Pharmacological disruption of the outer limiting membrane leads to increased retinal integration of transplanted photoreceptor precursors

Retinal degeneration is the leading cause of untreatable blindness in the developed world. Cell transplantation strategies provide a novel therapeutic approach to repair the retina and restore sight. Previously, we have shown that photoreceptor precursor cells can integrate and form functional photoreceptors after transplantation into the subretinal space of the adult mouse. In a clinical setting, however, it is likely that far greater numbers of integrated photoreceptors would be required to restore visual function. We therefore sought to assess whether the outer limiting membrane (OLM), a natural barrier between the subretinal space and the outer nuclear layer (ONL), could be reversibly disrupted and if disruption of this barrier could lead to enhanced numbers of transplanted photoreceptors integrating into the ONL. Transient chemical disruption of the OLM was induced in adult mice using the glial toxin, dl-alpha-aminoadipic acid (AAA). Dissociated early post-natal neural retinal cells were transplanted via subretinal injection at various time-points after AAA administration. At 3 weeks post-injection, the number of integrated, differentiated photoreceptor cells was assessed and compared with those found in the PBS-treated contralateral eye. We demonstrate for the first time that the OLM can be reversibly disrupted in adult mice, using a specific dose of AAA administered by intravitreal injection. In this model, OLM disruption is maximal at 72 h, and recovers by 2 weeks. When combined with cell transplantation, disruption of the OLM leads to a significant increase in the number of photoreceptors integrated within the ONL compared with PBS-treated controls. This effect was only seen in animals in which AAA had been administered 72 h prior to transplantation, i.e. when precursor cells were delivered into the subretinal space at a time coincident with maximal OLM disruption. These findings suggest that the OLM presents a physical barrier to photoreceptor integration following transplantation into the subretinal space in the adult mouse. Reversible disruption of the OLM may provide a strategy for increasing cell integration in future therapeutic applications.
1
Introduction
Retinal degeneration is the leading cause of untreatable blindness in the developed world. Current clinical treatments are limited, at best only slowing disease progression and very rarely restoring visual function. Cell transplantation offers a novel therapeutic approach, enabling the replacement of photoreceptor cells lost in the degenerative process. Photoreceptor transplantation may be more feasible than other types of neuronal transplantation, because photoreceptors are stimulated by light and their function is not, therefore, dependent on the reformation of complex afferent connections. Nevertheless, an efferent connection to host second order sensory neurons in the retina is essential for visual function and this is arguably best achieved if the transplanted photoreceptor is fully integrated into the host outer nuclear layer (ONL).
Transplanted whole retinal sheets, derived from either embryonic or neonatal sources, can survive and differentiate, but frequently fail to integrate and make functional connections within the host neural retina (Ghosh and Ehinger, 2000; Royo and Quay, 1959; Seiler et al., 1990; Zhang et al., 2003b). Conversely, dissociated neural stem cells, such as those derived from the adult hippocampus, can migrate extensively within the retina when transplanted into an adult or developing recipient, but rarely differentiate into mature retinal phenotypes (Takahashi et al., 1998; Young et al., 2000). Progenitor cells isolated from dissociated embryonic retinae can differentiate into retinal neurons after transplantation and express photoreceptor-specific markers (Ahmad et al., 2000; Chacko et al., 2000; Coles et al., 2004; Klassen et al., 2004; Qiu et al., 2005; Yang et al., 2002), but migration and integration of these cells into the laminar structure of the host neural retina has remained limited. Better integration has been achieved by transplanting cells into an immature, developing retina, such as that of the neonatal Brazilian opossum, which provides a foetal-like host environment. The ability of transplanted cells to integrate within the host opossum retina declines with host maturation (Sakaguchi et al., 2003, 2004). This also coincides with the maturation of glial elements, such as Müller cells, which form anatomical barriers within the host retina, including the outer limiting membrane (OLM) (MacLaren, 1996).
We have recently shown that a significant degree of integration of fully differentiated and functional photoreceptors can be achieved after transplantation into the adult retina, but only if donor cells are post-mitotic photoreceptor precursors. When transplanted into the subretinal space, these cells can migrate into the recipient ONL, form synaptic connections with downstream targets and are light-sensitive (MacLaren et al., 2006). However, while the number of integrated photoreceptor cells is sufficient to restore the pupillary light reflex, higher levels of integration are needed to improve visual acuity. Given that immature neurons and progenitor cells are intrinsically capable of migrating and differentiating (Komuro and Rakic, 1998a,b; Nadarajah and Parnavelas, 2002; Parnavelas et al., 2002; Pearson et al., 2005), it is likely that natural physical barriers, such as the OLM, impede migration. Transient disruption of these barriers at the time of transplantation might be one way of increasing the number integrating into the host retina.
The OLM comprises a series of zonula adherens junctional complexes, located between the plasma membranes of photoreceptor inner segments and the apical processes of Müller glia (Fig. 1a–c). These junctions seal off the light-sensitive photoreceptor inner and outer segments from the rest of the retina, limiting the diffusion of phototransduction cascade components. The OLM is first discernible by post-natal day 5 (P5) in the mouse (Uga and Smelser, 1973; Woodford and Blanks, 1989). Alpha-aminoadipic acid (AAA) is a glutamate analogue (Fig. 1d), which disrupts the OLM by inducing specific toxicity in Müller glial cells within the mammalian retina (Karlsen et al., 1982; Pedersen and Karlsen, 1979). Single intravitreal injections of AAA have been shown to disrupt the OLM irreversibly and to the extent whereby photoreceptors drop out of the ONL to reside amongst outer segments in the subretinal space (Ishikawa and Mine, 1983). In this study, we show that by using an appropriate dose and route of administration, AAA can produce a transient disruption in OLM integrity. Furthermore, OLM disruption can facilitate movement of cells in the opposite direction, significantly enhancing the number of donor photoreceptors integrated into the recipient ONL after transplantation into the subretinal space. These findings suggest that the OLM represents at least one important barrier to cell integration.
2
Materials and methods
2.1
Animals
C57Bl/6 and Nrl.gfp+/+ (Akimoto et al., 2006) mice were maintained in the animal facility at University College London. All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Mice defined as “adult” were at least 6 weeks, but not more than 3 months old. Nrl.gfp+/+ mice were used as donors to provide dissociated retinal progenitor cells for transplantation. Recipients were C57Bl/6 animals, unless otherwise stated.
2.2
α-Aminoadipic acid formulation and administration
dl-α-Aminoadipic acid (AAA; Sigma) was prepared in phosphate-buffered saline (PBS), adjusted to pH 7.5 and sterile-filtered prior to administration. Mice were anaesthetized with a single intra-peritoneal injection of 0.15 ml of a mixture of Dormitor (1 mg/ml medetomidine hydrochloride; Pfizer Pharmaceuticals, Kent UK), ketamine (100 mg/ml; Fort Dodge Animal Health, Southampton, UK) and sterile water for injections in the ratio of 5:3:42 for adult mice. AAA was administered by intravitreal, subretinal or subcutaneous injection.
For histological assessment, mice were sacrificed at various time points (3–4 mice per time-point) and the eyes were fixed in buffered formalin for 48 h at 4 °C. Retinal sections were prepared by overnight dehydration and paraffin embedding (Histocentre). Sections (5 μm thick) were cut and affixed to glass slides and stained using standard haematoxylin and eosin protocols.
2.3
Dissociation of retinal cells and transplantation
To investigate the role of AAA-induced disruption of the OLM on cell integration, C57Bl/6 mice received subretinal transplants of dissociated retinal precursor cells 72 h or 1 week (9 mice per time-point) after intravitreal injection (through the inferior pars plana) of AAA (test eye) or PBS (contralateral eye). Dissociated cells were prepared from P 2–5 Nrl.gfp+/+ mice, as described previously (MacLaren et al., 2006). Cells were dissociated using a papain-based kit (Worthington Biochemical, Lorne Laboratories, UK) and diluted to a final concentration of ∼4 × 105 cells/μl. Surgery was performed under direct ophthalmoscopy through an operating microscope, as previously described (MacLaren et al., 2006). Cell suspensions were injected (1 μl) slowly to produce a standard and reproducible retinal detachment in the superior hemisphere. Mice were sacrificed 21 days after transplantation and eyes were fixed in 4% paraformaldehyde (PFA) in PBS, for cell counts. Retinal sections were prepared by cryo-protecting fixed eyes in 20% sucrose, before cryo-embedding in OCT (TissueTek) and sectioning at 18 μm.
2.4
Histology and immunohistochemistry
Mice were sacrificed at various time points after AAA administration (3 mice per time-point) and eyes were fixed in 1% PFA in PBS, for immunohistochemistry. Retinal cryosections were permeablized in chilled acetone for 5 min. Sections were pre-blocked in Tris-buffered saline (TBS) containing normal goat serum (5%), bovine serum albumin (1%) and 0.05% Triton X-100 for 2 h before being incubated with primary antibody overnight at 4 °C. After rinsing with TBS, sections were incubated with secondary antibody for 2 h at room temperature (RT), rinsed and counter-stained with Hoechst 33342. Negative controls omitted the primary antibody. The following antibodies were used: rabbit anti-ZO-1 (kind gift of K. Matter) and rabbit anti-GFAP (Dako), with an anti-rabbit Alexa-546 tagged secondary antibody (Molecular Probes, Invitrogen). An apoptosis TdT DNA fragmentation kit (ApopTag Red Apoptosis Detection Kit, Chemicon, CA, USA), which stains apoptotic cells red, was used to perform an in situ TUNEL assay on the sections.
2.5
Electron microscopy
Mice were sacrificed at various time points after AAA administration. The eyes were fixed, the cornea and lens removed and the eye cups orientated and processed, as previously described (Tschernutter et al., 2005). Ultrathin sections were collected on copper grids, (100 mesh, Agar Scientific) contrast stained with 1% uranyl acetate and lead citrate and analysed using a JEOL 1010 transmission electron microscope (80 kV), fitted with a digital camera for image capture.
2.6
Confocal microscopy
Retinal sections were viewed on a confocal microscope (Zeiss LSM510), as previously described (MacLaren et al., 2006). Unless otherwise stated, images show: (i) merged Nomarski and confocal fluorescence projection images of GFP (green) and the nuclear counter stain Hoechst 33342 (blue), and (ii) the same region showing GFP signal only.
2.7
Integrated cell counts
Cells were considered to be integrated if the whole cell body was correctly located within the outer nuclear layer and at least one of the following was visible: spherule synapse, inner/outer processes and/or inner/outer segments, as previously defined (MacLaren et al., 2006). The number of integrated cells per eye was determined by counting all the integrated GFP-positive cells in alternate serial sections through each eye. This was doubled to give the mean number of integrated cells per eye. While it is unlikely that a photoreceptor cell body would be present across three sections, given that AAA causes cell swelling in Müller glia (Ishikawa and Mine, 1983), it is possible that it may also affect other cell types including photoreceptors. The total number of nuclei in the ONL in a volume of 2500 μm3 at the site of transplantation was determined in both control and AAA-treated eyes (72 h and 1 week prior to transplantation). There was no significant difference in the number of photoreceptor cell bodies between any of the treatment groups (P = 0.35, N = 6; ANOVA). Thus, AAA treatment is highly unlikely to lead to double-counting of integrated cells.
2.8
Apoptotic cell counts
The number of apoptotic cells was determined by counting all TUNEL-positive profiles in each layer of the retina in alternate serial sections. Only sections that encompassed the site of intravitreal injection were used and are thus not representative of apoptosis in the whole eye.
2.9
Statistics
All means are stated ±SEM (standard error of the mean), unless otherwise stated. The statistical test used was a two-tailed paired t-test with a significance threshold of P < 0.05. N, number of eyes; n, number of sections examined or cells counted, where appropriate.
3
Results
3.1
Dosage and route of AAA administration
We sought to determine whether it is possible to achieve a transient, reversible disruption of the OLM in the adult mouse by administration of the glial toxin, AAA. First, we assessed different routes of administration including: intravitreal (20 μg/μl, N = 4), subretinal (10 μg/μl, N = 4) and subcutaneous (0.7–2.7 mg/g body weight, N = 6) injection. The dose of AAA used for each route of administration was ascertained from previous published studies (Ishikawa and Mine, 1983; Pedersen and Karlsen, 1979; Rich et al., 1995) Retinae failed to recover normal histological morphology following subretinal injection, while subcutaneous injections resulted in variable morphological changes (data not shown). However, intravitreal administration caused modest and reversible morphological changes (see below). Therefore, AAA was administered to the retina via intravitreal injection, through the inferior pars plana towards the superior hemisphere of the eye, for the remainder of the experiments described.
In order to establish the optimum dose of AAA required to induce a transient disruption of the OLM, several doses (20, 100 and 320 μg/μl, N = 3, N = 4 and N = 4, respectively for each time point) were administered intravitreally and the retinae examined histologically at 6, 24 h or 3 weeks post-injection (Fig. 2) and compared with contralateral controls. At 6 h post-injection of all three doses of AAA, we observed vacuoles at the margin of the OLM that protruded into the inner/outer segment layer. These were possibly due to a swelling of the Müller cell apical processes and both the size and number of vacuoles present was dosage dependent (Fig. 2a,d,g). Upon morphological examination at 24 h post-AAA injection, eyes receiving the low (20 μg/μl) dose appeared normal (Fig. 2b). Conversely, recovery was not evident at this time following application of either 100 or 320 μg doses; vacuoles remained in both, and eyes receiving the highest dose displayed marked morphological abnormalities (Fig. 2e,h). Following administration of these higher doses, the OLM also appeared disrupted, as indicated by the presence of cell bodies in the segment layers (Fig. 2e,h). By 3 weeks post-administration, retinae receiving 20 μg/μl AAA appeared normal by morphological assessment, whilst those receiving 100 μg/μl AAA, exhibited a small number of remaining vacuoles (Fig. 2c,f). In eyes that received the highest dose (320 μg/μl) retinal morphology remained severely disrupted at 3 weeks, including a loss of retinal lamination, sustained disruption of the outer limiting membrane and retinal thinning (Fig. 2i). An apparent disruption of the inner limiting membrane was also noted in these eyes, but was not observed with lower doses of AAA.
Thus, the optimum dose of AAA required for transient OLM disruption by intravitreal injection was determined to be 100 μg/μl. This dose invariably resulted in OLM disruption, as determined by histological assessment, but with consistent recovery of OLM integrity and relatively normal retinal morphology by 3 weeks. The early morphological effects of AAA in the retina described here concur with the few published studies on subcutaneous and intravitreal injections in mice and rats (Pedersen and Karlsen, 1979; Rich et al., 1995). In addition, our findings also demonstrate that these effects can be induced in a reversible manner.
3.2
Window of OLM disruption
Having determined the optimal dose of AAA for a reversible and consistent OLM disruption (100 μg/μl, administered intravitreally), we also wished to identify the time point at which this disruption was maximal. We therefore examined retinal morphology, OLM integrity and apoptosis in treated retinae at 24, 48, 72 h, 1 and 2 weeks post AAA administration (N = 3 for each time point).
The effect of AAA pre-treatment on host photoreceptor morphology was examined in the adult Nrl.gfp+/+ mouse, which expresses GFP in rod photoreceptors (Akimoto et al., 2006; Mears et al., 2001; Swain et al., 2001). Disruption of the ONL was clearly identifiable by the movement of GFP-labelled cells into the inner and outer segment layers of the retina (Fig. 3a). At 24 h post AAA injection, photoreceptor morphology was largely normal, with correctly orientated inner and outer segments and only occasional photoreceptor cell bodies in the subretinal space. By 48 h, the laminar organization of the retina was disrupted and more photoreceptors were displaced from the ONL. Some retinal folds were evident at 72 h post-injection and the organization of the inner and outer segments was significantly disrupted. Recovery of the retina was first seen at 1 week post AAA injection, as lamination returned and photoreceptor inner and outer segments regained nearly normal orientation. Photoreceptor organization appeared largely normal by 2 weeks post injection.
The displacement of photoreceptor cell bodies into the subretinal space post AAA administration suggested that the OLM was disrupted. To confirm this, we used antibodies directed against ZO-1, an adherens junction protein located at the OLM (see Fig. 1b,c). Staining showed that the OLM was largely intact at 24 and 48 h post-AAA administration, with the exception of a few localized areas of disruption (Fig. 3b). The peak of OLM disruption was present at approximately 72 h post-administration, as demonstrated by a substantial lack of ZO-1 staining at sites where photoreceptors had dropped out of the ONL. By 1 week, the OLM had largely reformed, with disturbed but continuous adherens junctions seen. Normal staining was seen by 2 weeks post AAA administration.
While AAA is a glial-specific toxin (Karlsen et al., 1982; Pedersen and Karlsen, 1979), we wished to establish whether or not other cell types, particularly photoreceptors, might be affected by the changes induced by AAA (Fig. 3c). Retinal sections encompassing the area of AAA administration were stained with an ApopTag Red Apoptosis detection kit and the number of apoptotic nuclei per section quantified. Apoptosis staining revealed that at 24 and 48 h post AAA administration, the vast majority of apoptotic nuclei were present in the inner nuclear layer (25 ± 2 cells per retinal section at 24 h and 31 ± 5 cells per retinal section at 48 h, n = 15 sections), most likely Müller cells. Apoptosis was absent in the ONL at these time-points. By 72 h, however, the peak of morphological and OLM disruption, some apoptotic cells were present in the ONL (61 ± 5 cells per retinal section, n = 15). Less than 12 ± 2 apoptotic cells per retinal section remained in the ONL at 1 week, reducing to 2 ± 1 cells by 2 weeks post-administration (n = 12). Occasionally, apoptotic profiles were seen in the ganglion cell layer. However, these were most likely the result of the intravitreal injection procedure itself, as similar levels were seen in control PBS injected eyes. Note that little apoptosis was observed in any layers of the retina in the rest of the eye, away from the site of AAA injection, at all times points examined. Neuronal toxicity after d,l- AAA treatment is thought to be a secondary effect due to the loss/inhibition of the supporting glial cells in the retina or brain (Tsai et al., 1996). This concurs with our results, since apoptosis in the ONL was observed only after apoptosis of cells in the INL.
The impact of AAA administration on photoreceptor morphology and OLM integrity were further examined using electron microscopy. Retinae were examined 72 h and 1 week post AAA administration and compared with PBS injected controls (Fig. 4). In PBS injected control retinae, the OLM was intact and photoreceptor inner and outer segment morphology was normal at 72 h post-administration (Fig. 4a). Conversely, the integrity of the OLM was lost in many regions of the AAA treated retinae and photoreceptor nuclei were mislocalized in the outer segment layer. The photoreceptor inner and outer segments were significantly disturbed, vacuoles were present and there was a loss of outer segments (Fig. 4b). By 1 week post AAA treatment, retinae showed significant recovery of OLM integrity, together with fewer vacuoles in the inner segment region and recovery of inner and outer segment organization (Fig. 4c).
Together, these findings demonstrate that intravitreal administration of 100 μg AAA in the mouse causes a transient, reversible disruption of the OLM approximately 72 h post injection.
3.3
Cell integration with OLM disruption
We next sought to determine whether or not disruption of the OLM permits greater levels of integration of transplanted photoreceptor precursor cells. To test this, animals received a subretinal injection of early post-natal Nrl.gfp+/+ donor cells 72 h after intravitreal administration of AAA, when OLM disruption is maximal. To control for the intravitreal injection, the contralateral eye received a subretinal injection of donor cells 72 h after intravitreal administration of PBS. Three weeks post-transplantation, retinae were sectioned and the total number of Nrl.gfp cells integrated within the ONL was quantified. Nrl.gfp expression is restricted to rod photoreceptors (Akimoto et al., 2006; Mears et al., 2001; Swain et al., 2001) and provides genetic evidence that any transplanted integrated cells within the ONL are photoreceptors. We have previously demonstrated that these integrated donor cells are light sensitive and form functional synaptic connections with downstream targets in the recipient retina. Transplanted non-GFP-positive cells remained in the subretinal space, forming a cell mass. Very few integrated cells, that were not photoreceptors, have previously been observed in transplants using donor tissue from C57Bl/6 GFP+/− mice (MacLaren et al., 2006).
In all animals examined, the eyes that received pretreatment with AAA showed a significantly higher number of integrated donor cells within the ONL compared with their contralateral PBS-treated counterparts (AAA-treated 1088 ± 172.28 cells, vs. PBS control 523 ± 106.49 cells; N = 9, P = 0.009, paired t-test; Fig. 5a). The number of integrated photoreceptors was also increased compared to previous cell count data from wildtype mice (control 691 ± 209.50 cells; N = 5). To exclude variability in cell counts resulting from inter-animal variation, the ratio of the number of cells in the AAA treated eye compared with the contralateral control eye was also calculated for each individual mouse. This revealed an average three-fold increase in the number of transplanted photoreceptors for each animal as a result of AAA treatment (3.0 ± 0.75; N = 9; Fig. 5b,c).
To determine whether this statistically significant increase in the number of integrated cells coincided with OLM disruption, we also performed cell transplants at 1 week post-AAA administration when the OLM disruption had largely recovered. Transplantation at this stage showed a mean fold-difference of less than 1.3-fold between the AAA treated eye compared with the control PBS-treated eye (Fig. 5b) and the number of integrated cells was not significantly higher post-AAA administration (AAA-treated 363 ± 52.22 cells, vs. PBS control 527.78 ± 94.89; N = 9, P = 0.34, paired t-test; Fig. 5a).
We investigated whether AAA treatment enhanced reactive gliosis as this may affect cell integration. Previous studies have shown that AAA affects astrocytes in both the brain and eye (Ishikawa and Mine, 1983; Khurgel et al., 1996) and Rich et al. (1995) demonstrated up-regulation of GFAP in Müller cell processes, after chronic systemic administration of AAA during development (P3–9). GFAP is a marker of reactive gliosis and astrocytes, and we examined GFAP immunohistochemistry after AAA treatment and compared it with PBS injected controls. Here the doses of AAA used were much lower than those in the studies cited above and were administered as single intravitreal injections. We observed no difference in GFAP staining between the AAA and PBS treated eyes at either time point, indicating that AAA had little effect on Müller cell activation or astrocyte toxicity (Supplementary Fig. 1).
Pre-treatment with either AAA or PBS had no identifiable effect on the morphology of integrated photoreceptors. In both the PBS control and the AAA treated retinae, integrated photoreceptors appeared fully differentiated and morphologically indistinguishable from those we have described previously (MacLaren et al., 2006) (Fig. 5d).
4
Discussion
Here we demonstrate that the OLM in the adult mouse retina can be transiently disrupted by the intravitreal administration of AAA. OLM disruption is maximal approximately 72 h post administration. When combined with precursor cell transplantation, this time point correlates with a significantly enhanced level of transplanted photoreceptor cell integration into the recipient ONL, compared with sham-injected controls. These findings suggest that the OLM represents a natural barrier to the successful integration of photoreceptor precursor cells transplanted into the subretinal space. Consideration of the OLM may therefore be important in any future clinical photoreceptor transplantation strategies directed towards retinal repair.
4.1
Effect of AAA on Müller cells and the OLM
We show for the first time that the glutamate analogue, AAA, can be used to induce a transient disruption of both Müller glial morphology and OLM integrity in the adult mouse retina. AAA appears to disrupt the OLM by exerting a largely Müller glial-specific transient toxicity (Karlsen et al., 1982; Olney, 1982; Pedersen and Karlsen, 1979). The Müller glia recover well, with only low levels of cell death observed in the first week after AAA administration. Death of Müller glia following exposure to AAA has previously been observed in the carp retina, following an injection of much larger doses than those used in the present study (Sugawara et al., 1990). In addition to Müller cells, the other glial cell type of the retina, astrocytes, also exhibit toxicity in response to AAA in the brain and eye (Ishikawa and Mine, 1983; Khurgel et al., 1996). The mechanism of action of AAA on Müller glia is uncertain, although the morphological damage includes swelling and nuclear changes (Pedersen and Karlsen, 1979). Suggested modes of action include: inhibition of glutamate uptake, resulting in possible neuroexcitotoxicity (Tsai et al., 1996); inhibition of the cystine/glutamate transporter expressed by Müller glia, leading to reduced levels of intracellular glutathione and oxidative stress (Kato et al., 1993); and uptake of AAA itself by Müller glia, and subsequent cytotoxicity via metabolic stress (Chang et al., 1997; McBean, 1994). The downstream effects of AAA occur in a time specific manner, resulting first in the early gliotoxicity, followed by an apparent secondary neurotoxicity, seen with increased dose (Tsai et al., 1996).
4.2
OLM disruption and photoreceptor integration
Cells transplanted into the subretinal space of adult mice are capable of correctly integrating within the recipient ONL and forming functional, synaptically-connected photoreceptors, if these cells are at the appropriate post-mitotic precursor stage of development (MacLaren et al., 2006). However, the numbers integrating are below that likely to be required for a clinical therapy. Given that immature neurons and neural stem cells are intrinsically capable of migration (Hagg, 2005; Komuro and Rakic, 1998a; Nadarajah and Parnavelas, 2002; Parnavelas et al., 2002; Pearson et al., 2005), it is likely that barriers exist within the adult retina that impede greater numbers of donor cells from migrating and integrating, as demonstrated by the failure of graft integration in recipients where the host photoreceptor layer is largely intact (Zhang et al., 2003a). Extensive migration into the neural retina is largely restricted either to severely degenerated retina (Zhang et al., 2004) or, in wildtype animals, to areas where there is significant disruption to the ONL (Ghosh et al., 1999; Gouras et al., 1994; Zhang et al., 1999). Zhang and colleagues concluded that breaks in the OLM and/or loss of the photoreceptor component of the OLM were necessary for the formation of bridging fibres between graft and host tissues (Zhang et al., 2003a). Similarly, localized mechanical disruption of the retina may facilitate migration of dissociated cells from the subretinal space, as observed with neural stem cells (Nishida et al., 2000).
The OLM consists of unique heterotypic (involving Müller cells and photoreceptors) or homotypic (between Müller cells) adherens junctions (Paffenholz et al., 1999; Williams et al., 1990). We have shown that AAA causes marked morphological changes within Müller glia, leading to the disruption of these adherens junctions. Furthermore, a number of photoreceptor nuclei became displaced within the segment layer, outside the OLM, which normally acts to retain them (Rich et al., 1995). This suggests that if cells can exit the ONL following disruption of the OLM, the converse is also likely—cells in the subretinal space can migrate more readily into the ONL when the OLM is disrupted. Accordingly, pre-treatment with AAA leads to significantly greater numbers of donor cells integrating following transplantation, compared with controls. Importantly, this effect was only seen if cells were transplanted 72 h after AAA administration, i.e. only when OLM disruption is at its peak.
OLM disruption causes a significant increase in photoreceptor integration following transplantation. However, the increase (3-fold) is less than might be expected if the OLM were the only factor limiting donor cell migration, suggesting that manipulation of additional factors is likely to be required to optimize integration. A number of factors need consideration in relation to our findings. First, the donor cell population is heterogeneous; only a small proportion of cells in the early post-natal retina will be at the appropriate stage and specification for transplantation (i.e. photoreceptor precursors), so cell number may be a limiting factor. This could be augmented in the future by pre-selecting photoreceptor precursors, provided efficient cell sorting methods can be established. Second, Müller glia may actually facilitate donor cell migration into the ONL of the recipient retina or play a role in supporting rod differentiation, and the transient toxic effects of AAA may impede or reduce these supportive functions. Thus, while AAA may aid integration by disrupting the integrity of the OLM, it may conversely limit that enhancement by disturbing this supportive glial scaffold. Müller glia are also known preferentially to support rod process outgrowth (Kljavin and Reh, 1991). However, because all integrated photoreceptor cells in the AAA-treated retinas were morphologically identical to controls and those previously described (MacLaren et al., 2006), this suggests that Müller cells are at least partially dispensable, which is consistent with the observation that AAA-treatment does not affect normal photoreceptor development in early post-natal mice (Rich et al., 1995).
Finally, Müller glia up-regulate the expression of GFAP and other intermediate filament proteins under stress (Bignami and Dahl, 1979; Bjorklund et al., 1985). It is possible that the physiological changes induced by AAA could trigger aspects of the glial scarring pathway, which may subsequently impede the migration of transplanted cells. Recent work investigating retinal cell transplantation by Kinouchi et al. (2003) has demonstrated that cell migration, to the ganglion cell layer is enhanced in mice lacking GFAP and vimentin, two intermediate filament proteins found in reactive Müller glia and astrocytes. Müller cell morphology was stated to appear normal in these mice and spanned the entire retina. They did not, however, observe increased cell integration into other retinal layers, including the outer nuclear layer (Kinouchi et al., 2003).
4.3
Therapeutic implications
The results described here demonstrate a proof of concept; namely that disruption of the OLM increases the integration of transplanted photoreceptor precursor cells. However, the compound used, AAA or the l-AAA enantiomer have been shown to have other significant effects in the retina, albeit at higher doses than those used in this study. These include Müller cell necrosis, light insensitivity and suppression of the electroretinographic b-wave (Kato et al., 1990; Pedersen and Karlsen, 1979; Sugawara et al., 1990). Therefore, AAA is highly unlikely to be of therapeutic value. It will be of considerable interest to identify alternative reagents that can induce a specific, reversible disruption of OLM integrity without impacting on the function of the retina. It is important to note that cystoid macular oedema (CME) is a condition seen in the end stages of many diseases of the outer retina, such as retinitis pigmentosa and diabetic maculopathy. Microscopic examination of pathological specimens has shown that CME represents an intra-cytoplasmic swelling (oedema) of Müller cells in the foveal region (Yanoff et al., 1984) which is similar to the effects of AAA described in this study. Based on the results of our experiments, it is therefore not inconceivable that the OLM disruption resulting from CME may make the diseased human fovea a particularly favourable site for future retinal cell transplantation strategies.

outer limiting membrane
photoreceptor
mouse
cell integration
stem cells
müller cell
retinal transplantation


In-vivo demonstration of the effectiveness of thoracoscopic anterior release using the fulcrum-bending radiograph: a report of five cases

Thoracoscopic anterior release of stiff scoliotic curves is favored because of its minimally invasive nature. Animal and human cadaveric studies have shown that it can effectively improve spinal flexibility in non-scoliotic spines; however it has not been demonstrated to be effective in actual patients with scoliosis. The fulcrum-bending radiograph has been shown to accurately reflect the post-operative correction. To demonstrate that the flexibility was increased after the anterior release; five patients with idiopathic thoracic scoliosis who underwent staged anterior thoracoscopic release and posterior spinal fusion were assessed using the fulcrum-bending radiograph. The average number of discs excised was four. Spinal flexibility as revealed by the fulcrum-bending technique, was compared before and after the anterior release. The patients were followed for an average of 4 years (range 2.2–4.9 years). Fulcrum-bending flexibility was increased from 39% before the thoracoscopic anterior spinal release to 54% after the release (P<0.05). The average Cobb angle before the anterior release was 71° on the standing radiograph and 43° with the fulcrum-bending radiograph. This reduced to 33° on the fulcrum-bending radiograph after the release, and highly corresponded to the 30° measured at the post-operative standing radiograph and at the latest follow-up. Previous animal and cadaveric studies demonstrating the effectiveness of thoracoscopic anterior release did not have scoliosis. We are able to demonstrate in patients with adolescent idiopathic scoliosis, that thoracoscopic anterior spinal release effectively improves the spinal flexibility.
Introduction
In the management of patients with stiff thoracic scoliosis and kyphosis, anterior spinal release is helpful in increasing the spinal flexibility and therefore, the result of deformity correction. Excision of intervertebral discs through open thoracotomy is the preferred method in the past. However, cutting of the chest-wall muscles is associated with complications such as reduced ventilation, post-operative atelectasis, extensive and painful scars, blood loss, and prolonged hospital stay [7].
Video-assisted thoracoscopic surgery (VATS) for anterior release of the spine is becoming increasingly more popular due to its minimally invasive nature [1, 19, 25]. However, opponents do not believe that VATS is effective in improving the spinal flexibility as it is difficult to perform a radical discectomy, and to a lesser extent, rib-head excision by thoracoscopy [5, 23]. While VATS supporters have used animals and cadavers to demonstrate the effectiveness of thoracoscopic spinal release [6, 20], these studies were performed in spines that did not have scoliosis.
The fulcrum-bending radiograph, obtained with the patient lying sideways hinging over a fulcrum provides a simple and reproducible technique for the in-vivo assessment of spinal flexibility. It can accurately predict before surgery, the amount of correction that can be achieved by modern segmental spinal instrumentations [4, 11, 16, 17]. Using this method, one can assess the effectiveness of an anterior release by either directly comparing the spinal flexibility (as reviewed by the fulcrum-bending radiograph) before and after an anterior release, or indirectly by comparing the predicted correction by the pre-release fulcrum-bending radiograph and the actual correction achieved by the anterior release and posterior fusion. Either way, differences in the measured Cobb angle would be attributable to the effect of the thoracoscopic release.
In our institution, a number of thoracoscopic anterior release and posterior fusion surgeries for thoracic idiopathic scoliosis were staged. This gave the opportunity to perform the fulcrum-bending radiograph before and after the anterior release, thereby allowing direct assessments of flexibility changes as a result of the thoracoscopic procedure.
Materials and methods
Between 1997 and 1999, five patients with idiopathic scoliosis requiring anterior release for stiff thoracic curves were prospectively investigated. The authors define stiff curves as those which have a Cobb angle of more than 40° with the fulcrum-bending radiograph; this was arbitrarily used as we felt that the residual curves of over 40° gave unacceptable cosmetic results, and therefore a flexibility-modifying procedure would be indicated. The mean age at the time of operation was 23.0 years (range 13.9–35.3). According to the King’s classification [10], there were one type I, two type II, and two type III curves. For the patient with type I curve, both thoracic and lumbar curves were corrected and fused, and for the type II and III curves, only the thoracic curves were instrumented. According to Lenke’s classification [12], there were three type I, one type III, and one type V curve.
All the patients underwent an anterior thoracoscopic release, followed by a second-stage posterior instrumented correction, and fusion 1 week later. The instrumentations used for the posterior spinal fusion were Texas Scottish Rite Hospital (TSRH, n=2; Sofamor-Danek, Memphis, TN, USA), ISOLA (n=2; Acromed Corp., Cleveland, OH, USA), and CD-Horizon (CD-H: n=1; Sofamor-Danek, Memphis, TN, USA) systems. The mean number of thoracic discs resected was four, ranging from 3 to 5 (Table 1). Posterior fixation was done by a pre-dominant hook construct using one upper and two intermediate hooks, and either a hook or pedicle-screw at the lowest fusion level.
Table 1General patient dataCase 1Case 2Case 3Case 4Case 5King’s typeIIIIIIIIIIIAge (years) at operation21.513.925.435.219.1Levels releasedT7-T10T7-T11T6-T10T7-T12T6-T10Number of discs released34454Instrumentation used TSRHTSRHISOLACDISOLA
The thoracoscopic anterior release was performed with the patient under general anesthesia. The technique involved a discectomy by making a large annular window over the convex side of the scoliosis; the whole nucleus and cartilaginous end-plates were removed. Excision of the posterior annulus to the posterior longitudinal ligament is always attempted but is usually successful only at the apical discs. Rib-head excision was not performed.
The effectiveness of the thoracoscopic anterior release in increasing spinal flexibility was investigated by two methods. First by a direct comparison of the Cobb angle measured from a pre-release fulcrum-bending radiograph with that of the post-release fulcrum-bending radiograph. Second, by an indirect method, comparing the pre-release fulcrum-bending radiograph with the final post-operative correction. As the pre-operative fulcrum-bending radiograph has been reported to be able to accurately predict the post-operative coronal deformity correction for posterior surgery [4, 11, 17], the difference of the Cobb angles between the pre-operative fulcrum-bending radiograph (which predicted the correction for the posterior surgery alone) and the post-operative standing X-ray (which shows the actual correction achieved by combined anterior release and posterior instrumentation) will indirectly demonstrate the in vivo effect of the anterior thoracoscopic spinal release. Statistical analyses were carried out using the Student’s t-test, with a significance level of (P<0.05).
The “fulcrum flexibility”, a measure of the spinal flexibility as revealed by the fulcrum-bending radiograph [11, 17] was calculated based on the following formula:  where FB stands for fulcrum bending.
This relationship was used to directly assess the changes in flexibility as a result of the anterior release.
Results
The patients were followed for an average of 4 years (range 2.2–4.9 years). The mean fulcrum flexibility before the anterior release was 39%; it increased by 15–54% after the anterior thoracoscopic release (P<0.05).
The mean pre-operative Cobb angle on the postero anterior (PA) standing radiograph was 71°, the mean pre-operative fulcrum-bending angle was 43°, the mean post-release fulcrum-bending angle was 33°, and the actual mean Cobb angle after combined anterior thoracoscopic release and posterior surgery was 30°. The latter correlated with the post-release fulcrum-bending result (P=0.09), and was significantly different (P<0.05) from the pre-release fulcrum-bending result to suggest that the thoracoscopic anterior release can effectively improve the surgical correction of the coronal curve. On an average, four discs were excised per patient, and as the mean improvement in correction is 13°, it suggested that resection of one disc resulted in a mean improvement in correction by approximately 3° (Table 2, Fig. 1).
Table 2Measured Cobb angles for each case (For case 5, it was not possible to perform a post-release fulcrum-bending XR because of wound pain)Case 1Case 2Case 3Case 4Case 5MeanPre-operative AP standing 657675786171Pre-release fulcrum bending434145454043Post-release fulcrum bending35284030N/A33AP standing latest follow-up322830282629Fig. 1 aPre-operative anteroposterior standing radiograph of case 2, showing a 76°-curve from T6 to T12. b Pre-release fulcrum-bending radiograph showing a correction to only 41°. c Fulcrum-bending radiograph taken 1 week after an anterior thoracoscopic release of four levels, showing an improvement in the flexibility to 28°. d Post-operative standing radiograph taken 1 week after the posterior correction, showing the curve correction to 29°
The immediate post-operative Cobb angle of 30° (data not shown) was well predicted by the post-release fulcrum-bending radiograph of 33°. There was no significant change in this correction at the latest follow-up (Table 2).
Discussion
Anterior spinal release by open thoracotomy has been extensively used in the past to help improve spinal flexibility in stiff thoracic scoliosis. Its role has been increasingly taken over by VATS as the latter is associated with a lower morbidity [8, 21]. However, some surgeons feel that an anterior thoracoscopic release is not as effective as an open release. This is because they believe that a successful anterior spinal release requires a rib-head resection and a radical discectomy, which cannot be easily performed through VATS [5, 23]. Although studies using animals and cadavers have demonstrated that a thoracoscopic release can improve spinal flexibility [6, 20], it was not performed on actual patients with scoliosis. Thus, there is no definitive proof in human patients with scoliosis that the thoracoscopic spinal release is effective. Although some clinical studies have demonstrated that combined VATS and posterior spine fusion resulted in good scoliosis correction [2, 21, 22], they did not assess the actual flexibility of the scoliosis and therefore were not able to directly demonstrate that the thoracoscopic release added to the spinal flexibility.
The use of the fulcrum-bending radiograph, which reflects the spinal flexibility and accurately predicts the post-operative coronal deformity correction, provides an opportunity to assess the effectiveness of thoracoscopic release in vivo [24, 27, 28] The five cases included in this study had stiff curves, with a pre-operative fulcrum-bending angle of more than 40°, a mean pre-operative fulcrum flexibility of only 39%, and a mean Cobb angle of 71°. As these cases had staged surgery, we were able to obtain a post-release fulcrum-bending radiograph for comparison with the pre-release and post-operative Cobb angles. After anterior thoracoscopic release, flexibility was increased from 39 to 54%, thus representing a direct proof that the procedure can improve the spinal flexibility, in our cases by an average of 15%.
One patient (Case 5) was unable to lie on the fulcrum to perform a post-release fulcrum-bending radiograph due to wound pain. Nevertheless, case 5 has been included in the analysis, as a comparison of the pre-release fulcrum-bending radiograph with the post-operative radiograph will still provide an indirect evidence on the success of a thoracoscopic release (Table 2). For those who were able to lie on the fulcrum, we found the technique reliable. In this limited series of four cases, the post-release fulcrum-bending radiograph correlated well with the post-operative result.
It should be noted that the previous work on the fulcrum-bending radiograph are based on the use of hooks and hybrid systems only [4, 15]. The predictability of this method with reference to the use of pedicle-screw fixation is not known. It is widely believed that pedicle-screw systems give a superior degree of correction when compared to hook systems, and may obviate the need to perform anterior releases. However, there is no published data, which directly compares the two systems, taking into account the spinal flexibility.
The authors are aware of two different ways to measure Cobb angles before and after surgery. One method is to determine the Cobb angles in the pre-operative standing radiograph, and then to use the same levels throughout, although in the post-operative radiograph, the same measured levels may no longer be the most “tilted” vertebra. The alternative method is always a measure from the most “tilted” levels even though the levels may change between pre- and post-operative radiographs. While the authors prefer the former method, use of the latter method would not alter the results. Using the presented case as an example (Fig. 1), the pre-operative Cobb angle from T6 to T12 was 76°, the pre-release Cobb angle from T7 to T10 was 50°, the post-release Cobb angle from T8 to T10 was 40°, and the final Cobb angle from T7 to T10 was 40°.
The technique of anterior spinal release is different amongst different surgeons, while some do only a discectomy; others routinely remove the rib-heads, and even disrupt the posterior longitudinal ligament. The use of VATS limits the number of structures that can be easily released due to limitations in visualization and access to all disc levels. This study is not comparing the open thoracotomy and release with the thoracoscopic anterior release; this is because no data is available for the former. Moreover, an evaluation of open release versus thoracoscopic release using the present method is not possible, as patients undergoing the open release would still have a painful wound, which would prevent them from lying sideways over a fulcrum.
One potential pitfall of this study is that three different types of implants were used, while the original study on the fulcrum-bending radiograph was based on TSRH [4]. It maybe possible that different implants vary in their ability to correct scoliosis and therefore invalidate the indirect assessment. However, the authors feel that this is unlikely because it has been demonstrated by the same group that there was no significant difference in the ability of different instrumentation systems to correct thoracic scoliosis [18]. Moreover, the direct comparison of fulcrum flexibility before and after an anterior release would still stand.
This study was performed in the early part of our experience with thoracoscopic anterior release, hence relatively few discs were released and the surgeries were staged. However, it did serve the purpose of demonstrating that thoracoscopic anterior release does result in an improvement in spinal flexibility. With increasing experience, the authors tend to release five to six discs per patient and the posterior surgery is performed on the same day.
With the advent of new techniques and instrumentation, the indications for a thoracoscopic release may change in time. In particular, pedicle-screw systems appear to provide a better correction of large magnitude curves [9, 13, 26]. However, to date, there are no randomized studies comparing hooks versus screw systems, nor a correlation of the correction to spinal flexibility assessment, such as the fulcrum-bending correction index [17, 18]. Additionally, the use of anterior instrumentation systems may mean that such surgeries are carried out as a single-stage anteriorly, avoiding the need for posterior surgery [3, 14].
In summary, this is the first study to provide a direct in vivo evidence demonstrating the effectiveness of thoracoscopic anterior release in improving the spinal flexibility in patients with scoliosis.

anterior release
spinal flexibility
adolescent idiopathic scoliosis
video-assisted thoracoscopic surgery
thoracoscopy


Different perceptions of the burden of upper GI endoscopy: an empirical study in three patient groups

Background Few studies have evaluated patients’ perceived burden of cancer surveillance tests. Cancer screening and surveillance, however, require a large number of patients to undergo potentially burdensome tests with only some experiencing health gains from it. We investigated the determinants of patients’ reported burden of upper gastrointestinal (GI) endoscopy by comparing data from three patient groups.
Introduction
Opportunities for screening and surveillance of premalignant conditions have increased and will increase in the future. However, such interventions can be burdensome, and, as in any screening situation, the number of subjects exposed to this burden is often much higher than the number of subjects experiencing the beneficial health effects of the screening [1].
Upper gastrointestinal (GI) endoscopy is commonly used to diagnose and treat patients with a range of conditions and symptoms. Complications related to upper GI endoscopy are rare, and it is considered to be a safe procedure [2, 3]. Patients with Barrett esophagus (BE), a premalignant condition mostly without physical symptoms but associated with an increased risk of developing esophageal adenocarcinoma of 0.5% per year, are recommended to undergo regular biennial endoscopic surveillance for early detection of esophageal cancer [4]. All patients participating in surveillance experience the pain and discomfort of biennial upper GI endoscopy, whereas progression to adenocarcinoma occurs only in a minority of BE patients [5–8] and undisputable evidence that surveillance prolongs survival is still lacking [9–12]. Hence, the patients’ perceived burden of upper GI endoscopy testing needs to be taken in to account in evaluating the health benefits of surveillance of subjects with BE.
In some situations, there is a trade-off between the effectiveness of screening (or surveillance) and the test uptake. For example, colorectal cancer screening using sigmoidoscopy is more effective than faecal occult blood testing [13]. At present, this trade-off is not relevant for surveillance of BE because a less burdensome test than upper GI endoscopy is not available, but the recognition that upper GI endoscopy is burdensome may prompt a reconsideration of the frequency of surveillance. Ongoing studies aim to identify groups of BE patients at lower risk of developing esophageal cancer than others, so that offering less frequent surveillance may be warranted [14].
At the patient level, empirical data on perceived burden of upper GI endoscopy can be used in the process of informing subjects with BE who consider participation to a surveillance programme. In a general sense, empirical data of the patients’ perceived burden of testing may contribute to subjects’ informed decision-making on participation (or non-participation) to screening or surveillance and hence, to quality of health care [15].
Studying the determinants of patients’ perceived burden of upper GI endoscopy, e.g. by comparing data from different patient groups, may allow for the identification of patient groups who are likely to experience more pain or discomfort than others. This information can be used in practice guidelines, e.g. on provision of sedation to prevent pain and discomfort, or other types of patient support. Studying determinants of patients’ perceived burden is of additional interest from the perspective of evaluation research. If patients’ perception of the burden of endoscopy differs by the context of e.g., surveillance or diagnostic work-up, the generalisability of data from one context to another is limited.
Our previous work [16] has shown that BE patients under regular surveillance perceive upper GI endoscopy as burdensome. They experienced anxiety and discomfort, but hardly reported pain or symptoms. We analysed potential determinants of the perceived burden of upper GI endoscopy by comparing BE patients with two additional patient groups, i.e., patients with non-specific upper GI symptoms (NS) and patients with a recent diagnosis of cancer of the upper GI tract (CA).
Methods
Ethics approval
The Medical Ethical Review Board of Erasmus MC—University Medical Center Rotterdam, The Netherlands, approved of the study (MEC 03.1064; October 9, 2003).
Patients
Patients undergoing upper GI endoscopy for surveillance of BE were participants of an ongoing trial (CYBAR), whose endoscopic burden was previously reported [16]. Inclusion criteria were: BE segment of 2 cm or more confirmed by a histological diagnosis of intestinal metaplasia, absence of high-grade dysplasia and carcinoma, willingness to adhere to endoscopic surveillance, ability to read the Dutch language and informed consent.
Patients with non-specific upper GI symptoms (NS) were referred for endoscopy by their respective GPs because of non-specific upper GI symptoms. They needed to be able to read the Dutch language, provide informed consent, not to have “alarm symptoms” such as hematemesis, melena, or dysphagia, and not be diagnosed with BE previously.
Patients with a recent diagnosis of upper GI cancer (CA) were referred for upper GI endoscopy plus ultrasonography (EUS) to determine therapeutic options. Ability to read the Dutch language and to give informed consent was also required in these patients. Patients were recruited from one academic and two regional hospitals for BE, two regional hospitals for NS and in one academic hospital for CA.
Endoscopic procedure
BE and NS patients underwent endoscopy with adult endoscopes (Olympus GIF-Q160, Zoeterwoude, The Netherlands). In the group of cancer patients, a combined endoscopy and EUS was performed with a Olympus GF-UM160. More than 95% of patients received oral anaesthetics (Xylocain 10% spray, Astra Zeneca, Zoetermeer, The Netherlands) preceding the introduction of the endoscope. Additional sedation with 2,5-5 mg midazolam (Roche, Woerden, The Netherlands) intravenously was offered as a standard procedure to all cancer patients, but was only administered with explicit patient consent. In BE and NS patients this was not standard, but it was administered on a patient’s request. Practice variations between and within countries in the use of sedation for upper GI endoscopy are common [17].
Hypotheses
Perceived burden of endoscopy was operationalised as pain and discomfort during the procedure, symptoms afterwards and psychological distress over time. We hypothesized that subjects who had previous endoscopies may get used to it to some extent and hence report less burden. Demographic characteristics (age, sex, educational level, employment status, etc) were considered as potential confounders. We expected that BE patients may get used to regular endoscopy to some extent, and that they adhere to surveillance expecting that the test result will be reassuring. Therefore, we expected BE patients to report less discomfort and burden than the patients with non-specific GI symptoms, who had less endoscopy experience. We also expected the BE group to report less burden from the endoscopy than the cancer patients, due to the endoscopy itself (combined with EUS in the cancer patients) and the fact that cancer patients were aware of their generally bad prognosis. Table 1 shows the potential determinants of perceived burden of endoscopy between patient groups.
Table 1Potential determinants of perceived burden of endoscopy for three patient groupsDeterminantBENSCA Indication for endoscopyRegular endoscopic surveillance for early detection of cancerTo find out if symptoms can be explained by e.g. a hiatal hernia or duodenal ulcer; a life-threatening diagnosis is not expected.To determine whether intentionally curative therapy is possible or notEndoscopy experienceYes, under endoscopic surveillance Limited, ranging from 0 to some Yes (by definition: at least one previous endoscopy needed to diagnose the cancer)Generic health statusGenerally healthy Mild impairment (aspecific gastrointestinal symptoms)Seriously impaired (cancer diagnosis)AgeIn-betweenYoungestOlderSexMore males than females Equal sex distributionPredominantly maleEndoscopy procedureNormal endoscopy procedure, sedation only on patient requestNormal endoscopy procedure, sedation only on patient requestEndoscopy combined with ultrasonography; sedation routinely offered
Questionnaires and measurements
Patients were asked to complete questionnaires at different time points, i.e., one week before the endoscopy (baseline), at the day of endoscopy (just before undergoing it), one week and one month after endoscopy [16]. In order to minimize the questionnaire load for CA patients they received only two questionnaires: the first on the day of endoscopy and the other one week afterwards. Some baseline items had to be included in the ‘endoscopy day’ questionnaire in the CA group. The content of the questionnaires is described below.
Pain and discomfort
Separate items in the questionnaire one week after endoscopy were used to assess pain and discomfort, respectively, as experienced during the procedure, for four steps of the procedure: the introduction of the endoscope, the endoscopy itself, the removal of the endoscope, and the period directly after endoscopy. Subjects were offered three response options (‘no’, ‘quite’ and ‘very’ painful or discomforting, respectively). Additionally, patients rated the overall burden of undergoing the endoscopy (very, somewhat, not burdensome) [16].
Symptoms
We compared the prevalence of 10 symptoms experienced in the week after endoscopy with the prevalence at baseline. For CA patients, the baseline questions were asked at the day of endoscopy. Presence of throat ache, heartburn, regurgitation, flatulence or feeling bloated, vomiting, hematemesis, dysphagia for solid foods or for of liquids, diarrhea, and constipation, was assessed using four response options (not at all, one day, 2–3 days, 4 or more days) [16].
Psychological distress (BE and NS patients)
We assessed general distress using the Hospital Anxiety and Depression scale (HAD) at all time points [18, 19]. Anxiety and depression scores of this scale range from 0–21, with scores of 11 or over indicating clinical, and scores between 8 and 10 indicating borderline anxiety or depression [18, 19]. We analysed the pattern of scores across measurements, assuming scores to return to normal after endoscopy. Scores from a Dutch general population sample (n = 1901; mean age = 61 year; 51% female) were available for comparison [19].
At baseline and at one week we also measured specific distress with the Impact of Event Scale (IES) [20, 21]. At baseline we assessed intrusive and avoiding thoughts regarding the endoscopy itself, and at one week regarding the communication of the final test result. The total scale ranges between 0 and 75, with scores of 26 or over indicating a high risk of developing a stress disorder [22].
Psychological distress (CA patients)
For CA patients we omitted the HAD and the IES measures regarding the endoscopy itself, because we expected that distress in these patients was already at the top of the scale, making any additional distress caused by the procedure itself indiscernible. The IES to assess specific distress regarding the endoscopy result was included in the questionnaire at the day of endoscopy, because these patients received the endoscopy results earlier than the next questionnaire.
Demographics and other data
Demographic data were collected at baseline (at the endoscopy day for CA patients). The EQ–5D self-classifier results in a patient’s classification of own health on five domains: mobility, self-care, usual activities, pain, and anxiety and depression (3 response options: no, some, severe/ complete limitations) and a summary score [23–25]. We asked BE and NS patients whether this was their first, second or a later endoscopy. Whether sedation was used during endoscopy was recorded separately.
Analyses
Differences in demographic and treatment characteristics between patient groups were analysed by Chi-square tests for categorical variables or one-way analysis of variance (ANOVA) for continuous variables.
The items for pain and discomfort were combined into summary scores to enable adjustment for confounders and analysis of determinants, by adding up the item responses (0, 1, 2, respectively) of the 4 items (range of pain and discomfort summary scores: 0 (no pain or discomfort) to 8) [16]. The response to the single item rating of overall burden was also treated as a summary score, with a range from 0 (no burden) to two [16]. Because these summary scores had a limited number of possible values and because the data were not distributed normally, we chose to analyse them with proportional odds models [26]. These models produce odds-ratios (ORs) for cumulative probabilities of the outcome variables. Proportional odds models are a variant of simple logistic regressions, but now ORs for dichotomies at all possible cut-off levels are estimated. E.g., for a variable with three possible outcomes 1, 2, and 3, ORs are estimated for (1 + 2) vs. 3 and for 1 vs. (2 + 3). The OR presented represents an overall OR, that is assumed to be similar across cut-off levels. Because some of the outcome variables and determinants had 10–15% missing data, and there were no reasons for selective missing data, we used multiple imputation (function AregImpute in Splus 6.0) [27] so that all available information in our dataset was used. In multivariate analysis of the determinants of patients’ perceived burden with the proportional odds model, we first adjusted for confounders (age, sex and employment status). Subsequently we evaluated the potential effects of the following determinants on discomfort, pain and overall burden, respectively:patient group (BE, NS or CA). This variable combines the differences in the endoscopy procedure (with or without EUS, sedation) and the indication to undergo the endoscopy. For BE and NS patients, this analysis was refined by additional separate analysis of the effect of the number of previous endoscopies (continuous, truncated at ≥20).baseline generic health status (EQ 5D summary score).whether sedation was administrated or not.baseline HAD anxiety score (not available for the CA patients).The prevalence of symptoms before and after endoscopy was compared using a method analogous to the Wilcoxon test. Responses were ranked and ANOVA was applied to the differences in these ranks [16].
The continuous HAD and IES scores were compared over time in SAS version 8.2 with repeated-measures ANOVA, using ‘Proc Mixed’ with REML and a compound symmetry covariance structure. Models comprised main effects of time (the measurements), confounders, determinants and interactions between determinants and time.
Proportional odds models were estimated with Splus 6.0. All other analyses were conducted in SPSS version 11.0.1.
Results
Patients and response
In total, 684 patients were eligible for inclusion: 192 BE, 365 NS and 127 CA patients. The overall response rate was 70% with 476 patients completed at least one questionnaire. The response differed by patient group; it was 180/192 (94%) in BE patients, 214/365 (59%) in NS patients and 82/127 (65%) in CA patients. Most BE patient had no dysplasia (78%), 22% had low-grade dysplasia [16]. NS patients were diagnosed with hiatal hernia (45%), non-specific gastritis (25%), reflux esophagitis (20%) and some other diagnoses (e.g. ulcer, polyps; 10%). CA patients underwent endoscopy and EUS for staging of esophageal carcinoma (72%), gastric cancer (26%) or lymphoma (2%).
Differences between groups in mean age, sex and employment status were statistically significant (P < 0.001) (Table 2). We therefore considered these variables as confounders and controlled for them in further analyses.
Table 2Patient characteristicsaBENSCADifferbNGroup18021482N.A.476Mean age (sd)62 (12)54 (16)64 (10)<0.001474Sex: male119 (66%)101 (47%)66 (80%)<0.001476EmploymentPaid employment59 (34%)85 (44%)25 (36%)<0.001438Retired87 (50%)65 (34%)38 (54%)Unpaid/unemployed29 (17%)43 (22%)7 (10%)Civil status Married/ together134 (77%)137 (69%)57 (80%)0.034444Never married/ tog.13 (7%)26 (13%)3 (4%)Divorced10 (6%)23 (12%)4 (6%)Widowed18 (10%) 12 (6%)7 (10%)EducationPrimary35 (20%)37 (19%)16 (23%)0.498435Secondary95 (56%)122 (63%)43 (61%)Tertiary40 (24%)35 (18%)12 (17%)HospitalAcademic center (1)37 (21%)082 (100%)N.A.476Regional hospital (3)143 (79%)214 (100%)0Sedation: yes43 (27%)18 (9%)56 (77%)<0.001419Endoscopy numberFirst1 (1%) 99 (59%)Unknown<0.001338Second26 (15%)38 (23%)Third or later144 (84%)30 (18%)EQ–5D summary score0.85 (0.18)0.73 (0.22)0.77 (0.21)<0.001433N.A., Not assessedaData for the BE group were published previously [16]bχ- test (categorical variables) or F-test (continuous) for differences between patient-groups
About 84% of the BE patients had had two or more previous endoscopies [16], compared with 18% of the NS patients (P < 0.001). Seventy-seven per cent of the CA patients received sedation during endoscopy, compared with 27% of the BE and 9% of the NS patients (P < 0.001). The differences in the mean EQ–5D summary score were in the expected direction (P < 0.001) (Table 2).
Pain and discomfort
Tables 3–5 show that the patient groups differed significantly in reported discomfort, pain and overall burden of endoscopy. The p-values shown for the summary scores relate to univariate analysis of differences between the patient groups before adjustment for confounders.
Table 3Discomfort during upper GI endoscopy as reported by patientsaDiscomfortNotQuiteVerynDifferbIntroducing the endoscope141 (34%)177 (42%)99 (24%)417P < 0.001    NS42 (24%)76 (43%)58 (33%)176    BE64 (37%)81 (47%)27 (16%)172    CA35 (51%)20 (29%)14 (20%)69Undergoing endoscopy166 (40%)162 (39%)89 (21%)417P = 0.024    NS62 (35%)63 (36%)51 (29%)176    BE75 (44%)72 (42%)25 (15%)172    CA29 (42%)27 (39%)13 (19%)69Removing the endoscope290 (70%)90 (22%)35 ( 8%)415P < 0.001    NS97 (55%)52 (30%)26 (15%)175    BE144 (84%)24 (14%)3 ( 2%)171    CA49 (71%)14 (20%)6 ( 9%)69Period immediately after317 (78%)71 (17%)20 ( 5%)408P = 0.348    NS132 (77%)29 (17%)11 ( 6%)172    BE136 (81%)29 (17%)4 ( 2%)169    CA49 (73%)13 (19%)5 ( 8%)67Discomfort summary score (range: 0–8)Mean (sd)    All2.35 (2.10)406P < 0.001    NS2.92 (2.36)171    BE1.88 (1.69)168    CA2.07 (1.99)67aData for the BE group were published previously [16]bSignificance of differences between three groups as determined by Chi-square test (categorical variables) or proportional odds models for ordinal response data (summary score). No correction for confoundersTable 4Pain during upper GI endoscopy as reported by patientsaPainNotQuiteVerynDifferbIntroducing the endoscope332 (80%)68 (16%)17 (4%)417P = 0.050    NS135 (77%)29 (17%)12 (7%)176    BE145 (85%)24 (14%)2 (1%)171    CA52 (74%)15 (21%)3 (4%)70Undergoing endoscopy320 (77%)77 (19%)17 (4%)414P < 0.001    NS141 (81%)23 (13%)10 (6%)174    BE137 (81%)28 (17%)5 (3%)170    CA42 (60%)26 (37%)2 (3%)70Removing the endoscope365 (88%)39 (9%)11 (3%)415P = 0.098    NS152 (87%)16 (9%)6 (3%)174    BE157 (92%)11 (6%)3 (2%)171    CA56 (80%)12 (17%)2 (3%)70P = 0.454Period immediately after350 (84%)52 (13%)15 (4%)417    NS147 (84%)23 (13%)6 (3%)176    BE145 (85%)22 (13%)4 (2%)171    CA58 (83%)7 (10%)5 (7%)70Pain summary score (range: 0–8)Mean (sd)    All0.86 (1.60)413P = 0.02    NS0.91 (1.81)173    BE0.66 (1.35)170    CA1.20 (1.60)70aData for the BE group were published previously [16]bSignificance of differences between three groups as determined by Chi-square test (categorical variables) or proportional odds models for ordinal response data (summary score). No correction for confoundersTable 5Overall burden of upper GI endoscopy as reported by patientsaOverall burdenNotQuiteVerynDifferbEndoscopy in general137 (34%)204 (51%)58 (15%)399P = 0.007    NS48 (30%)82 (50%)32 (20%)162    BE68 (41%)87 (52%)12 ( 7%)167    CA21 (30%)35 (50%)14 (20%)70Overall burden summary score (range: 0–2)Mean (sd)    All0.80 (0.67)399P < 0.001    NS0.90 (0.70)162    BE0.67 (0.61)167    CA0.90 (0.71)70aData for the BE group were published previously [16]bSignificance of differences between three groups as determined by Chi-square test (categorical variable) or proportional odds models for ordinal response data (summary score). No correction for confounders
Table 6 shows these adjusted differences in summary scores for pair wise comparisons between the groups, and how these are affected by the determinants. NS patients reported significantly more discomfort than BE patients, as demonstrated by the significant OR of 1.69. After adjusting for differences in the number of previous endoscopies, the difference in reported discomfort between NS and BE patients was no longer significant. Similarly, the difference in reported discomfort between NS and BE patients could also be explained by differences regarding the administration of sedation. The differences between the NS and BE groups in the baseline EQ–5D summary score and in baseline anxiety scores did not explain the differences in reported discomfort: the ORs remained significant. Reported pain during upper GI endoscopy did not differ between NS and BE groups. The difference in reported overall burden was significant (OR = 1.64, P = 0.03). This difference became also insignificant after adjustment for the number of previous endoscopies and for sedation.
Table 6Differences in discomfort summary score, pain summary score and overall burden score, pair wise between patient groups (after correction for age, sex and employment status as confounders); and effects of other determinants than patient group on these differencesDeterminantDiscomfort scorePain scoreOverall burdenORa95% CIbP-valueORa95% CIbP-valueORa95% CIbP-valueNS compared to BE (BE = reference group)Patient group: NS versus BE1.691.15–2.47< 0.011.090.70–1.710.701.641.05–2.550.03    + Number of previous endoscopies1.490.98–2.270.061.190.73–1.940.501.560.96–2.530.07    + Baseline EQ–5D1.511.02–2.230.040.830.53–1.320.441.490.96–2.330.08    + Baseline anxiety1.631.12–2.390.011.040.66–1.640.861.591.02–2.490.04    + Sedation1.420.96–2.090.081.080.68–1.720.731.400.88–2.220.15CA compared to BE (BE = reference group)cPatient group: CA versus BE1.220.75–1.990.422.691.51–4.77<0.012.371.38–4.07<0.01    + Baseline EQ–5D1.070.66–1.740.792.321.29–4.18<0.012.101.21–3.66<0.01    + Sedation2.061.16–3.640.012.711.40–5.27<0.013.101.68–5.74<0.01aOdds ratios were calculated by proportional odds models for ordinal response data. They show the differences between the patient groups, corrected for confounders and the determinant mentionedb95% confidence intervalcNumbers of previous endoscopies and baseline anxiety were not available for CA patients
CA patients reported significantly more pain (OR = 2.69, P < 0.01) and overall burden than BE patients (OR = 2.37, P < 0.01; Table 6). The differences in reported pain could not be explained by differences in baseline EQ–5D summary scores or whether sedation had been administrated or not (all ORs remained significant, Table 6). CA and BE patients did not differ in reported discomfort (OR = 1.22, P = 0.42), but after taking differences in the provision of sedation into account, the difference in reported discomfort became significant (OR = 2.06, P = 0.01).
Symptoms
After endoscopy, throat ache was the only symptom that was reported more often than before the procedure (51 vs. 23%; P < 0.001). Other symptoms did not increase in frequency. Compared to BE patients, the increase in throat ache was smaller for NS patients and larger for CA patients (P < 0.001); 31% of NS patients reported throat ache before and 46% afterwards, compared to12% and 47% of BE patients, and 12% and 70% of CA patients, respectively.
Psychological distress
Figure 1 shows unadjusted mean anxiety and depression scores (HAD—not available for CA patients) by patient group over time.
Fig. 1Differences between BE and NS groups in Hospital Anxiety and Depression (HAD) scale scores for general distress before and after upper GI endoscopy (mean scores, no adjustment for confounders)
After adjusting for confounders (repeated measures ANOVA), anxiety levels were similar between the BE and NS groups across measurements, but the pattern differed significantly between them (interaction effect of ‘group’ with ‘time’, P = 0.01): BE patients reported lower anxiety levels at the start and slightly higher at the end. Determinants (number of previous endoscopies, baseline EQ–5D summary score, sedation) did not influence this pattern of anxiety over time (no significant interaction effects with ‘time’). Anxiety scores of both NS and BE patients were significantly higher at all time points than reported by a general population sample (score = 3.9; P < 0.001 for each group at each measurement) [19].
At all measurements, depression scores were lower in BE than in NS patients (P < 0.001). This difference was significantly larger before than after endoscopy (interaction effect of ‘group’ with ‘time’, P = 0.01). The number of previous endoscopies affected the pattern of depression over time (interaction effect of ‘number of previous endoscopies’ with ‘time’ P = 0.046) making the pattern of the two groups more similar. Depression scores differed from those reported by the general population sample: BE patients reported significantly lower levels at all measurements, while baseline NS scores were significantly higher (norm score = 3.7, P < 0.001 for each comparison).
Specific distress (IES) scores regarding the endoscopy itself and its outcome were lower in BE patients than in NS patients (mean scores at baseline measurement and 1 week measurements are BE 5.5 (sd 9.5), NS 12.9 (sd 14.7), and BE 3.5 (sd 7.7), NS 9.4 (sd 14.3) respectively, P < 0.001). The determinants did not affect this difference. In both BE and NS patients, specific distress regarding the endoscopy (IES, baseline measurement) was higher than regarding the test result (IES, one week measurement) (P < 0.001). High IES-distress scores regarding the endoscopy were seen in 51 patients (14%). CA patients (mean IES score 22.3 (sd 17.8)) had significantly higher distress levels (IES) regarding the test-result than the other patient groups (P < 0.001).
Discussion
This study is the first to investigate determinants of patients’ perceived burden of upper GI endoscopy. Patients undergoing endoscopy for different reasons reported a different burden from the procedure. BE patients who underwent endoscopy as part of regular surveillance, reported the lowest discomfort, pain and overall burden, confirming our hypotheses in this respect. Patients with non-specific GI complaints reported more discomfort from the procedure, while those diagnosed with cancer experienced more pain and both groups reported more overall burden than patients under surveillance for BE. These differences remained significant after adjustment for confounders (age, sex, employment status). Differences in baseline anxiety scores or in baseline general health (EQ–5D) did not explain the differences in reported discomfort or pain. Differences in the number of previous endoscopies, and in whether sedation was provided during endoscopy or not, explained part of the differences in reported discomfort between NS and BE patients. Whether sedation was provided or not did not explain the differences in reported pain and overall burden between BE and CA patients.
The study also confirms that that upper GI endoscopy is burdensome for all groups of patients: two-thirds of the total group of patients reported discomfort and overall burden from the procedure, and patients were distressed beforehand. These results may however underestimate the actual burden because this empirical study was limited to patients who actually underwent upper GI endoscopy, hence excluding patients who refrained from undergoing endoscopy because of past or anticipated adverse experiences. Another potential limitation of our study results from the differences in response rates between the groups.
Differences between patient groups were also found for symptoms resulting from the endoscopy. Of all symptoms explored, only throat ache increased after upper GI endoscopy. CA patients reported a higher increase in throat ache than BE patients and NS patients. As upper GI endoscopy hardly caused any symptoms, we considered an investigation into determinants of these differences to be less interesting and therefore omitted those analyses.
Furthermore, BE and NS patients differed in the levels of generic (HAD) and specific (IES) distress they reported. Specific distress (IES) was significantly higher in NS patients than in BE patients, both regarding the endoscopy itself and its result. General distress (HAD) also differed between groups: BE patients reported less depression across all measurements and the pattern of anxiety and depression across measurements was different. However, general distress is not necessarily related to the endoscopy. The persistent higher depression scores across different time points suggest that NS patients have more depressive symptoms in general but that this was not related to the endoscopy. The different pattern of anxiety levels before and after endoscopy, however, suggests that the patient groups also differed in endoscopy-related distress. The pattern corroborated the findings of the specific (IES) distress scores: NS patients were more distressed than BE patients before the endoscopy. The investigated determinants did not explain the differences between groups in specific distress or general distress pattern, except for the number of previous endoscopies explaining part of the difference in the depression scores.
BE patients thus reported less distress and also less pain or discomfort than other patient groups. This was not caused by differences in patient characteristics (age, sex, employment status, baseline anxiety, baseline general health). There are several potential reasons why the reported burden differs. Firstly, BE patients are under regular surveillance and may get used to or adapt to the procedure decreasing its burden. As the number of previous endoscopies explained the lower distress, discomfort and overall burden reported in the BE group, we conclude that getting used, or adapting to endoscopy plays a role.
Secondly, patients who perceive a greater benefit of the test may weigh its burden differently and consequently report less burden. BE patients potentially have more to gain from early discovery of adenocarcinoma than NS patients, who are usually referred for endoscopy to detect potential explanations for their symptoms, and also more than CA patients for whom endoscopy and ultrasonography are only part of the procedure to determine their treatment options and prognosis. As we did not measure perceived expected benefit of the endoscopy we are not able to determine whether this mechanism is part of the explanation.
Thirdly, the endoscopic procedure was slightly different for the different patient groups. CA patients received sedation more often. Adjusting for this difference into the analysis did not explain the differences in pain and overall burden, whereas the difference in reported discomfort became significant after adjustment for sedation. These results suggest that differences in the proportions of patients receiving sedation during endoscopy did not explain the differences between the groups, and that sedation was provided to those patients who really needed it. The procedure for CA patients also differed; they underwent upper GI endoscopy combined with ultrasonography, and for the combined procedure an endoscope with a slightly larger diameter is used. Our data did not allow us to test separately whether this affected perceived pain and overall burden. Finally, most CA patients had esophageal carcinoma, and this disease may make passing the endoscope through the esophagus more difficult and therefore more painful.
We measured general psychological distress (HAD) at different time points; assuming that a pattern of higher distress levels before compared to after endoscopy indicates that the procedure causes distress. As discussed in a previous paper [16], this may be debated for the reason that lower distress levels afterwards may also result from a reassurance effect of patients receiving a negative test result (no serious disease present). Nevertheless, the fact that the specific distress (IES) score relating to the endoscopy was higher than the IES score relating to the test outcome led us to conclude that the prospect of undergoing upper GI endoscopy does indeed increase distress levels.
Even if upper GI endoscopy causes HAD anxiety and depression scores to be increased before the endoscopy, the relevance of these increased distress levels can be questioned. Anxiety may be a relevant problem with 20% of patients having scores indicating clinical anxiety levels at baseline, while the depression scores are less worrisome (6%). Endoscopy-specific distress (IES) was high in 14% of patients and higher than the distress related to the outcome. Anxiety scores in our study were increased compared to general population scores at all time points. Especially the fact that NS patients remained at increased levels one month after endoscopy makes the comparability of our scores with the population scores questionable [19]. General population scores are not available for procedure specific-distress (IES), as this can only be measured in patients. Considering the cut-off values for clinical scores, the prospect of endoscopy causes moderate distress.
The observation that patients under regular endoscopic surveillance may adapt to this invasive procedure should not result in an underestimation of the burden of regular endoscopic surveillance. The search for less invasive surveillance tests should continue, and frequency of surveillance should preferably be established by evidence-based individualized estimates of risk of progression.

barrett esophagus
endoscopic surveillance
discomfort
anxiety
distress
upper gastrointestinal endoscopy
perceived patient burden