Canada) weighing 280–310 g were implanted bilaterally with stainless steel guide cannulae (19 gauge, 15 mm) under anesthesia induced by xylazine (7 mg/kg) and ketamine hydrochloride (100 mg/kg) delivered intraperitoneally. Cannulae were implanted 1 mm below dura immediately above the NAc [in millimeter: +1.7 anteroposteriorly (AP) and ±1.1 mediolaterally (ML) from bregma] or MD striatum (+1.2 AP, 2.5 ML) and secured with dental acrylic and four stainless steel screws. Stylets maintained patency of the cannulae until probe implantation. An additional sham-cannula was embedded within the back half of the acrylic head cap for purposes of habituation (see Behavioral apparatus and training).
Immediately after surgery, rats were moved to a reverse light-cycle (lights on 7 a.m.to 7 p.m.) colony room maintained at 20°C and housed individually in plastic bins lined with corncob bedding. Novel objects (e.g., egg cartons and paper rolls) were placed weekly in the cages to promote exploratory and play behavior. Rats were handled and weighed by the experimenter on a daily basis. Four days after surgery, rats were placed on a food restriction schedule for the duration of the experiment, maintaining their body weight at ∼85% of their free-feeding weight. The daily ration of food (20–25 g Purina Rat Chow) was given to the rats in their home cages following each day’s operant responding session. Water was available at all times except during the operant training and testing sessions.
Behavioral apparatus and training
Training and experiment sessions were conducted in a Plexiglas chamber (30×23×23 cm) with wire-mesh flooring. It was equipped with a retractable lever, a dispenser that delivered 45 mg Noyes pellets, and a light source (1 W, 12 V) located on the wall opposite the location of the lever. Pellets were dispensed into a photocell-equipped magazine located just left of the lever. The chamber was enclosed in a sound-attenuated ventilated box (Colbourn Instruments; Allentown, PA, USA) which had a small hole in the ceiling to allow passage of dialysis lines.
Training began 8 days into the food-deprivation schedule. Before a rat was placed in a testing chamber, a stainless steel coil was attached to the sham-cannula. This allowed the rat to habituate to the weight and feel of being tethered to the stainless steel coil which normally sheathes microdialysis lines during experiments. Each session began with rats being placed in operant chambers under baseline conditions (coil attached, lever retracted, and light off), the length of which was varied from day to day. Illumination of the light and insertion of the lever signaled the availability of pellets contingent on lever presses. A computer was used to control the equipment and record the number of lever presses and nose poke entries into the magazine.
At the start of the study, rats were trained to approach the magazine to retrieve 30 pellets delivered on a random-time 60-s schedule. Instrumental training sessions began the next day (day 1) on a RI 2-s schedule of pellet delivery. The schedule was changed to RI 15-s on day 2 and then to RI 30-s on day 3, and remained so for the remainder of the training and microdialysis sessions. Each instrumental training session was terminated when rats had responded for 30 pellets, except on days 4 and 15 when rats were allowed to press for 60 pellets (see Fig. 2). To minimize the effect of stress before microdialysis tests, rats remained overnight in the chambers under baseline conditions with water, after the training session on day 2.
Microdialysis probes and high pressure liquid chromatography system
Microdialysis probes were constructed as previously described (Ahn and Phillips 1999; Fiorino et al. 1997). They were concentric in design with a 2-mm semipermeable membrane (340 μm outer diameter, 65 kD MW cut-off, Filtral 12, Hospal; Neurnberg, Germany) with PE50 inlet and silica outlet tubing. A probe assembled in this manner typically had, at 21°C, in vitro recoveries of ∼22% DA. Once a probe was implanted in the brain through the guide cannula (see “Microdialysis experiments” section below for implant coordinates), a cylindrical brass collar secured the probe in place. The inlet tubing was connected to a liquid swivel (Instech 375s; Plymouth Meeting, PA, USA) which was mounted on top of the Colbourn box. Both inlet and outlet tubing were encased in a protective stainless steel coil which extended from the liquid swivel to the brass collar. A 2.5-ml gas-tight syringe (Unimetrics) and syringe pump (Model 22, Harvard Apparatus; South Natick, MA, USA) were used to perfuse a modified Ringer’s solution (in millimolar: 10 sodium phosphate buffer, 1.3 CaCl2, 3.0 KCl, 1.0 MgCl2, 147 NaCl, pH 7.4) through the probe at 1 μl/min.
DA content in microdialysis samples were separated by high pressure liquid chromatography (HPLC) and quantified by electrochemical detection. The HPLC system consisted of, in sequence of flow, a Bio-Rad pump (Hercules, CA, USA), an SSI pulse damper (State College, PA, USA) a Valco Instruments two-position auto-Injector (EC10W; Houston, TX, USA), a Beckman reverse-phase column (Ultrasphere, ODS 5 μm, 15 cm, 4.6 mm internal diameter; Fullerton, CA, USA), an ESA guard cell (Model 5020; Chemlsford, MA, USA), an ESA analytical cell (Model 5011), an ESA Coulochem II Electrochemical detector, and a dual channel chart recorder. The electrochemical detection parameters were: +450 mV for the oxidation channel, −300 mV for the reduction channel, and −450 mV for the guard cell output. The mobile phase (in millimolar: 83 sodium acetate buffer, 27 EDTA, and 1.30 sodium octyl sulfate at pH 3.5, 10% methanol) flowed through the system at 1 ml/min.
Microdialysis experiments
In all experiments, each rat was tested with microdialysis on days 5 and 16. The side of probe implantation (i.e., left or right brain structure) was counterbalanced for each experiment day. The two microdialysis sessions were conducted using an identical protocol. The day before an experiment, rats were implanted unilaterally with probes in the NAc (exposed membrane spanned −6.0 to −8.0 mm DV from dura) or MD striatum (−4.0 to −6.0 mm DV from dura) after the day’s training session and kept overnight (∼16–18 h) in the test chamber with water under baseline conditions. The next morning, microdialysis samples were collected at 10-min intervals (10 μl volume) and analyzed immediately with HPLC. Baseline conditions were continued until four consecutive samples showed stable DA levels (i.e., <10% fluctuation between samples; times 1–4).
In “Experiment 1”, the baseline period was followed by the concurrent illumination of the chamber and insertion of the lever. Rats were then allowed a 30-min period (times 5–7) during which they could lever press for food on a RI 30-s schedule. The session concluded with the retraction of the lever and a 40-min time-out period with the lights off (times 8–11). In “Experiment 2”, a 30-min extinction phase was incorporated (times 5–7) before a food reward phase. All aspects of the extinction phase were identical to previous rewarded training sessions except that lever presses did not lead to food delivery. A 20-min time-out period under baseline conditions (times 8–9) preceded a 10-min priming period when the light was on and five pellets were delivered randomly and noncontingently (time 10). The start of a food-rewarded phase (times 11–13) was marked by the insertion of the lever into the already illuminated chamber. During this period, rats were allowed to lever press for pellets on a RI 30-s schedule for 30 min. An experiment concluded with an additional 40-min postsession baseline period (times 14–17).
Histology
Rats were deeply anesthetized with chloral hydrate and intracardially perfused first with 0.9% NaCl and then phosphate-buffered formalin (3.7% formaldehyde). The removed brains were stored in 15% (w/v) sucrose in formalin for at least 24 h before being prepared as 50 μm coronal sections on 2% gel-coated slides. Cresyl violet staining was used to help verify placement of probe tracts. Only data from those rats with tracts in the shell/core region of the NAc (16 of 18 rats) and MD striatum (six of seven rats) of both hemispheres were included in the statistical analyses.
Data analyses
Neurochemical data were transformed into percentage of change from baseline (i.e., 0% representing the average concentration of the three samples preceding the final 4th baseline sample). Neurochemical data were analyzed using either a one-way (time) or two-way (day × time) repeated measures ANOVA followed by the Dunnett method of multiple comparisons, using the final baseline sample (time 4) as the control sample. The Huynh–Feldt correction for nonsphericity was applied to the degrees of freedom for all within-subject analyses. Comparisons between two means were assessed using paired t tests. A coefficient of determination (R2) was computed based on a linear regression of a scatter plot between lever presses/10 min (y-axis) and corresponding percent changes in DA efflux (x-axis), for data obtained on test days 5 and 16. Statistical analyses were performed using Systat or SPSS statistical packages.
Results
Experiment 1
Changes in extracellular DA levels in the NAc were compared after limited and extended training sessions, while rats lever pressed for food reward on a RI 30-s schedule. As shown in Fig. 1, the rates of responding approximately doubled from days 5 to 16, even though rats obtained and consumed a similar number of rewards during the two sessions [43.3 pellets (∼2.0 g) on day 5 and ∼46.2 pellets (∼2.1 g) on day 16]. The number of magazine entries also did not differ between test days. Separate one-way ANOVAs revealed a significant main effect of time on DA efflux on day 5 (F7,49=12.038, p<0.001) and day 16 (F7,49=5.325, p<0.044). Further analyses indicated that on both days, there was a significant increase in DA efflux above their respective baselines that remained elevated for the 30-min duration of instrumental responding for food pellets (Dunnett’s, p<0.05). Despite the doubling of response rates from days 5 to 16, the pattern and magnitudes of DA efflux on the two test days were not statistically different (maximal increase of 69±16% on day 5 and 71±28% on day 16), as a two-way repeated measures ANOVA failed to show a significant interaction of day × time on DA efflux (F7,98=0.598, p=0.528). However, a paired t test showed that during the first 10 min (time 5), DA efflux was significantly higher on day 16 than day 5 (68±24 vs 30±12%, respectively; p<0.05). Basal values of DA in the NAc (uncorrected for recovery) were 2.72±0.61 and 2.19±0.29 nM for days 5 and 16, respectively, and were not statistically different from each other.
Fig. 1Change in DA efflux in the NAc (line graph) during instrumental conditioning on day 5 (left panel) and day 16 (right panel) in the same group of rats (n=8). Dotted lines highlight the Reward phase in which food pellets were delivered on a RI 30-s schedule (times 5–7). Lever presses (gray bars) and magazine entries (black bars) are shown per 10 min bin. Data are represented as mean±SEM. * indicates significant difference from the final baseline value (time 4) according to Dunnett’s method of multiple comparisons, p<0.05. † indicates significant difference from the corresponding data point on day 5 according to a paired t test, p<0.05
Experiment 2
Instrumental behavior As shown in Fig. 2, all rats in the NAc and MD striatal microdialysis groups learned to lever press for food pellets on a RI 30-s schedule and to retrieve the pellets, as indicated by the number of magazine entries. Training data for one rat in the MD striatal group was lost and not included in the following analyses. Instrumental behavior became more efficient through the initial days of training, and during the third and fourth training sessions, rats made significantly more lever presses than magazine entries. This increase in ratio of lever presses to magazine entries, from limited to extended training experience, may be explained by proposing that rats learned to use the auditory click made by the dispenser with the delivery of a pellet. Thus, rats learned to enter the magazine only when they heard the click. On days (4 and 15) before microdialysis tests, rats were allowed to press for 60 pellets, rather than the normal 30 pellets available on other days, and accordingly increased the rate of lever presses on these days. The mean number of lever press responses per training session across the RI 30-s schedule tended to be higher in the NAc group (from 327 to 835) than in the MD striatal group (from 367 to 436). However, an ANOVA test revealed no main effect of group (F1,11=2.414, p=0.149). ANOVA of magazine entry data similarly indicated that counts were comparable between the NAc and MD striatal groups (F1,11=0.366, p=0.557).
Fig. 2Instrumental performance of rats in the NAc or MD striatal group over 16 training sessions. Rats were trained to lever press for food pellets on a RI 2-s schedule the first day, RI 5-s the next day, and then RI 30-s for the remainder of the study. Each training session terminated upon delivery of 30 pellets (exception: *60 pellets delivered on days before test). Test days (day 5 and 16) were composed of two phases, Extinction and Reward, which lasted 30 min each. Lever presses (gray and striped bars) and magazine entries (black bars) are shown per training session. Data are represented as mean+ SEM
On test days, rats were tested for instrumental responding in extinction and then with food reward. During the extinction phase, the pellet dispenser was disconnected from the magazine, but still produced an auditory click according to the RI 30-s schedule. During the rewarded phase, all rats consumed every pellet delivered during the lever press sessions. During the rewarded component of the experiment, rats consumed ∼45.7 pellets (∼2.1 g) on day 5 and ∼46.2 pellets (∼2.1 g) on day 16. The similarity in food consumption and difference in magnitude of DA efflux (see below), across days 5 and 16, again supports the view that DA response is not a function of food reward. In both the NAc and MD striatal groups (Figs. 3 and 4), rate of instrumental performance approximately doubled from day 5 to day 16, during both the extinction and rewarded phases of the sessions. Over the 30-min extinction phase, rats displayed a typical decline in rate of lever presses, whereas over the 30-min reward phase, there was a slight increase in rate of responding. In the NAc group, the number of lever presses was significantly higher on day 16 than on day 5 during the first 10 min of the extinction (t test, p=0.003) and rewarded (t test, p=0.002) phases. In the MD striatal group, the lever press rates during the first 10 min of the extinction phase were comparable on days 5 and 16, but differed significantly between the 2 days during the first 10 min of the rewarded phase (p=0.038). In both the NAc and MD striatal groups, an ANOVA indicated that the number of magazine entries did not differ significantly between days 5 and 16.
Fig. 3Change in DA efflux in the NAc (line graph) during instrumental conditioning on day 5 (left panel) and day 16 (right panel) in the same group of rats (n=8). Dotted lines highlight the Extinction phase during which an auditory CS+ was activated on an RI 30-s schedule in the absence of food pellet rewards (times 5–7) and a Reward phase in which food pellets were delivered on a RI 30-s schedule (times 11–13). P represents the period during which five priming pellets were delivered noncontingently. Lever presses (gray bars) and magazine entries (black bars) are shown per 10 min bin. Data are represented as mean±SEM. * Indicates significant difference from the final baseline value (time 4) according to Dunnett’s method of multiple comparisons, p<0.05. † Indicates significant difference from the corresponding data point on day 5 according to a paired ttest, p<0.05. § Indicates significant difference from the final baseline value (time 4; ttest, p<0.05)Fig. 4Change in DA efflux in the MD striatum (line graph) during instrumental conditioning on day 5 (left panel) and day 16 (right panel) in the same group of rats (n=6). See Fig. 3 for explanation of symbols. No statistically significant results were observed for neurochemical data set. † Indicates significant difference from the corresponding behavioral score on day 5 according to a paired ttest, p<0.05
DA efflux Basal values of DA in the NAc (uncorrected for recovery) were 2.58±0.32 and 2.16±0.16 nM for days 5 and 16, respectively, and were not statistically different from each other. Basal values of DA in the MD striatum (uncorrected for recovery) were 2.71±0.23 and 2.95±0.34 nM for days 5 and 16, respectively, and also did not differ statistically.
In the NAc, the overall pattern of DA efflux across the different phases of the microdialysis test on day 5 appeared comparable to that observed on day 16 (Fig. 3), but statistical analyses revealed several key differences. Accordingly, an ANOVA identified a significant day × time interaction on DA efflux (F13,182=2.760, p=0.022), with a significant simple main effect of Time on day 5 (F13,91=8.690, p<0.001) and day 16 (F13,91=14.649, p<0.001). On both days, there was an increase in NAc DA levels during the initial 10 min of responding in extinction (10±7% above baseline on day 5 and 19±6% on day 16), but this increase was only significant after extended training on day 16 (paired samples t test, p=0.005). DA efflux then returned to baseline for the remainder of the extinction phase and time-out period. During the 10-min period preceding the rewarded responding phase, five pellets were noncontingently dispensed into the magazine; the purpose of this was to prime the rats to lever press again for food reward. During this period, DA levels did not differ from baseline values on day 5 (4±9%) but by day 16, were increased significantly above baseline (27±7%; Dunnett’s test, p<0.05). The reward phase of the session was accompanied by significant elevation of DA efflux on both days (maximal increase of 37±12% on day 5 and +83±15% on day 16; Dunnett’s test, p<0.05) that remained elevated after retraction of the lever and cessation of instrumental responding. DA levels then gradually declined towards baseline values over the remaining 60 min of the test session. During the initial 10 min of the reward phase (time 11), the magnitude of change on day 16 (83±15%) was significantly greater than the change in efflux observed on day 5 (37±12%; t test, p=0.013).
In the MD striatum, there were no statistically significant changes in DA levels during the entire instrumental responding session on both days 5 and 16 (Fig. 4). Despite performing lever presses and magazine entries at rates comparable to the NAc group, DA levels in this group showed only slight fluctuations around baseline.
Correlation between response rate and DA efflux
Based on a linear regression of a scatter plot between lever presses/10 min and percent change in DA efflux, R2 values of 0.0014 for data from day 5 and 0.0065 on day 16 indicated that <1% of the variation in lever presses could be explained by a linear correlation between lever presses and DA levels, on both test days (Fig. 5).
Fig. 5Correlation between instrumental response rates and NAc DA efflux. Scatter plot of lever presses/10 min (y-axis) and corresponding percent change in DA efflux (x-axis) for data obtained on day 5 (left panel) and day 6 (right panel). Shown on each graph is the best fit linear regression line and R2 value
Histology
The locations of all microdialysis probes are presented in Fig. 6. The 2-mm lengths of the dialysis membrane were in the NAc (shell-core boundary) or medial aspect of the MD striatum (just dorsal and lateral of the anterior commissure).
Fig. 6Location of microdialysis probes in the NAc and MD striatum. Black bars represent 2 mm length of dialysis membranes. Numbers beside each plate correspond to millimeter from bregma. Coronal drawings were modified from Paxinos and Watson (1997)
Discussion
The present study examined the role of DA activity in the NAc in behaviors maintained by instrumental and Pavlovian incentive learning. DA efflux in the NAc was increased significantly during both early and later training stages of an instrumental response for food on a RI-30-s schedule of reinforcement (Figs. 1 and 3). It is important to note that this pattern of results was observed whether a period of extinction preceded a 30-min period of instrumental responding reinforced by food pellets. As such, the present findings confirmed previous reports of increased DA release in the NAc during lever pressing for food, employing fixed interval or ratio schedules of reinforcement (Salamone et al. 1994; Richardson and Gratton 1996; Cousins et al. 1999). Response actions during the early phase of training on interval schedules (i.e., in rats having received as few as 120 outcomes) have been characterized as goal- or outcome-directed (Adams and Dickinson 1981; Balleine and Dickinson 1992; Dickinson et al. 1995) and accordingly may represent instrumental incentives, as distinct from Pavlovian incentive processes related to incentive motivation (Parkinson et al. 2002). Continued performance of these instrumental actions leads to habitual responding which, unlike action–outcome learning, is impervious to outcome devaluation or contingency degradation (Dickinson et al. 1995). We failed to observe a selective increase in DA efflux when rats had limited as compared to extended training experience, as might be expected if dopaminergic activity in the NAc is related to instrumental incentive learning. Indeed, the magnitude of DA efflux was significantly greater after extended training when behavior is said to be no longer controlled by incentive learning and is based instead on habit. These increases in DA efflux were site-specific, as no significant changes in medial MD striatal DA efflux were observed throughout the different phases of this experiment (Fig. 4).
Performance on instrumental tasks is often conducted under nonrewarded or extinction conditions to evaluate the control of behavior by Pavlovian incentive stimuli, unconfounded by unconditioned reward stimuli. In the present study, a significant increase in DA efflux in the NAc was observed only during the initial 10-min sample of responding in extinction on training day 16, but not on day 5 (Fig. 3). The specific CS+ present in this experiment was a distinct auditory “click” of the pellet dispenser which occurred on a RI 30-s schedule, by itself during extinction or accompanied by delivery of food pellets into the magazine during rewarded responding. As such, these data are consistent with previous reports of increased DA efflux in the NAc elicited by a CS+ (Phillips et al. 1993; Datla et al. 2002). In a Pavlovian to instrumental transfer protocol, a CS+ previously paired noncontingently to food reward can facilitate the acquisition of an instrumental response (Parkinson et al. 2002). Systemic administration of DA receptor antagonists during Pavlovian pairings of a CS+ with food reward blocks Pavlovian to instrumental transfer (Beninger and Phillips 1981; Dickinson et al. 2000). Damage to the shell or core of the NAc spares the acquisition of Pavlovian to instrumental transfer, but disrupts the potentiation by intra-NAc amphetamine on responding for the CS+ (Parkinson et al. 2002). Together, these findings suggest that a phasic increase of DA in the NAc, shown to occur after treatment with amphetamine (Taepavarapruk and Phillips 2003; Brebner et al. 2005), may mediate the facilitatory effects of a Pavlovian CS+ on instrumental responding.
It is also of interest to note that the inclusion of an extinction session before a reinforced phase of instrumental responding attenuated the magnitude of DA efflux in the NAc observed when food reward was available on day 5, but not on day 16. This finding may be attributed to attenuation in the secondary reinforcement property of the CS+ associated with the delivery of food reward or possibly, an influence of frustrative nonreward engendered by extinction. In either case, it is apparent that these effects of extinction are restricted to the early phase of instrumental training.
In earlier studies, Salamone et al. (1994) proposed that an important aspect of dopaminergic activity in the NAc is related to behavioral activation, exertion of effort, and possibly cost benefit analyses relating effort to value of reward stimuli (Salamone et al. 2003, 2005). In support of this hypothesis, consumption of large quantities of freely available food pellets or lab chow was not accompanied by increased DA efflux. It must be noted in passing that consumption to satiety of a large meal of a palatable food such as fruit loops, onion rings (Ahn and Phillips 1999), or sucrose (Hajnal and Norgren 2002), is accompanied by a significant increase in DA efflux in both the NAc and medial prefrontal cortex. Salamone et al. (1994) also observed a significant relationship between response rates of individual rats and the magnitude of DA efflux in the NAc. Specifically, an increase in DA release was only observed in rats that responded at medium to high rates of responding, whereas in rats with low response output, this measure did not differ from controls.
Our data are also relevant to the relationship between response output and DA activity. In both Experiments 1 and 2, there was no evidence of a simple relationship between response rates and magnitude of DA efflux. As shown in Fig. 1, although the rate of lever presses were twice as high on day 16 compared to day 5, the magnitude of DA efflux did not different significantly between the 2 days. In Fig. 3, on day 5, initial response rates during the extinction and reward phases of the test were comparable during the first 10 min (times 5 and 11), yet the corresponding magnitude of DA efflux during the reward phase was three times greater than the extinction phase. A similar pattern was observed on day 16. Thus, different rates of responding were associated with similar magnitudes of DA efflux, and similar rates of responding were associated with different magnitudes of DA activity. Accordingly, no positive correlations between rate of lever press responding and magnitude of DA efflux in the Nac were observed after limited and extended training (Fig. 5). Finally, with respect to the appealing hypothesis that dopaminergic activity in the NAc is related to behavioral activation (Salamone et al. 2003, 2005), it must be emphasized that although the present data challenge this hypothesis, it cannot be refuted simply on the basis of the lack of a correlation between magnitude of DA efflux and intensity or degree of behavioral activation.
The failure to observe a significant increase in DA efflux in the MD striatum during either action–outcome or habit-based instrumental responding provides a clear indication that DA transmission in this region of the striatum is not involved in instrumental conditioning or stimulus–response habit formation. These data are consistent with the finding that neither excitotoxic lesions nor reversible inactivation of the anterior MD striatum had any effect on acquisition or expression of action–outcome associations in instrumental conditioning (Yin et al. 2005). In contrast, lesions or inactivation of the MD striatum, posterior to the probe placements in the present study, impaired instrumental performance based on outcome–expectancy (Yin et al. 2004). Blockade of NMDA receptors in the dorsomedial striatum also disrupted action–outcome learning consistent with a role for glutamate-mediated synaptic plasticity in the encoding of action–outcome associations (Yin et al. 2005). The MD (“associative”) striatum receives inputs from association cortices (e.g., prelimbic region of the prefrontal cortex and premotor areas), as well as the basolateral amygdala, which appears to mediate the assignment of incentive value to the consequences of instrumental actions (Corbit and Balleine 2005).
Integrity of the dorsolateral striatum has been shown to be required for habit formation in instrumental learning, and furthermore, rats with damage to this region of the striatum reverted to a state in which instrumental actions were goal-directed (Yin et al. 2005). This finding implies that the system involving the dorsolateral striatum responsible for habit formation can inhibit the circuit that mediates action–outcome or goal-directed instrumental actions. This in turn raises the possibility that the increase in NAc DA efflux observed in the present study after extended training provides a representation of instrumental incentive learning that is held in check by activity in the dorsolateral striatum.
In conclusion, the present findings provide neurochemical evidence in support of previous data questioning the role of DA in the NAc in coupling incentive value to representations of instrumental outcomes (de Borchgrave et al. 2002). The data showing elevated DA efflux in the NAc during extinction in the presence of a Pavlovian CS+, in turn are consistent with a role for the NAc in incentive motivation (Fibiger and Phillips 1986; Robbins et al. 1989; Phillips et al. 1993; Balleine and Killcross 1994; Berridge and Robinson 1998). Rats received an apportionment of ∼60 food pellets during microdialysis test on days 5 and 16, yet the magnitude of DA efflux was significantly greater after extended training sessions. These data refute the hypothesis that dopaminergic activity in the NAc is a reflection of either reward value or reinforcement of instrumental responses (Wise 2004). The present “within subject” design revealed a hitherto unappreciated effect of extended training of instrumental responding with an interval schedule on the magnitude of DA efflux in the NAc. For reasons discussed above, this does not appear to be related to motor responding per se. Rather, we speculate that this effect may reflect the specific condition of a random or variable interval schedule of outcome presentation, in which extended training is necessary to appreciate that the probability of receiving a beneficial outcome at any particular time in the 30-min test session is always unpredictable. This degree of uncertainty may be highly compatible with the optimal conditions for activating midbrain DA neurons (Fiorillo et al. 2003), which in turn would result in a sustained increase in DA release in the NAc throughout a period of random reinforcement. This pattern of DA release could play an important role in maintaining a high level of motivation at the service of a variety of response strategies available to ensure access to objects essential for survival.

dopamine
nucleus accumbens
habit
food reward
action–outcome
microdialysis
instrumental learning
uncertainty
mediodorsal striatum


The mouse complement regulator CD59b is significantly expressed only in testis and plays roles in sperm acrosome activation and motility

In mouse, genes encoding complement regulators CD55 and CD59 have been duplicated. The first described form of CD59, CD59a, is broadly distributed in mouse tissues, while the later identified CD59b was originally described as testis specific. Subsequent studies have been contradictory, some reporting widespread and abundant expression of CD59b. Resolution of the distribution patterns of the CD59 isoforms is important for interpretation of disease studies utilising CD59 knockout mice. Here we have performed a comprehensive distribution study of the CD59 isoforms at the mRNA and protein levels. These data confirm that expression of CD59b is essentially restricted to adult testis; trace expression in other tissues is a consequence of contamination with blood cells, shown previously to express CD59b at low level. In testis, onset of expression of CD59b coincided with puberty and was predominant on the spermatozoal acrosome. Ligation of CD59b, but not CD59a, markedly reduced spermatozoal motility, suggesting a specific role in reproductive function.
1
Introduction
CD59, a broadly distributed glycosyl phosphatidylinositol (GPI)-anchored protein, is the principal regulator of complement membrane attack complex (MAC) assembly on cell membranes (Meri et al., 1990). A decade ago, we identified the mouse analogue of CD59 and showed that it was broadly distributed on cells and tissues (Powell et al., 1997). Deletion of the gene encoding mouse CD59 caused minimal disturbance in unchallenged animals but markedly enhanced susceptibility to complement-driven pathologies (Holt et al., 2001; Turnberg et al., 2003, 2004; Lin et al., 2004; Mead et al., 2004; Williams et al., 2004). Others demonstrated that the gene encoding CD59 is duplicated in the mouse; the protein products of these two genes were then termed CD59a for the first described, and CD59b for the product of the new gene (Qian et al., 2000). In this first report, it was stated that CD59b message was significantly expressed only in testis. We developed monoclonal antibodies specific for CD59a and CD59b, enabling us to confirm the broad distribution of CD59a and testis restricted expression of CD59b at the protein level; we concluded that CD59a was the principle regulator of MAC assembly in mouse tissues (Harris et al., 2003). This conclusion was challenged in a re-analysis of the pattern of distribution of mRNA encoding the two forms of CD59; these studies suggested that both CD59a and CD59b were broadly expressed in the mouse and contended that the latter was the major regulator of MAC in the mouse (Qin et al., 2003). These contentions were bolstered by the demonstration that deleting the gene encoding CD59b in mice caused a severe, spontaneous phenotype with anaemia, platelet dysfunction and fertility problems. In light of these data we reassessed the distribution patterns at protein and mRNA levels using a combination of specific monoclonal antibodies and shared primers for PCR in high sensitivity assays (Baalasubramanian et al., 2004). These analyses confirmed our earlier work showing that CD59a was broadly expressed while significant expression of CD59b was restricted to testis. Trace amounts of CD59b message and protein were detected in erythrocytes but quantitative assays of protein expression showed that CD59b expression on erythrocytes was less that 5% that of CD59a. Further, we confirmed that CD59a was dominant in protection of erythrocytes from MAC lysis. The findings of these painstaking studies have recently been challenged yet again (Qin et al., 2006). The appropriateness of the techniques used and specificity of probes and primers have all been questioned. These authors concluded that CD59b was broadly expressed and an important regulator of MAC assembly on erythrocytes and in tissues.
We, and others, are using CD59a knockout mice in disease models to explore roles of MAC based on our evidence that CD59a is the principal regulator of the MAC in most tissues. If CD59b is indeed widely distributed then the value of studies in CD59a knockouts is in question. It is therefore essential that we test the evidence. Here using multiple methods we revisit these published studies and undertake new analyses to explore the expression patterns of CD59a and CD59b at the mRNA and protein level. We conclude that expression of CD59b at the mRNA and protein level is essentially absent in all tissues other than testis. Low level expression on blood cells was confirmed and trace detection of mRNA in tissues was shown to be likely due to blood contamination. We further analysed expression of CD59b in testis and showed that expression coincided with onset of puberty and was restricted to spermatozoa and their immediate precursors. Ligation of CD59b on spermatozoa with monoclonal antibody markedly inhibited sperm motility, suggesting a specific role in reproductive function.
2
Materials and methods
2.1
Mice
Adult (8–16 weeks) and infant (1–5 weeks; pre-puberty) male mice, and embryos (day 5, 10 and 15) from C57BL/6(H-2b) background mice were used in our investigation. All experimental procedures were performed in compliance with Home Office and local ethics committee regulations. cd59a−/− mice generated as described previously (Holt et al., 2001), and back-crossed 10 generation onto the same background were used as controls.
2.2
Antibodies and reagents
Rat anti-mouse CD59a (CD59a.1; IgG1) was generated in house (Harris et al., 2003). Mouse anti-mouse CD59b (CD59b.2; IgG1) was also made and characterized in house (Baalasubramanian et al., 2004). Separate aliquots of CD59a.1 and CD59b.2 were labelled with NHS-biotin (Sigma–Aldrich, Gillingham, Dorset, UK) and FITC-NHS (Perbio Science UK Ltd., Cramlington, UK) according to manufacturer's protocols. As negative controls, rat IgG1 and mouse IgG1 (purified in-house) were labelled in the same manner. FITC-labelled streptavidin was purchased from DAKO (Ely, Cambridgeshire, UK). HRPO-labeled donkey anti-rat IgG and HRPO-labeled donkey anti-mouse IgG were purchased from Jackson ImmunoResearch Europe (Newmarket, Suffolk, UK).
2.3
Semi-quantitative RT-PCR
Total RNA was purified from all investigated mouse tissues using GenElute kit (Sigma–Aldrich) and controlled for DNA contamination by RT-PCR without using reverse transcriptase and employing a β-actin specific primer pair (Table 1). Only samples that did not show amplification were used in all analyses. Aliquots of these RNAs (1 μg each) were reverse transcribed using random hexamers and multiscribe reverse transcriptase according to the manufacturer's instructions (Applied Biosystems, Warrington, UK). Primer pairs specific for either CD59a or CD59b were as described by Qin et al. (2006), and a new primer pair common for both CD59a and b and amplifying a sequence in exon 4 comprising 199 bp for CD59a and 162 bp for CD59b, were used in the subsequent amplifying reactions for detection of CD59a and CD59b mRNAs (Table 1). Either 25 or 35 amplification cycles were used, except where attempting simultaneous detection of both mRNAs when 40 cycles were performed. Amplified products were separated either in 1% agarose or in 5% poly-acrylamide gels (PAAG). To obtain semi-quantitative data for expression of the two genes, we used as templates either 2 μl of the original cDNA, or a series of dilutions as indicated in figures. Gels were scanned and band intensity was measured by densitometry using Quantity One 4.3.0 software (BioRad, Hemel Hempstead, Hertfordshire, UK). The quantifications were carried out in triplicate and means and errors calculated.
2.4
Quantitative real-time PCR analysis
The cDNAs from testis and liver prepared as described for the semi-quantitative RT-PCR, were subject to a Taqman assay as described previously (Baalasubramanian et al., 2004). One of the primer pairs was as described previously (Baalasubramanian et al., 2004), a perfect match for the sequence of CD59a mRNA and with a single internal nucleotide mismatch to CD59b mRNA; the second set was a perfect match for the same sequence in CD59b and with a single internal nucleotide mismatch to CD59a (Table 1).
To measure the relative number of mRNA copies for CD59b in liver, perfused liver and testis we used the primer pair reported by Qin et al. (2006) to specifically amplify CD59b in a quantitative PCR (QPCR). Because these primers were not designed specifically for this assay, they did not meet the optimal annealing temperature requirements. To correct for this anomaly, the standard amplification programme was modified to include a pre-annealing step at 56 °C for 20 s prior to the extension phase.
Primers were designed to β-actin (Table 1) as an internal control for normalization of starting cDNA levels. Quantitative PCR was performed on ABI PRISM 7000 using either TaqMan Universal PCR Master Mix or SYBR Green PCR Master Mix according to the manufacturer's instructions (Applied Biosystems) with 50 cycles of amplification.
2.5
Preparation of tissue lysates
To obtain tissue lysates, freshly harvested mouse organs were immediately chilled and homogenized with ice-cold lysis buffer (PBS containing 2% NP40, 1 mM phenylmethylsulfonyl fluoride, 10 mM EDTA, 1 μg/ml leupeptin, and 1 μg/ml pepstatin; 1 g of tissue/1.5 ml of buffer) and incubated for 60 min on ice. Insoluble debris was removed by centrifugation (5000 × g, 15 min at 4 °C) and the supernatants stored in aliquots at −80 °C until use. In some experiments the mouse was perfused with saline at the time of sacrifice to reduce blood contamination in harvested organs.
2.6
SDS-PAGE and Western blot analysis
Lysates were mixed 1:1 with sample buffer for SDS-PAGE and separated under non-reducing conditions in 15% gels. Separated proteins were transferred onto nitrocellulose membranes (Schleicher & Schuell, London, UK), and membranes blocked with 5% (w/v) non-fat milk in PBS (PBS-M). Membranes were then probed with the primary mAb diluted in PBS-M, washed in PBS containing 0.1% Tween-20 (PBS-T), then probed with HRPO-conjugated donkey anti-rat Ig in PBS-M to detect the rat anti-CD59a or HRPO-conjugated donkey anti-mouse Ig (Jackson) to detect the mouse anti-CD59b. After further washing in PBS-T, bands were developed using ECL (Perbio Science UK Ltd.) and captured on autoradiographic film (Kodak Ltd., Hemel Hempstead, Hertfordshire, UK).
2.7
Spermatozoa preparation and analysis
Motile cauda epididymal spermatozoa were obtained by a ‘swim-up’ technique as previously described (Mizuno et al., 2004, 2005). Briefly, two cauda epididymi from an adult C57BL/6 mouse were roughly minced in 1 ml of DMEM (Invitrogen, Paisley, UK). This suspension was carefully overlayered with 1 ml DMEM and incubated at room temperature for 15 min. The upper layer was removed and spermatozoa pelleted by centrifugation (1000 × g) for 5 min at room temperature. Cells were washed twice by gentle centrifugation as above. For immunofluorescence studies, the re-suspended cells were smeared onto glass slides, air-dried immediately, fixed in acetone at room temperature for 1 min and stored at −20 °C until use. To prepare lysate, the swim-up spermatozoa from 4 cauda epididymi were pelleted and incubated with mixing in 100 μl of lysis buffer for 30 min on ice. Insoluble debris was removed by centrifugation (15,000 × g, 15 min at 4 °C) and the supernatant stored in aliquots at −80 °C.
To compare CD59b expression in unactivated and acrosome-reacted spermatozoa, the acrosome reaction was induced essentially as described (Mizuno et al., 2004). Briefly, swim-up spermatozoa (106 cells/ml of DMEM) were incubated for 1 h at 37 °C with the calcium ionophore A23187 (Sigma–Aldrich) at 1 μM to induce the acrosome reaction. Control cells were incubated without ionophore. Acrosome-reacted and control spermatozoa were smeared on glass slides and immediately air-dried. To observe CD59b distribution, the smears were incubated with FITC-labeled CD59b.2. Nuclei were counterstained with DAPI (4′-6-diamino-2-phenylindole-2 HCl; 100 ng/ml final concentration; Sigma–Aldrich). On each slide, at least 100 cells were counted and the assay was carried out in triplicate.
2.8
Functional inhibition assay of CD59a and CD59b in mouse sperm
Spermatozoa harvested by swim-up from two epididymi, either from wild type or cd59a−/− mice, were washed and suspended in 2 ml DMEM. Paired aliquots (200 μl) were incubated at 37 °C with mAbs CD59a.1, CD59b.2, or isotype-matched control mAb, each at 10 μg/ml. After 4 h of incubation, the sample was immediately placed on a glass haemocytometer slide and the total number of sperm and percentage remaining motile were counted under light microscopy. This experiment was performed in triplicate.
2.9
Statistical analysis
All values are expressed as mean ± standard error (S.E.M.). The statistical analysis was performed by one-way ANOVA. When significant differences were observed, statistical analysis was further carried out using unpaired t-test between two groups. Significance between two groups was claimed when P < 0.05.
3
Results
3.1
CD59b mRNA is highly expressed only in testis
We have previously demonstrated that CD59a is the primary regulator of MAC assembly in mouse (Baalasubramanian et al., 2004). However, in a recent work (Qin et al., 2006), abundant expression of CD59b mRNA in a number of tissues was reported. In an attempt to clarify this issue, which is of key importance for complement studies in mice, we repeated some of the experiments carried out by Qin and co-authors using the primer pairs designed by them. Under routine conditions (25 cycles of amplification), CD59a mRNA was detected in all tissues tested while CD59b mRNA was found only in testis (Fig. 1A). Increasing the number of amplification cycles to 35 (Fig. 1B) revealed weak expression of mRNA for CD59b in liver, blood cells, heart and lung. Amplified sequence was confirmed to be CD59b by sequencing.
We next re-visited the primers used in our previous quantitative PCR analysis to assess expression of the two CD59 isoforms in different mouse tissues (Baalasubramanian et al., 2004). We had designed a primer pair that annealed to both isoforms and Taqman probes specific for either CD59a or CD59b to enable accurate quantitation. Qin et al. (2006) criticised these experiments on the basis that, while both primers used in our assay matched perfectly CD59a, each had a single internal nucleotide mismatch compared to the CD59b sequence (Table 1). They suggested, without evidence, that this mismatch favoured amplification of CD59a and caused our inability to detect CD59b in tissues other than testis and bone marrow. There is a large primer design literature that shows clearly that internal mismatches, unlike those at the 3′ end, do not significantly influence amplification (Sommer and Tautz, 1989; Kwok et al., 1990; Christopherson et al., 1997; Löffert et al., 1998). Nevertheless, we addressed further this issue by repeating our previous QPCR investigation but including a second primer pair from the same sequence matching perfectly CD59b mRNA but with a single mismatch to CD59a mRNA in the same position as in the original primer set (Table 1). Using the original primer pair that matched perfectly CD59a we obtained very similar results to those we previously published (Baalasubramanian et al., 2004). When we used the pair matching CD59b, there was a small increase in threshold cycle (Ct) for both CD59a and CD59b, likely a result of altered primer annealing temperature, but there was no difference in the calculated relative amounts for CD59a and CD59b mRNA in testis compared to the original primer set (Table 2). Of note, QPCR with either primer set did not detect any CD59b mRNA in liver, suggesting that the high cycle number PCR described above was detecting very small amounts of mRNA in these tissues. In summary, the data shows an 8-fold greater expression of CD59a mRNA in liver compared to testis, a 5-fold greater expression of CD59a mRNA compared to CD59b in testis and undetectable expression of CD59b in liver, regardless of primer set used.
In light of these results we developed a semi-quantitative PCR to quantify the low levels of CD59b mRNA detected in tissues. In order to be able directly to compare the expression of both isoforms, we designed a new primer pair within exon 4 (Table 1), which is 100% homologous to both mRNAs. Amplification of DNA-free mRNA with these primers will result in bands of 199 bp for CD59a and 162 bp for CD59b. In testis we detected both isoforms as expected (Fig. 2A). Densitometric comparison of band intensities at higher dilutions of template, when the amplification reaction is in the linear range, showed approximately 5-fold higher expression of CD59a mRNA as compared to CD59b mRNA in testis, a result compatible with the QPCR data (Table 2) and our published results (Baalasubramanian et al., 2004). However, in liver we failed to detect presence of CD59b even after 40 cycles of PCR (Fig. 2B). We reasoned that the large excess of CD59a mRNA in liver out-competes trace amounts of CD59b mRNA for limited reagents in the initial cycles of amplification, thereby reducing the chance for amplification of the low abundance CD59b mRNA. To test this reasoning we performed semi-quantitative PCR in separate tubes, using the primers specific for either CD59a or CD59b mRNA (Qin et al., 2006). Firstly, to optimise the annealing and amplification efficiency for both primer pairs, we performed this reaction for testis mRNA (Fig. 3A). Similar band intensities for CD59a and CD59b were obtained for 5 × 105-fold and 105-fold template dilution respectively (Fig. 3A and B). This indicated an approximate 5-fold higher expression of CD59a mRNA compared to CD59b mRNA, supporting our results described above (Table 2) and published (Baalasubramanian et al., 2004), and confirming similar amplification efficiency for the specific primer pairs. Amplification of liver mRNA using these primers, and comparison of band intensities in dilutions of templates showed that expression of CD59b mRNA in liver was approximately 105-fold less compared to that of CD59a mRNA (Fig. 3C and D similar band intensities for CD59a and CD59b at 5 × 105 and 5-fold dilution respectively). In these latter experiments we used liver perfused with saline at the time of sacrifice to remove the bulk of entrapped blood; nevertheless, the trace mRNA detected might be from residual blood cells, which clearly express CD59b protein at low level (Baalasubramanian et al., 2004; Qin et al., 2006). To clarify this issue, we compared mRNA from perfused and non-perfused liver by QPCR using CD59b-specific primers and found the amount of CD59b mRNA in liver was reduced 12-fold by perfusion (Fig. 3E). For comparison, expression of CD59b in testis was 2200-fold higher than in unperfused liver and approximately 25,000-fold higher than in perfused liver. Taken together these results strongly suggest that the detectable traces of CD59b mRNA in liver and likely other organs are a consequence of contamination with blood cells.
3.2
Expression of CD59b protein is testis restricted
We have previously reported, using immuofluorescence staining, that CD59b protein was highly expressed in testis but absent from all other organs tested (including liver, lungs, spleen, kidney and heart) (Baalasubramanian et al., 2004). We here extend these studies by Western blotting of testis, liver, lungs, plus aorta, chosen because of the suggested role of CD59 in vascular disease (Qin et al., 2004) (Fig. 4). Each organ was perfused with saline prior to preparation of protein lysates. CD59a was strongly detected in all the tissues; however, CD59b was detected only in testis lysates. These data confirm our published contention that organ expression of CD59b protein is restricted to testis.
3.3
Expression of CD59b mRNA in testis coincides with puberty and plays a role in spermatozoal motility
We previously reported that CD59b expression in testis was restricted to developing and mature spermatozoa (Baalasubramanian et al., 2004). To confirm this germ cell restricted pattern we examined expression of CD59b mRNA in testis from pre-pubertal mice. CD59b mRNA was absent in testes harvested from mice at days 10 and 20 post-partum and appeared only from day 30 on, coincident with puberty (Fig. 5A). In contrast, CD59a mRNA was present in testes at all timepoints. RNA extracted from embryos at days 5, 10 and 17 were all positive for CD59a but negative for CD59b mRNA (Fig. 5A). These observations were confirmed by Western blotting (Fig. 5B). Expression of CD59b protein was highest in spermatozoa, intermediate in adult testis and absent from infant testis. CD59a was present in all the lysates, albeit at low level in spermatozoa and with a reduced apparent molecular mass compared with the testis protein, suggesting modification during sperm maturation.
We previously reported that CD59b on spermatozoa was focussed on the head region in a highly granular pattern (Baalasubramanian et al., 2004). Here we have explored the expression pattern of CD59b in more detail. In freshly isolated spermatozoa, more than 80% showed this head/granular staining pattern, a minority showing a more diffuse staining on the head region (Fig. 6). CD59b was weakly expressed on spermatozoal tails and, in about two thirds, strongly in the mid-piece. Initiation of the acrosome reaction (with A23187) caused a precipitous loss of CD59b expression on sperm heads, more than 60% of cells being negative by 30 min and 90% by 180 min post-initiation, indicating that CD59b was shed with the outer acrosomal membrane (Fig. 6). These data suggested that CD59b might play a role in acrosome function, an essential component of sperm capacitation for fertilisation. Therefore, we next investigated whether ligation of CD59b with antibody influenced spermatozoal mobility, a surrogate marker for fertilisation capacity (Fig. 7). Ligation of CD59b caused a significant suppression of motility of spermatozoa harvested from both wild type and cd59a−/− mice. As a control, CD59a was similarly ligated but did not alter spermatozoal motility compared with the effect of a control antibody. The data suggest that CD59b but not CD59a has a role in regulating spermatozoal motility. These findings are of particular relevance in that a major feature of the CD59b knockout mouse was spermatozoal dysfunction that included diminished motility and infertility (Qin et al., 2003).
4
Discussion
We have recently examined the distribution patterns of the two isoforms of CD59 in the mouse and concluded, based upon its broad distribution, that CD59a is the primary regulator of MAC assembly in mouse (Baalasubramanian et al., 2004). We were unable to detect the expression of CD59b, either at mRNA or protein level, in brain, lungs, heart, liver, spleen and kidney. These data have recently been questioned by Qin et al. (2006) who first used a BLAST search of the mouse EST database and found ESTs matching the CD59b sequence from several tissues and organs. These in silico data were supported by RT-PCR analyses using primer sets specific for CD59a and CD59b, respectively that showed abundant expression of CD59b mRNA in all tissues tested, indeed, expression in testis was lower than in other organs. The apparent absence of CD59b mRNA in the CD59a knockout mouse in these published data was unexplained.
Here we designed a number of quantitative and semi-quantitative assays to comprehensively address this controversial issue. Our data unambiguously demonstrated that expression of CD59b is essentially restricted to testis (Figs. 3 and 4). A comparative quantitative analysis of expression of CD59b mRNA in perfused and unperfused liver (Fig. 3) showed a 12-fold decrease in expression of CD59b following perfusion, strongly suggesting that the trace detection of this mRNA was a consequence of contamination with blood cells. This would explain why Qin et al. (2001) were able to detect CD59b in multiple mouse tissues using Northern analysis in which they loaded 10 μg of mRNA in each lane, a huge excess for this sensitive procedure. The contamination likely also explains the presence in multiple tissues of CD59b-specific EST sequences.
We further demonstrated that the testis expression of CD59b coincides with puberty, supporting our findings of expression only on spermatozoa and their immediate precursors (Fig. 5). We found that CD59b was released from spermatozoa heads upon acrosome activation (Fig. 6), an observation that strongly support a role for this protein in functioning of acrosome. Ligation experiments with antibodies showed that CD59b but not CD59a was involved in spermatozoa motility (Fig. 7), providing support for an earlier report describing decreased motility and viability of sperm in cd59b KO mice (Qin et al., 2005).
5
Concluding remarks
The data presented here, obtained using a broad panel of reagents and methods designed to give unbiased and unequivocal results, show that CD59b expression is limited. CD59b mRNA is abundantly expressed only on male germ cells and present in trace amounts in bone marrow and blood cells, but is absent from other organs where trace detection of mRNA is likely due to blood contamination. CD59b protein is abundant only on developing and mature spermatozoa. Erythrocytes express CD59b at low levels that we have previously quantified as less than 200 molecules per cell, irrelevant for protection from complement when CD59a is present at 2500 molecules per cell (Baalasubramanian et al., 2004). We show a unique distribution pattern of CD59b protein on spermatozoa, the precipitous loss of this protein with outer acrosomal membranes and effects of CD59b ligation on sperm motility. We conclude that CD59a is indeed the principal regulator of MAC expressed in the mouse and that the CD59a knockout is an appropriate model for studying the roles of MAC and its regulation in disease models. The CD59b knockout mouse might prove of value for studies of fertility; however, it should be noted that the mouse described by Halperin and co-workers is, for unexplained reasons, also markedly deficient in CD59a (Qin et al., 2006), limiting its utility for studying specific roles of CD59b in vivo.

mouse
complement
cd59b
acrosome
cd59a
reproductive immunology


Family history is neglected in the work-up of patients with colorectal cancer: a quality assessment using cancer registry data

In the diagnostic work-up of hereditary non-polyposis colorectal cancer (HNPCC, Lynch syndrome), high-risk patients can be identified using information from the family history on cancer (‘Amsterdam criteria’ and ‘Bethesda guidelines’). To investigate to what extent the medical specialists apply these criteria to patients with colorectal carcinoma and a suspicion of HNPCC, we collected information on diagnostic work-up of 224 patients of seven hospitals in the region of the Comprehensive Cancer Centre West in Leiden, The Netherlands. These patients were diagnosed with colorectal cancer between 1999 and 2001 and satisfied at least one of the Bethesda guidelines. A complete family history was recorded for 38 of the 244 patients (16%). Patients with a complete family history were more likely to be referred to the Clinical Genetic Centre than those with an incomplete or absent family history (53% vs. 13% and 4%, respectively; P < 0.0001), and more likely to be analyzed for microsatellite instability (MSI), which is a characteristic of HNPCC (34% vs. 6% and 1%, respectively; P < 0.0001). We conclude that the family history is neglected in the majority of patients with colorectal cancer and MSI-analysis is only performed in a small proportion of the patients that meet the guidelines for this analysis.
Introduction
Colorectal cancer (CRC) is one of the most common forms of cancer in Western society. Every year 9,500 patients in the Netherlands are diagnosed with the disease and almost half of them die from it (Dutch Cancer Registry, 2002). It is estimated that in around 20% of the patients with a colorectal tumour genetic factors play a role in the aetiology [1]. About 1–5% of the patients with CRC are thought to have hereditary non-polyposis colorectal cancer (HNPCC, Lynch Syndrome); a dominant hereditary disease, which is caused by a defect in one of the DNA-Mismatch Repair (MMR) genes [2]. The most important clinical characteristics of CRC associated with HNPCC, are the relatively young age at which patients are diagnosed with the disease (average <45 years old) and the proximal localisation of the tumour in the colon. Besides an increased risk of developing a tumour in the colon, there is an increased risk of developing a tumour elsewhere in the body, especially in the endometrium (lifetime risk: 50%), the small intestines, the ovaries, the brain, the urinary tract, the biliary tract and the development of a keratoacanthoma or a carcinoma of the sebaceous glands of the skin [3].
The identification of patients with hereditary colorectal carcinoma is of great importance for the patient, because the treatment and follow-up of the tumour differ from those with non-hereditary colorectal carcinoma [4]. Furthermore, the identification of these patients is important, because it offers efficient manners for the prevention of colorectal carcinoma and other forms of cancer for the patient himself as well as his family [5]. It has been shown that a colonoscopy every three years can lead to a decline in mortality of at least 65% [6].
The genetic defect in HNPCC can be detected by immunohistochemic staining of the MMR proteins in tumour cells [7]. Another method to select families for mutation analysis of the MMR genes is analysis of errors in repetitive DNA-sequences, i.e., Micro Satellite Instability (MSI) [8]. MSI is found in around 15% of the non-selected CRC and in more than 95% of the colorectal tumours associated with HNPCC [9]. In 1996, guidelines were formulated for patients whose tumours should be analyzed for MSI [10]. These so-called Bethesda-guidelines describe practically all situations, where there is a suspicion of HNPCC. Recently these guidelines have been revised (Table 1) [11].
Table 1Guidelines for the performance of MSI-analysis of colorectal tumour [11]Revised Bethesda-guidelinesA person with colorectal carcinoma diagnosed at age ≤50A person with colorectal carcinoma and MSI-associated pathologya<60 yearsA person with colorectal carcinoma and a HNPCC associated tumourbA person with colorectal carcinoma and a first degree relative with a colorectal or HNPCC associated tumour; at least one of the tumours is diagnosed before the age of 50Three relatives diagnosed with colorectal carcinoma or a HNPCC associated tumour, diagnosed at any age; one patient needs to be a first degree relative of the other twoaThe presence of tumor-infiltrating lymphocytes, so called, "Crohn's like lymfocyte reaction", mucinous or signet ring cell carcinoma differentiation or medullary growth patternbCarcinoma of the endometrial tissue, stomach, small intestines, pancreatic gland, biliary tract, urinary tract, ovaries, brain, keratoacanthoma and carcinoma of the sebaceous glands
Before the discovery of the MMR-genes, the most common approach in the diagnostic work-up for HNPCC was to use the ‘Amsterdam criteria’. These criteria are met if there are, within one family, three individuals with a colorectal (or another HNPCC-associated kind of) tumour, of whom one person is a first degree family member of the other two and at least one carcinoma is diagnosed before the age of fifty [12]. To evaluate the Amsterdam criteria in patients with CRC, a complete history on cancer in the patient’s family has to be obtained. Until now it is not known whether an adequate family history is taken of all patients with a colorectal carcinoma. We also do not know to what extent medical specialists use the above mentioned clinical Bethesda guidelines and if the tumours of all the patients who match the criteria are tested for MSI. The objective of the present study was to answer these questions using data of the Cancer Registry of the Comprehensive Cancer Centre West (CCCW) in Leiden, The Netherlands.
Patients and methods
We selected patients who were diagnosed with a primary and invasive colorectal tumour in the period 1999–2001 from the Cancer Registry (CR) of the Comprehensive Cancer Centre West (CCCW) in The Netherlands. The patients had to satisfy one of the following two Bethesda guidelines: the patient had to have more than one tumour, i.e., one colorectal carcinoma and a second one (colorectal cancer or another HNPCC-associated kind of tumour), or the patient had to be fifty years or younger at diagnosis. The selected patients were considered to have an indication for the performance of MSI-analysis and/or referral to the Clinical Genetic Centre (CGC). Patients with a carcinoma in situ or a carcinoïd of the appendix were not included in the analysis.
Between 1999 and 2001, 434 patients who complied with the above mentioned criteria were diagnosed with CRC in one of the twelve hospitals in the CCCW-region. Seven hospitals gave permission for the collection of information concerning family history, MSI-analysis and referral to the CGC. We extracted this information from the various (electronic) medical reports. The family history was considered complete if the medical records reported on cancer in the family, and if so, information about the age at the time of the diagnosis, the type of cancer and the occurrence of cancer within first-degree and second-degree family members.
Data were collected of 244 patients. Of these patients, 120 patients had multiple tumours, 109 patients were fifty years or younger at the time of diagnosis, and 15 patients had both characteristics. For comparisons between patients with multiple tumours and patients who were young at diagnosis, those with both characteristics were allocated to the “multiple tumours” group. The data were analyzed using SPSS statistical software (version 12.0.1). Univariate comparisons of proportions between patient groups were performed by Chi-squared test. Multivariate logistic regression analysis was used to study whether the presence of a complete family history or referral to a CGC could be explained by age, sex, inclusion criterion (multiple tumours or young age at diagnosis), hospital or type of medical specialist.
Results
The study group consisted of 244 persons, who complied with one of the Bethesda guidelines and therefore were considered to be referred for MSI-analysis and/or genetic counselling. The male:female ratio was 49:51 and did not differ between the groups selected on the basis of multiple tumours or age ≤50 years at diagnosis.
A complete family history was recorded in the medical records of 38 (16%) of the 244 patients. For 136 patients (55%) limited information on the family history was available, and for 70 (29%) patients no information on the family history was found in the medical records. In the seven participating hospitals, a family history was reported for 38–91% of the patients.
Of the 38 patients with a complete family history, 20 (53%) were referred to the CGC. This percentage was higher than that of patients with an incomplete family history (13%) and that of patients without any information on family history (4%) (P < 0.0001, Table 2). MSI-analysis was performed more often in the patients with a complete family history: 34% of patients with a complete family history compared to 6% of patients with an incomplete family history and 1% of patients without any family history (P < 0.0001) (Table 2). Presence of a complete family history and the performance of MSI-analysis were not associated with age, sex, inclusion criterion (multiple tumours or young age at diagnosis), hospital or type of medical specialist (multivariate logistic regression analysis; data not shown).
Table 2Diagnostic work-up for HNPCC in 244 patients with colorectal cancer, by completeness of the family history as reported in the medical recordsDiagnostic workupFamily history complete (n = 38)Family history incomplete (n = 136)Family history absent (n = 70)Referred to CGC20 (53%)17 (13%)3 (4%)MSI-analysis performed13 (34%)8 (6%)1 (1%)Results of MSI-analysis3 MSI, 10 stable7 stable, 1 unknown1 stableDiagnosis of HNPCC6 (16%)3 (2%)1 (1%)
Discussion
We used the Bethesda-guidelines to select a group of patients with a suspicion of HNPCC. These patients were diagnosed with colorectal cancer between 1999 and 2001, a period during which MSI-analysis and the Bethesda guidelines were already available. Therefore we expected that for these patients, physicians would have examined and reported their patients’ family history and that MSI-analysis would have been performed. In our study group, however, the family history of the patients diagnosed with colorectal carcinoma was not sufficiently examined and reported in the medical records. For this reason, we believe the Bethesda-guidelines were not sufficiently applied by the physicians. As a consequence, MSI-analysis was performed on a small proportion of the tumours. More patients with a complete family history in their medical records were referred by their physicians to the CGC than patients without such a family history. MSI-analysis was also performed more often in this group. We expect that in a low-risk population, i.e., patients with colorectal cancer who do not meet the Bethesda guidelines, these results would be even more dramatic.
On the one hand, our results may appear to be better than they actually are. We collected our data using medical records from various medical specialties, while the treating physician will not have this overview in practice. On the other hand, it is possible that when a physician examined a family history and none of the family members was diagnosed with cancer, he did not report it in the medical records. In this case, the family history was considered as absent, although it in fact was examined. Nevertheless we expect that if MSI-analysis was performed or the patient was referred to the CGC, this would have certainly been reported.
We found that the attention for HNPCC in the diagnostic workup for CRC differed widely. For the seven participating hospitals, the proportion of patients with a reported family history on cancer ranged from 38% to 91%. Furthermore, only half of the approached hospitals were willing to cooperate. For these reasons, we cannot generalise our results for the whole CCCW-region. Nevertheless, we conclude that the family history appears to be neglected in the majority of patients with colorectal cancer in our study period, and that MSI-analysis was only performed in a small proportion of the patients that meet the guidelines for this analysis. Possibly, the attention for identification of patients with HNPCC has increased in more recent years. Our findings underscore the importance of implementation of family history and Bethesda guidelines in the physician education.


Elimination of daily routine chest radiographs in a mixed medical–surgical intensive care unit

Objective To determine the impact of elimination of daily routine chest radiographs (CXRs) in a mixed medical–surgical intensive care unit (ICU) on utility of on demand CXRs, length of stay (LOS) in ICU, readmission rate, and mortality rate.
Introduction
Chest radiographs (CXRs) are frequently obtained as a complement to physical examination of critically ill patients [1, 2]. There are two different schools of thought regarding the utility of CXRs in the intensive care unit (ICU): The CXRs should be made on indication only, specifically when there is a sound reason to obtain a film (so-called on demand CXRs); or CXRs should be obtained routinely every day, that is, without any specific reason (so-called daily routine CXRs). Argument for the latter strategy is the high prevalence of findings on CXRs of ICU patients [3]; however, interpretation of studies on the usefulness of daily routine CXRs is hampered because of major differences in methodology [4]. Importantly, most studies did not attempt to discriminate between clinically relevant and irrelevant findings. We recently demonstrated that daily routine CXRs hardly ever reveal potentially important abnormalities and seldom result in a change in therapy [5].
While it can be recommended to discontinue a daily routine CXR practice in ICU patients, elimination of these CXRs may have several disadvantages. Firstly, eliminating daily routine CXRs bears the risk that the number of on demand CXRs increases. In addition, elimination of daily routine CXRs might result in on demand CXRs being obtained more frequently during off-time hours, which may cause an inverse rise of costs. Secondly, length of stay (LOS) in ICU, readmission rate and mortality rate might be negatively influenced by this change in CXR practice.
To evaluate the impact of elimination of daily routine CXRs we determined the change in on demand CXR practice in our multidisciplinary ICU, where a daily routine CXR strategy was applied until performance of this study. In addition, we evaluated the diagnostic and therapeutic value of on demand CXRs before and after this intervention. Finally, LOS in ICU, readmission rate, and mortality rate during a daily routine CXR strategy were compared with those during an on demand CXR strategy.
Materials and Methods
Subjects
A prospective, nonrandomized, controlled design with an intervention was used for this study. Of all patients, all CXRs taken in the adult ICU department of the Academic Medical Center in Amsterdam, Netherlands, from 1 March 2004 to 31 July 2004 and from 1 September 2004 to 31 January 2005 were studied. This department is a closed-format tertiary care, referral, 28-bed multidisciplinary ICU. The patient population consists of cardiothoracic surgery patients, medical patients (including cardiology patients and pulmonary disease patients), and surgery patients (including trauma patients and neurosurgery patients). Patients who were admitted during the period in between phases 1 and 2, as well as patients that were readmitted, were not analyzed. The study protocol was approved by the local ethics committee.
Protocol
The study period was divided into two parts: phase 1, a 5-month phase before the intervention during which the daily routine CXR strategy was practiced; and phase 2, a 5-month phase which began 1 month after the intervention. The intervention consisted of a change in the ordering practice of CXRs: no standing orders for daily routine CXRs; each (on demand) CXR required a clinical indication, such as admittance to the ICU, insertion of central venous lines, intra-aortic balloon pump or tracheal and chest tubes, an increase in oxygen requirement, or a change in pulmonary secretions with or without fever (see Table E1).
For phases 1 and 2, CXR volume data were collected prospectively. Type of, and reason for, admission was registered for all patients. Severity of illness was scored by means of acute physiology and chronic health evaluation (APACHE) II for all patients. Data on LOS in ICU, readmission to ICU as well as ICU, and hospital mortality rate, were collected from the National Intensive Care Evaluation (NICE) database [6]. The LOS was calculated from day and time of arrival at, and discharge from, ICU. The total number of hours in ICU were divided by 24 to determine the exact LOS in ICU in days.
Diagnostic and therapeutic value of on demand CXR
Diagnostic and therapeutic value of on demand CXRs was determined as described previously for daily routine CXRs [5]. In short, the attending physician completed a specially developed data sheet on radiological abnormalities which was printed on the back of the normal CXR request form. It was to be ticked whether a certain finding was expected, and whether it was “old” (i.e., already present on preceding CXR) or “new” (i.e., not present on preceding CXR). All CXRs were interpreted by an independent radiologist on the day the on demand CXR was performed. Similar to the ICU physicians, the radiologist structurally interpreted these on demand CXRs for each patient (i.e., the radiologist ticked whether radiological abnormalities were absent or present and, if an abnormality was present, whether it was judged to be an “old” or “new” finding).
If a predefined finding was unexpectedly found, then we determined whether any action was taken because of the new unexpected finding. To do this, two of us (M.G. and M.J.S.) and two independent observers carefully read the medical records, checked the patient data management system (Metavision, iMDsoft, Sassenheim, The Netherlands) and searched the hospital information system for the following: orders for sputum cultures or performance of a bronchoalveolar lavage for culture, or start of, or a change in, antimicrobial therapy in case of unexpected infiltrates on the CXR; repositioning of tubes in case of malposition of orotracheal tubes (ignoring planned extubations); ultrasound of the thorax in case of pleural effusion on the CXR, start or change in medication (diuretics); insertion of a pleural drain; and repositioning of devices in the case of malposition of medical devices other than orotracheal tubes (ignoring planned changes such as removal of intravenous lines). The observers were not involved in the daily care of the patients, and ICU physicians were not aware of this part of the observation. As a consequence, the clinical relevance of the predefined abnormalities could not be evaluated in some cases, specifically in cases of large atelectasis and severe pulmonary congestion, since start of physiotherapy, changes in levels of positive end-expiratory pressure, and the use of diuretics might have been triggered by other (clinical) findings.
Statistical analysis
All data are expressed as means (± SD), or medians (interquartile ranges). A Mann-Whitney U-test was used for analyzing continuous variables. A chi-square test was used to compare the groups in phase 1 and phase 2. The incidences of expected and unexpected findings, and clinically important abnormalities, were compared by chi-square test. A p-value < 0.05 was considered to be statistically significant. All calculations were performed using SPSS version 12.0.1 software (SPSS, Chicago, Ill.).
Results
Study population
We evaluated 1376 patients over the two periods. Patient profiles on entering this study are summarized in Table 1. A total of 3894 CXRs were obtained from 754 patients in phase 1; these included 2457 daily routine CXRs and 1437 on demand CXRs. A total of 1267 CXRs were obtained from 622 patients in phase 2. These CXRs were, by definition, all on demand CXRs.
Table 1Demographic data. APACHE-II Acute Physiology and Chronic Health Evaluation II, CXRs chest radiographs, CI confidence intervalPhase 1Phase 2Significance (p)No. of patients 754 622Age (years; mean, SD) 60 (16) 62 (16)0.02Gender (male; n) 475 (63%) 398 (64%)0.70CXRs while patients being mechanically ventilated (n)a3194 (82%)1115 (88%)< 0.001APACHE-II score 16.4 ± 6.9 16.4 ± 7.21.00Patient subgroups Cardiac surgery (n) 317 (42%) 306 (49%)0.01 Medical (n) 197 (26%) 119 (19%) Surgical (n) 144 (19%) 131 (21%) Neurosurgical/neurology (n) 69 (9%) 46 (7%) Other (n) 27 (4%) 20 (3%)Length of stay in ICU (days; median IQR)  1.9 (1.0–4.6)  1.9 (0.9–4.6)0.95Mortality ICU (n) 94 (12%) 62 (10%)0.49 Hospital (n) 132 (18%) 104 (17%)0.70 Predicted hospital mortality (%) 181 (24%) 155 (25%)0.69 Observed/predicted ratio (95% CI)  0.73 (0.59–0.90)  0.67 (0.53–0.83)aAll patients were mechanically ventilated at any time during stay in ICU. Expressed is the percentage of CXRs during which patients were on the ventilator while the CXR was performed
Utility of CXRs
The number of CXRs per day for the whole ICU declined from 22.6 ± 4.9 to 8.2 ± 3.2 (p < 0.05; Fig. 1). Adjusting for patient volume, the ratio of CXRs per patient day decreased from 1.1 ± 0.3 to 0.6 ± 0.4 after the intervention (p < 0.05). The median number of CXRs per patient for the complete stay in ICU declined from 3 (range 2–5) during phase 1, to 1 (range 1–2) after the intervention. The number of on demand CXRs increased minimally after the intervention, and the distribution over 24 h, did not change (see ESM, Fig. E1).
Fig. 1Number of CXRs/day during the study. Phase 1: daily routine CXR strategy, i.e., a daily routine CXR was made every morning, from March to July; phase 2: on demand CXR strategy, i.e., each CXR required a clinical indication, from September to January. Open bars: mean number (± SD) of on demand CXRs/day; closed symbols: mean number (± SD) of all CXRs/day
Diagnostic and therapeutic value of on demand CXRs
The diagnostic and therapeutic value of on demand CXRs increased with elimination of daily routine CXRs (Tables 2, 3). Before intervention, 38 expected predefined abnormalities were found (2.6% of all on demand CXRs in 4.9% of all patients), and after the intervention 64 expected predefined abnormalities were found (5.0%; p < 0.05) in 9.5% of cases (p < 0.05). All these findings led to a change in therapy. Before intervention, 147 unexpected predefined abnormalities were found (10.2% of all on demand CXRs in 15.9% of all patients), of which 57 (4.0 in 6.4%) led to a change in therapy. After intervention 156 unexpected predefined abnormalities were found (11.6% of all on demand CXRs in 19.1% of all patients), of which 64 (4.8 in 9.5%; p < 0.05) led to a change in therapy. Subgroup analysis revealed no differences between phases 1 and 2, except for medical patients, in which there was a significant rise in the number of on demand CXRs that showed an unexpected predefined major abnormality (p < 0.05 vs phase 1; see ESM, Table E2).
Table 2Expected and unexpected findings on on demand chest radiographsPhase 1 (n = 1437)Phase 2 (n = 1267)AbnormalitiesExpectedExpected+foundUnexpected+foundExpectedExpected+foundUnexpected+foundLarge atelectasis 37 (2.6) 2 (0.1) 13 (0.9) 49 (3.9) 3 (0.2) 15 (1.2)Large infiltrates 57 (4.0) 3 (0.2) 21 (1.5) 69 (5.4) 5 (0.4) 27 (2.1)Pulmonary congestion 98 (6.8) 8 (0.6) 25 (1.7)104 (8.2)14 (1.1) 22 (1.7)Pleural effusion 41 (2.9) 3 (0.2) 17 (1.2) 43 (3.4) 4 (0.3) 27 (2.1)Pneumothorax or pneumomediastinum 68 (4.7) 4 (0.3) 17 (1.2) 39 (3.1)c 3 (0.2) 12 (0.9)Malposition of invasive devices350 (24.4)18 (1.3) 54 (3.8)392 (30.9)c35 (2.7)c 52 (4.1)Total no. of abnormalities6513814769664c155Total no. of CXRs with abnormalitiesa641 (44.6)38 (2.6)133 (9.2)384 (30.3)c63 (5.0)c147 (11.6)cTotal no. of patients with CXRs with abnormalitiesb580 (76.9)37 (4.9)120 (15.9)223 (35.9)c58 (9.5)c119 (19.1)Numbers in parentheses are percentagesa Absolute number of chest radiographs (CXRs; percentage of all daily routine CXRs)b Absolute number of patients (percentage of all patients with on demand CXRs)c p < 0.05 vs phase 1Table 3Unexpected findings on on demand chest radiographs resulting in a chance in therapy. ND not definedPhase 1 (n = 1437)Phase 2 (n = 1267)AbnormalitiesResulting in a change in therapyResulting in a change in therapyLarge atelectasisNDNDLarge infiltrates10 (0.7%)14 (1.1%)Pulmonary congestionNDNDPleural effusion11 (0.8%)12 (0.9%)Pneumothorax or pneumomediastinum11 (0.8%) 9 (0.7%)Malposition of invasive devices25 (1.7%)29 (2.3%)Total no. of abnormalities5764Total no. of CXRs with abnormalitiesa56 (3.9%)61 (4.8%)Total no. of patients with CXRs with abnormalitiesb48 (6.4%)59 (9.5%)ca Absolute number of chest radiographs (CXRs; percentage of all daily routine CXRs)b Absolute number of patients percentage of all patients with on demand CXRs)c p < 0.05 vs phase 1
LOS in ICU, readmission rate and mortality rate
The LOS in ICU was not different in phase 1 as compared with phase 2 (Table 1). Total readmission rate was similar (8.4% in phase 1 vs 7.6% and phase 2, risk difference 0.8% (95% CI: 2.1–3.7%, P = 0.6), and did not change with the intervention for the different subgroups. There were no statistically significant differences in ICU and hospital mortality rates before and after the intervention (Table 1).
Discussion
The present study demonstrates the impact of elimination of daily routine CXRs in a mixed medical–surgical ICU. We found a sharp decline in the total number of CXRs, while only a minimal increase in the number of on demand CXRs was observed. In addition, the number of CXRs in off-hours was similar between the two periods. Elimination of daily routine CXRs did neither affect LOS in ICU and readmission rate nor ICU and hospital mortality rate.
Although the diagnostic and therapeutic value of on demand CXRs was significantly higher after the intervention, we considered this difference clinically irrelevant. When one considers the increase in diagnostic and therapeutic value of on demand CXRs after elimination of daily routine CXRs indirect proof of the “value” of daily routine CXRs, one must also recognize its futility regarding the therapeutic value. Indeed, the percentage of CXRs with unexpected findings that truly led to a change in therapy was similar in the two study phases. Since readmission rate and mortality rate remained unchanged after the intervention, we conclude that the true value of daily routine CXRs in our multidisciplinary ICU is very low. Interestingly, only in medical patients did the number of CXRs that showed an unexpected predefined major abnormality increase after elimination of daily routine CXRs. The reason for this finding remains unexplained. The distribution of abnormalities encountered on CXRs of these patients was similar in the two study phases; however, neither readmission rate nor differences in raw or risk-adjusted ICU and hospital mortality rates of medical patients was affected by the change in CXR practice.
One interesting finding was the decrease in abnormalities presumed to be present on CXRs. Indeed, a 30% reduction in expected predefined findings was observed in phase 2. This finding remains unexplained and we can only speculate on its cause. Firstly, it may be that physicians learned from experience that many of their expectations proved to be untrue during the actual carrying out of the study. This may have caused them to be more reluctant in scoring for expected findings. Alternatively, physicians may have become less enthusiastic about the study, which might have resulted in failure to comply with study rules at some moments (i.e., they did not fill in the back of the formal CXR request form); however, there was no change in expectations of physicians regarding abnormalities that truly led to a change in therapy. More importantly, if the backside of the formal forms were not filled out, as a rule the CXR was simply not obtained. Indeed, collection of data was complete regarding this issue, there were no on demand CXRs without a completed form.
Our study has, at least partially, overlap with two other studies [7, 8]. Price et al. performed a nonrandomized controlled study on the financial impact of elimination of daily routine CXRs [7]. They showed that elimination of daily routine CXRs in a pediatric ICU resulted in decreased variability in ordering practice, fewer CXRs per patient, and an accompanying cost savings, while not influencing LOS. In addition, cost reduction with the change in radiology policy was significant in their study. This is in line with our results, since we found a substantial decline in radiology costs (see ESM). Besides the fact that this study was performed in a pediatric ICU, making generalization of study results difficult, their study did not include all patient categories. Indeed, postoperative cardiovascular surgery patients continued to receive daily routine CXR. We specifically included this patient group in our study because cardiovascular patients form one of the largest categories in many adult ICU. Krivopal et al. performed a randomized controlled trial to determine whether there is any difference in diagnostic, therapeutic, and outcome efficacy between protocols utilizing daily routine CXRs and those utilizing on demand CXRs in mechanically ventilated patients [8]. In their study a daily routine CXR strategy compared with an on demand CXR strategy was not associated with a negative effect on LOS or mortality; however, this study was small, including not more than 94 patients.
We did not collect information on less evident findings on CXRs. Less evident findings (such as atelectasis less than two lobes, infiltrates less than one lobe, or small pleural effusions [5]), however, might still influence daily management of ICU patients. Since LOS in ICU was not altered for the whole group, readmission rate and mortality rate remained unchanged after the intervention, we suggest that changes of less evident CXR findings are not at all important, at least in our ICU. In other ICUs, such as open-format ICUs, less evident findings might be of more clinical importance, however; therefore, our results must be interpreted with caution, it might be that our results are not easily translated to other types of ICU.
Several important drawbacks of our study must be mentioned. Firstly, our study did not include a strict method for tracking complications as a result of elimination of daily routine CXRs. Indeed, several abnormalities might have been missed (or discovered too late) which might (or do) have impact on clinical outcome. Examples of these types of abnormalities include pneumothorax causing weaning problems, the malposition of devices such as central venous lines, causing extravasation of fluid, or orotracheal tubes, potentially causing injury to the vocal cords. Considering these examples, such a strict method may mandate a daily check of all invasive devices. Although possible complications of elimination of daily routine CXRs could be discussed in daily bedside rounds, daily radiology conferences and daily multidisciplinary meetings during the performance of our study, no clinically important complications were reported as the result of elimination of daily routine CXRs; thus, although we assume that the elimination of daily routine CXRs does not cause any complications, we cannot be certain that this was truly the case. Secondly, as mentioned previously, it is of importance to realize that results that come from one center may simply not be similar for other centers: differences in staffing; especially during off-hours, and differences in case mix may be of great influence on outcome when abandoning daily routine CXRs. Thirdly, as mentioned previously, we found a reduction in expected predefined finding in phase 2. We assumed that the cause of this reduction might be that the physicians became less enthusiastic about the study, which might be seen as a limitation of the study.
Conclusion
In conclusion, in our mixed medical–surgical ICU elimination of daily routine CXRs leads to a sharp decline in the total number of CXRs, while only minimally increasing the number of on demand CXRs. Although we cannot be certain whether we missed important findings by abandoning daily routine CXRs, its elimination did neither affect LOS in ICU, nor readmission rate and ICU and hospital mortality rates.
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