Non-breeding habitat preference affects ecological speciation in migratory waders

Models of ecological speciation predict that certain types of habitat should be more conducive to species diversification than others. In this study, I test this hypothesis in waders of the sub-order Charadrii using the number of morphological sub-species per species as an index of diversity. I classified all members of this clade as spending the non-breeding season either coastally or inland and argue that these represent fundamentally different environments. Coastal mudflats are characterised by high predictability and patchy worldwide distribution, whilst inland wetlands are widespread but unpredictable. The results show that migratory species that winter coastally are sub-divided into more sub-species than those that winter inland. This was not the case for non-migratory species. I argue that coastal environments select for more rigid migratory pathways, whilst inland wetlands favour more flexible movement patterns. Population sub-division could then result from the passive segregation of breeding sites or from the active selection for assortative mating of ecomorphs.
Introduction
Models of ecological speciation show that the propensity of a species to diverge is affected by the shape of the fitness landscape generated by its environment (Gavrilets 2004). This predicts that some environments should be more conducive to speciation than others. Recent comparative studies have supported this prediction (Funk et al. 2006; Phillimore et al. 2007). However, these studies do not discuss aspects of habitats that promote or inhibit speciation.
In this study, I test this prediction for wading birds of the sub-order Charadrii. This group was chosen because its species can be divided into those that are adapted to coastal habitats and those that feed mostly inland (Piersma 1997, 2003). On a year-to-year basis, coastal habitats offer a more predictable environment than inland wetlands (Roshier et al. 2001). Inter-tidal mudflats and ocean beaches will be located in the same place each year and offer a highly predictable pool of food resources. They are distributed patchily around the globe, and patches differ in their food resources and other conditions (Piersma et al. 1993a). On the other hand, the inland wetlands favoured by other waders are unpredictable and include waterbodies in arid regions that flood infrequently. Flooded areas are often large, but may be located in different areas between years and shift within a single non-breeding season, promoting extensive movements of birds that rely on these habitats (Roshier et al. 2002). The suitability of grasslands in semi-arid regions varies from year to year due to the differences in rainfall level. Little is known about the composition of the food resources used by waders in these inland environments, but it seems likely that these may be similar on broad spatial scales. I thus predict that coastal habitats should be more favourable to species diversification than inland habitats.
Many of the wader species that utilise coastal or inland habitats for the greater part of the year migrate to very different habitats, such Arctic tundras or boreal swamps, to breed. Whilst the reasons for this are beyond the scope of this study (but see Piersma 1997), it means that in these species reproduction is spatially separated from the feeding habitat to which they are adapted. Red knot Calidris canutus illustrate this point. For 10 months of the year, this species feeds on bivalves on mudflats around the world. The species has evolved a range of adaptations to feeding on bivalves, including a muscular stomach and sensitive bill tip (Piersma et al. 1993b, 1998). Nevertheless, it breeds exclusively on high Arctic tundras where feeding on abundant insect larvae and berries require few special adaptations. In such cases, local adaptation to, for example, temperate or tropical inter-tidal mudflats requires that birds from the same non-breeding area mate assortatively on the breeding grounds (Webster et al. 2002).
Investigating ecological speciation in this group of birds thus adds two new dimensions to the general pattern found by Funk et al. (2006) and Phillimore et al. (2007). First, as argued above, I have a priori reasons to expect that one type of habitat (coastal) should be more conducive to speciation than the other (inland). Second, many of the species investigated reproduce away from the feeding grounds to which they are adapted and speciation thus requires differential migration to achieve assortative mating. To test these ideas, I use the number of sub-species per species as a measure of diversification. Thus, I treat sub-species as ‘incipient species’. Morphological sub-species are considered useful in estimating the patterns of divergence among populations (Phillimore et al. 2007). Evidence that wader sub-species represent phylogenetically distinct groups is available for Dunlin Calidris alpina (Wenink et al. 1993), and to a lesser extent, for Red knot (Buehler and Baker 2005). Because older species could have differentiated into more sub-species than younger species, I investigated whether the number of sub-species was correlated to species age.
Materials and methods
Data collection
I collated data on the number of sub-species, migratoriness, breeding and non-breeding habitat from Del Hoyo et al. (1996) for all 215 species of the sub-order Charadrii (see Table 2 of S1). Crude estimates of species age were obtained from Thomas et al. (2004). Due to high levels of polytomy in parts of the phylogeny, some of these are likely to be over-estimates; however, no better estimates are available at present. I adopted the number of sub-species from one reputable source rather than search for the more recent updates because some of these revisions are still controversial. Migratoriness was scored as non-migrant, partial migrant or migrant (>80%, between 20% and 80% and <20% of non-breeding range overlaps with breeding range, respectively). Breeding habitat was scored as one of seven classes: tundra, boreal/temperate, mountains, steppe, (sub)tropical wetlands (either inland or coastal), (sub)tropical forest or oceanic. Non-breeding habitat was scored as coastal, inland or pelagic. For species that regularly use both coastal and inland habitats, I scored the habitat considered by Del Hoyo et al. (1996) to be used by the largest part of the population.
Statistical analysis
I first investigated whether the dependent variable (number of sub-species) showed phylogenetic auto-correlation (i.e. whether closely related species resembled each other more in their tendency to form sub-species than expected by chance) using the phylogenetic topology from Thomas et al. (2004). I used runs test as implemented in the programme Phylogenetic Independence (Abouheif 1999) to compare the observed pattern to that generated by randomly redistributing the data over the tips of the phylogeny 1,000 times. For this analysis, the number of sub-species per species was dichotomised as 1 (i.e. only the nominate form) or >1. Finding no evidence for phylogenetic autocorrelation (see the “Results” section), I treated species as independent data points in further analysis.
As the number of sub-species per species followed a Poisson distribution, I analysed the data using generalised linear models with a Poisson error distribution and a log link function. GLMStat (Beath 2001) was used to generate glms. Significance was tested by dropping each term from the fuller model and comparing the resulting change in deviance to a chi-square distribution.
Results
The results of runs test showed that there was no evidence for auto-correlation in the phylogenetic distribution of the number of sub-species (runs test P = 0.46). In fact, 461 of the 1,000 randomly generated runs averages were greater than that of the real data set, indicating a non-significant tendency towards over-dispersion.
A glm containing migratoriness, non-breeding habitat, breeding habitat and species age and their interactions showed no significant effects of three- and four-way interactions. However, there was a significant two-way interaction between migratoriness and non-breeding habitat (Table 1), indicating that the pattern of covariance between non-breeding habitat and sub-species richness differed between migrants and non-migrants. Individual models revealed a significant effect of non-breeding habitat on the number of sub-species in migrants, but not in non-migrants or partial migrants (Table 1). These results are illustrated in Fig. 1: species wintering coastally are on average sub-divided into more sub-species than inland wintering species, but only in migrants. The results are not confounded by differences in species age (Table 1). Whilst there was a significant association between breeding and non-breeding habitat among migrants (, P = 0.009), this did not result in an association between breeding habitat and the number of sub-species (Table 1).
Fig. 1Mean number of sub-species per species for migratory and non-migratory waders. Pelagic wintering species (N = 2) and partial migrants (N = 31) not shown. Error bars represent the Poisson standard errorsTable 1Results of the generalised linear model with the number of sub-species per species as independent variable Estimate ± SEDeviance (df = 1)P valueMinimal adequate model    Migratoriness−0.10 ± 0.08 0.23 Non-breeding habitat−0.16 ± 0.19 0.39 Breeding habitat0.06 ± 0.04 0.17 Species age−0.01 ± 0.007 0.11Migratoriness × non-breeding habitat0.27 ± 0.124.920.03Separate models    Non-breeding habitat     Migrants0.42 ± 0.157.390.007  Partial migrants0.13 ± 0.100.070.79  Non-migrants−0.21 ± 0.211.070.30Non-significant interaction terms were removed. P values for the main effects were obtained by using the parameter estimate divided by its standard error as the test statistic. Also shown are the results of separate models for each category of migratoriness.
Discussion
The prediction that coastal habitats should be more conducive to diversification in waders than freshwater habitats is supported, but only for migratory species. This is remarkable, given that the reduction in gene flow between locally adapting populations requires an extra step in these species. In non-migrants, all that is needed to mate with a locally adapted partner is not to disperse. In migrants, however, all individuals migrate away from the non-breeding grounds to breed, often to the other end of the globe. To mate with a partner from the same non-breeding grounds, the non-breeding populations must either have segregated breeding grounds or have a very sophisticated individual recognition system. There is good evidence for a number of coastally wintering migrants that the former is true (Wennerberg 2001; Atkinson et al. 2005; Buehler et al. 2006), whilst the latter has not been investigated.
I suggest that the predictability of coastal non-breeding grounds favours rigid migratory pathways to and from the non-breeding grounds. Returning to the same non-breeding ground each year would save individuals from wasting time on searching for suitable non-breeding areas and allow them to benefit from learned aspects of local conditions (such as food distribution). The rigidity of the migratory pathways could then result in spatial segregation on the breeding grounds because individuals would be genetically predisposed to favour the same breeding grounds each year. The formation of distinct sub-species would then be a by-product of the spatially discontinuous distribution of non-breeding grounds. By contrast, many of the species that spend the non-breeding season on inland habitats have to track spatially and temporally variable resources (Roshier et al. 2001, 2002), which should select for flexible movement patterns during the non-breeding season. This could translate into flexible migration patterns, perhaps through a genetic correlation between migratory and non-migratory movements, and flexible movement patterns on the breeding grounds. If true, this would promote gene flow and inhibit diversification.
Alternatively, the divergent ecology of different coastal non-breeding grounds may actively select for assortative mating of corresponding ecomorphs. For example, it may be costly for waders wintering on tropical mudflats to pair with those that winter on temperate mudflats because the resultant hybrids would be poorly adapted to either habitat. Support for this interpretation comes from the fact that many sub-species of coastal waders differ from each other in bill length, which is an ecologically relevant trait (Engelmoer and Roselaar 1998). By contrast, the unpredictability of inland wetlands would not allow specialisation to the specific conditions presented by any particular area of habitat.
The question remains why the difference in diversification between species occupying coastal and inland habitats is not reflected in the non-migratory species that breed there? Given that migrant species often greatly outnumber resident species at individual feeding areas during the non-breeding season, migrant species may have greater effective population sizes per non-breeding area than non-migrants. This would allow more efficient response to selection and increase the potential for local adaptive change in migrants relative to non-migrants. Given the arguments above, such change is most likely in coastal areas.

non-breeding habitat
speciation
waders
sub-species


Simultaneous expression of Oct4 and genes of three germ layers in single cell-derived multipotent adult progenitor cells

Future application of adult stem cells in clinical therapies largely depends on the successful isolation of homogeneous stem cells with high plasticity. Multipotent adult progenitor cells (MAPCs) are thought to be a more primitive stem cell population capable of extensive in vitro proliferation with no senescence or loss of differentiation capability. The present study was aimed to find a less complicated and more economical protocol for obtaining single cell-derived MAPCs and understand the molecule mechanism of multi-lineage differentiation of MAPCs. We successfully obtained a comparatively homogeneous population of MAPCs and confirmed that single cell-derived MAPCs were able to transcribe Oct4 and genes of three germ layers simultaneously, and differentiate into multiple lineages. Our observations suggest that single cell-derived MAPCs under appropriate circumstances could maintain not only characteristics of stem cells but multi-lineage differentiation potential through quantitative modulation of corresponding regulating gene expression, rather than switching on expression of specific genes.
Introduction
Bone marrow mesenchymal stem cells (MSCs) are the most-studied adult stem cells and have been widely used owing to their advantages in autografting and multi-lineage differentiation potential, which is also referred to as the plasticity of stem cells [1–4]. However, some studies in recent years indicated that MSCs might have no plasticity or only have a limited plasticity [5, 6] and that the in vivo multi-lineage differentiation of MSCs might be a result of their fusion with the host cells [7, 8]. That is why, recently, people have been suspicious about the plasticity of MSCs. Some researchers think that the previously separated MSCs are actually a mixed cell population containing several kinds of stem cells with different differentiation potentials [9]. Therefore, how to isolate homogeneous MSCs with high plasticity becomes a ready task of researchers.
Isolation of MAPCs from adult bone marrows was firstly reported by Verfaillie’s team [10–12]. Compared with MSCs, these cells were thought to be a more primitive cell population with partially overlapping ontogeny. Moreover, MAPCs have been proven to be capable of extensive in vitro proliferation with no senescence or loss of differentiation capability. A few researchers held that when embryonic stem cells differentiate into different somatic germ layers and tissue cells, some pluripotent cells remain in specific tissues for future use [13]; judging from their differentiation property, MAPCs should belong to this type of cell. Although the above hypothesis needs to be further verified, this subpopulation of pluripotent stem cells provides a new thought and may serve as a new cell source for tissue engineering.
Based on the above understanding, we started our experiment with isolation of MAPCs from bone marrow of adult rats and then fully studied their differentiation potentials. MAPCs were isolated using a modified protocol of Verfaillie’ team [10]. By screening for cells highly expressing Oct4 from clone-like clusters, we successfully obtained a comparatively homogeneous population of MAPCs with multi-lineage differentiation potential. The present study was aimed to find a less complicated and more economical protocol for obtaining single cell-derived MAPCs and understand the molecule mechanism of multi-lineage differentiation of MAPCs. Our observations confirmed that single cell-derived MAPCs were able to transcribe Oct4 and genes of three germ layers simultaneously, and differentiate into multiple lineages.
Materials and methods
Culture and clone expansion of MAPCs
Bone marrow was obtained from 4-week-old male Sprague–Dawley (SD) rats. With fat and red blood cells removed, the remaining cells were seeded at a density of 1 × 106 cells/cm2 in 100 mm dishes coated with 10 ng/ml fibronectin (FN; Sigma-Aldrich, USA) and incubated at 37°C with 5% CO2 in a fully humidified atmosphere. The expansion medium of MAPCs consisted of 60% DMEM-LG (Gibco-Invitrogen, USA) and 40% MCDB-201 (Sigma), with 2% fetal bovine serum (FBS; Gibco-Invitrogen), 1× insulin/transferring/selenium (ITS), 1× linoleic acid bovine serum albumin (LA-BSA), 10−9M dexamethasone (Dex), 10−4M ascorbic acid 2-phosphate (all from Sigma-Aldrich), 100 U/ml penicillin, 1,000 U/ml streptomycin (Gibco-Invitrogen), 10 ng/ml EGF (Sigma-Aldrich), 10 ng/ml PDGF-BB (R&D Systems), and 10 ng/ml LIF (Chemicon International, USA). The nonadherent cells were removed by medium change at 24 h and every 3 days thereafter until 100% confluence was reached. Cells were passaged once with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA; Gibco-Invitrogen) and the passage 1 (P1) cells were replated at a 1:2 dilution under the same culture condition. After being passaged for at least four generations (P4), cells were seeded on a 96-well plate coated with 10 ng/ml FN at a density of 0.5 cell/well by limited-dilution method in expansion medium. The wells containing more than one cell under a phase-contrast microscope were discarded. When cells grew to 40–50% confluence, they were pipetted from one well, seeded to one of the 24 wells, and then serially reseeded to a six-well plate, 60 mm dish, and 100 mm dish in the presence of 0.25% trypsin-EDTA. When cells reached a density of 4–8 × 103 cells/cm2 in 100 mm dish, they were replated at a 1:3 dilution, and the fastest-growing clone was selected after being passaged for 20 times.
Total RNA isolation and RT-PCR analysis
Total RNA was extracted from the clonal cells using RNA Mini kit (Huashun, China). First-strand cDNA was synthesized with total RNA using a mixture of oligo (dT)12–18 and random hexamers (Roche Diagnostics, Germany) primers with AMV (Promega, USA) and Superscript II RNase H Reverse Transcriptase (Invitrogen, Germany). Polymerase chain reaction (PCR) was conducted with approximately 50 ng cDNA for amplification of the following marker genes: pluripotent marker Oct-4, endodermal marker lanosterol-14a-demethylase (CYP 51), mesodermal marker SM22α and GATA-4, and ectodermal marker N-methyl-d-aspartic acid (NMDA) glutamate binding protein. The housekeeping gene encoding 18S was used as an internal control. The PCR primers are listed in Table 1. After initial denaturation at 98°C for 3 min, PCR amplification was performed with Sawady Taq-DNAPolymerase (Takara Bio, Japan) at 95°C for 40 s followed by 28–35 cycles, with each cycle consisting of annealing at 55–60°C for 40 s, and a final extension at 72°C for 40 s. PCR products were subjected to 1.5% agarose gel electrophoresis, and the bands were visualized by ethidium bromide and photographed with Chemi Doc XRS (Bio-Rad Laboratories, Fudan, China). Each experiment was performed in triplicate.
Table 1Specific marker genes of three germ layers [14–16]GenesPrimers(5’-3’)Product size(bp)LocationLayerOct-4Forward: CGCCAGAAGGGGAAAAGA Reverse: CAGGAAAAGGGACCGAGTAG176  CYP 51Forward:ATCCTGACCGCTACCTACA Reverse: GGCTTCCCTGAAATCCTA461LiverEndodermSM22αForward:GTCCACAAACGACCAAGC Reverse: GTTCTCAGGCACCTTCACT279MuscleMesodermNMDAForward: TGAGCCCACCAGAAAAGG Reverse: ACTGCTTTGCCCTCCACC625BrainEctodermGATA-4Forward: TCCCCACAAGGCTATCCA Reverse: CCGAAGAAGGTCACGAGGT321HeartMesoderm18SForward: CAAGAACGAAAGTCGGAGGTTCG Reverse: TTATTGCTCAATCTCGGGTGGCTG460
Immunocytofluorescence and fluorescence-activated cell sorter analysis
Clonal cultured MAPCs were fixed with 4% paraformaldehyde for 4 min at room temperature. After being blocked with phosphate-buffered saline (PBS) containing 2% BSA, cells were permeabilized with 0.1%Triton-X100 for 10 min. Slides were incubated sequentially overnight at 4°C with the following primary antibodies: CD71 (1:50, Santa Cruz, USA), Vimentin (1:200, DAKO, USA), α-SMA (1:200, Boster Biotechnology, China), and SSEA-1(1:50, Santa Cruz, USA); then cells were incubated for 40 min at room temperature with fluorescein isothiocyanate (FITC) or Cy3-coupled IgG secondary antibody (1:500, Jackson), stained with DAPI (Sigma–Aldrich), and observed under a fluorescence microscope (BX41TB, Olympus, Japan).
For fluorescence-activated cell sorter (FACS) analysis, cells pelleted were incubated on ice for 30 min with FITC- or PE-conjugated monoclonal antibodies against CD34, CD44, CD45 (Becton–Dickinson, CA), and MHC-I (Abcam, UK). After two washes with cold PBS, the labeled cells were analyzed by a FACStar flow cytometer (Becton Dickinson, CA, USA).
In vitro differentiation of MAPCs
To induce differentiation, clonal cultured MAPCs were cultured in the media containing no FCS, EGF, PDGF-BB, or LIF unless otherwise indicated. All the experiments were repeated at least three times.
Adipogenic differentiation of MAPCs MAPCs were seeded at a density of 3 × 104 cells/cm2 in six-well plates, allowed to grow confluence, and cultured for another 3 days. Subsequently, cells were allowed to differentiate for 14 days in the expansion medium of MAPCs containing 0.5 mM 3-isobutyl-1-methylxanthine (Serva Electrophoresis, Germany), 100 nM Dex, 60 μM indomethacin, and 10 μg/ml insulin. At the end of the culture, cells were fixed with 10% formaldehyde (Shenggong, China) for 10 min and were stained with fresh Oil Red O (Sigma-Aldrich) for 2 h.
Osteogenic differentiation of MAPCs MAPCs, at a density of 2 × 104 cells/cm2, were induced with osteogenic medium containing 10–8M Dex, 2 × 10−4 M ascorbic acid and 7 × 10−3 M glycerolphosphate for 2 weeks. NBT/BCIP staining was used to analyze alkaline phosphatase (AKP; all from Sigma-Aldrich) expression during MAPCs differentiation. Meanwhile, cells were immunocytochemically stained with osteopontin to detect osteogenic differentiation of MAPCs.
Neuroectodermal differentiation of MAPCs MAPCs were plated at a density of 1 × 104 cells/cm2 on FN in a basal medium containing 100 ng/ml basic fibroblast growth factor (bFGF; R&D Systems). The cells were allowed to differentiate for 14 days and then immunofluorescently stained for GFAP (1:100) and TAU (1:100, both from Boster Biotechnology, China).
Hepatocyte differentiation of MAPCs MAPCs were plated at 2.2 × 104 cells/cm2 on plastic chamber slides coated with 2% Matrigel in a basal medium supplemented with 10 ng/ml FGF-4 and 20 ng/ml HGF (both from R&D Systems). After 14 days, immunofluorescent staining for albumin (1:500, DAKO, USA) was performed as described above.
Results
Stable in vitro proliferation and expansion of clonal cultured MAPCs
On the third day of primary culture, very few triangle-like cells adhered to the culture surface under a phase contrast microscope, coexisting with plenty of hematopoietic cells, which were round in shape and small in size, shining under the microscope (Fig. 1a, P0-d3). During the fourth to seventh day of culture, the triangle-like cells gradually increased and had a typical colony growth, with an obvious three-dimensional appearance and growth circle. The colony cells grew confluent and became morphologically homogenous after 7–10 days (Fig. 1a, P0-d10). After treatment with trypsin, the cultured cells no longer grew in clusters, but in a homogeneous manner (Fig. 1a, P4-d3).
Fig. 1Morphology of isolated MAPCs. a Phase-contrast micrographs of MAPCs at day  3 (d3), day 10 (d10) of primary culture (P0) and P4 at day 3 (d3) after passages (original magnification 100×). b MAPCs cloned by limiting dilution. A single cell adherent to the plate in one well proliferated in the primary culture for 2 days (D2), 4 days (D4), and 8 days (D8; original magnification ×400)
Single-cell clonal culture was performed by infinite dilution technique. Thirty percent of single cell-derived MAPCs formed larger clones during culture (Fig. 1b), and six cell clones (7F#, 4G#, 6E#, 4F#, 7C#, and 10D#) were obtained in the following expansion. When being cultured at an extremely low density (250 cells/cm2), this six-cell population still demonstrated strong capability of proliferation, with the doubling time being about 24–36 h. P20 cells still possessed the typical properties of MAPCs: polygonal-shaped, with larger nuclei, vacuoles around the nuclei, and sparse cytoplasm (Fig. 1b, D2, D4).
Expression of Oct4 and genes of three germ layers in single cell-derived MAPCs
The expression of self-renewal gene Oct4 was detected in the six cell clones after being passaged for more than 20 times. All the six cell clones were found to have different levels of Oct4 expression (Fig. 2a). Specific markers of the three germ layers were detected in the three cell clones (7F#, 4G#, and 10D#), which strongly expressed Oct4. The specific markers included CYP51 (present in the liver of adults) for endoderm, SM22α and GATA4 (present in the skeleton muscle the heart of adults) for mesoderm, and NMDA (present in the brain of adults) for neuroectoderm. As for the markers for the three germ layers, 4G# clone had a very weak expression of CYP51 and all the rest clones had high expression of their corresponding markers (Fig. 2b), especially for single cell-derived MAPCs-10D#. Therefore, we subjected single cell-derived MAPCs-10D# to further experiments.
Fig. 2RT-PCR analysis of expression of Oct-4 and specific markers for three somatic germ layers. a Oct-4 was highly expressed in six cell clones. b CYP 51, SM22α, GATA4, and NMDA were positive in three cell clones. NEG Negative controls, POS positive controls for CYP 51 (fetal liver), SM22α (fetal smooth muscles), GATA4 (fetal heart), and NMDA and Oct-4 (fetal brain tissues; n = 3)
Identification of molecular markers in single cell-derived P20 MAPCs-10D#
Single cell-derived P20 MAPCs-10D# was chosen for immunofluorescence assay. It was found that it expressed CD71 (a surface antigen of stem cells), Vimentin (a stromal cell skeleton protein), α-SMA (a marker of early smooth muscle cells), and SSEA-1 (a marker for more primitive stem cells; Fig. 3a), suggesting that it still harbored the surface antigen of stem cells, the stromal cell skeleton protein, and the marker of early smooth muscle cells.
Fig. 3a Expression of cell markers in the cloned MAPCs. Immunofluorescence analyses of single cell-derived P20 MAPCs-10D# were positive for CD71, Vimentin (with nuclear counterstained by Hochest33342), α-SMA and SSEA-1 (with nuclear counterstained by DAPI, all original magnification ×200). b FACS analysis: P4 MAPCs were negative for CD34, CD45, CD44, and MHC-I antigen
The results of flow cytometry showed that 10D# clone did not express CD45 or CD34 (markers for hematopoietic stem cells). For the expression of CD44, the stromal cell marker, and MHC I, the major histocompatibility complex class I antigens, MAPCs were all negative (Fig. 3b).
Differentiation potential of single cell-derived P20 MAPCs-10D# into three somatic germ layers consisting of osteoblasts, adipocytes, neurons, and hepatocytes
Single cell-derived P20 MAPCs were seen to have strong refractive oil drops around the nuclei on as early as the eighth day under the adipogenic condition, and the oil drops gradually increased and fused into larger ones with the prolongation of the induction time (Fig. 4a, 1). Twenty-one days after induction with osteoblastic medium, AKP staining of MAPCs showed purple-bluish deposits in the cytoplasm (Fig. 4a, 2), and immunocytochemistry staining was positive for osteopontin (Fig. 4a, 3). Fourteen days after MAPCs were directed to differentiate into neuronal cells in vitro, cell morphology underwent obvious changes: the cytoplasm was shrinking, and bipolar or multipolar processes were seen growing out from the spindle-shaped cells (Fig. 4b, 1). Immunofluorescence staining showed the presence of Tau protein, the specific marker of neurons (Fig. 4b, 2), and GFAP, the specific marker of glia cells (Fig. 4b, 3) in the cells after neurogenic induction. Fourteen days after MAPCs were programmed to differentiate into hepatocytes, the spindle-shaped cells (Fig. 4c, 1) turned into round or oval shape and became wider and larger, with narrowed intercellular junctions and appearance of cell patches (Fig. 4c, 2). Immunofluorescence analysis showed that the induced cells expressed albumin, the marker of hepatocytes (Fig. 4c, 3). The above findings suggested that clonal cultured MAPCs were able to differentiate into osteoblasts, adipocytes, neuronal cells, and hepatocytes under proper culture conditions.
Fig. 4Differentiation of cloned 10D# MAPCs into three somatic germ layers. a Differentiation into adipogenic-like cells and osteogenic-like cells. 1 differentiated MAPCs were positive in oil red O staining counterstained with hematoxylin; 2 and 3 differentiated MAPCs were positive for alkaline phosphatases (AKP staining) and osteopontin (immunocytochemical staining). b Differentiation into neuronal-like cells. 1 Under phase microscope, apparent branches were observed to grow out from the spindle-shaped cells. 2 and 3 Immunofluorescence staining showed the presence of GFAP and Tau protein. c Differentiation into hepatocytes-like cells.1 DAPI counterstaining showed that the MAPCs untreated with hepatogenic medium retained the spindle shape. 2 MAPCs turned from spindle into round or oval after hepatogenic induction. 3 Immunofluorescence analysis showed expression of albumin in differentiated MAPCs (all original magnification ×200)
Discussion
There is increasing evidence that isolation strategies of stem cells differ for different source tissues and different stem cell types; even though the tissue is from the same source, different isolating protocols may obtain different stem cell types [17]. For example, stromal stem cells screened out with different molecular markers demonstrated certain varieties in growth and plasticity, and were therefore given different names, including MSCs [18–19], MAPCs [10–12], MPCs (mesodermal progenitor cells) [20], and marrow-isolated adult multi-lineage inducible cells (MIAMI) [21]. In the present study, we used culture conditions similar to those of Verfaillie’s team and successfully obtained a comparatively homogeneous population of MAPCs through the following protocol: isolating mononuclear cells from rat total bone marrow by direct adherence, eliminating non-adherent cells after three to four passages, subjecting the remaining cells to single cell clonal culture by limiting dilution technique, plating the cells at a low density of 250 cells/cm2 for further passage, and finally screening for clone-like cells. We found that single cell-derived MAPCs did not express CD34, CD45, CD44, and MHC-I, which was consistent with the results of Verfaillie’s team, who found that MAPCs were able to reserve their pluripotency when plated at a low density, and unable to do so when plated at a much higher density. With pluripotency decreasing, CD44 and MHC I turned from negative to strongly positive in MAPCs, and therefore could serve as a marker for the loss of pluripotency. It should be notified that our protocol for MAPC isolation, which involved direct adherence, passage selection, single cell clonal culture and low density passage, is less complicated and more economical than that of Verfaillie’s team, who employed the technique of immunomagnetic microbeads. The reliability of our protocol is verified by its results. Single cell-derived MAPCs retained their multi-potential differentiation capability even after being passaged for 20 generations, and could not only give rise to adipocytes and osteoblasts (mesoderm) but to neurons, glia cells (ectoderm), and hepatocytes (endoderm).
It has been demonstrated that the isolation method and cultivation condition are vital to the isolation of bone marrow stem cells and the maintenance of stem cell property [16]. The latest research showed that in vitro culture of human MSCs with autologous serum is superior to FBS for the amplification and for maintaining the pluripotency of MSCs [22], and the original culture strategy might lead to the loss of differentiation potential of MSCs. Moreover, some researchers found that serum-free culture could upgrade the differentiation potential of MSCs [23]. So recently, researchers have made persistent efforts to optimize the isolation method of MSCs. For example, people have isolated another population of pluripotent cells, called marrow-isolated adult multilineage inducible (MIAMI) cells, from human bone marrow by plating whole bone marrow cells initially in media containing 5% FBS and subsequently maintaining them in the medium containing 2% FBS in fibronectin-coated dishes. These cultures were then maintained in hypoxic conditions at 1,300–1,400 cells/cm2 [20]. MIAMI cells still expressed telomerase, Oct-4, and Rex-1 even after being expanded for more than 50 population doublings. In the present study, our medium contained a low level of serum, but was supplemented with growth factor and LIF, which promoted the proliferation of MAPCs to some extent and retained their dedifferentiated state. Single cell clonal culture and low density passage may help to isolate a more homogenous cell population with strong proliferation capability.
As the marker for pluripotent stem cells, Oct-4 is closely linked with the pluripotency of MAPCs [24]. Our results showed that single cell-derived MAPCs not only highly expressed Oct-4 but expressed the early markers of endoderm, mesoderm, and ectoderm: CYP51, SM22αA [14], GATA4 [25], and NMDA [26], which is the molecular basis for their differentiation into three germ layers. Woodbury’s team [16] demonstrated that MSCs expressed germline, ectodermal, endodermal, and mesodermal genes before neurogenesis. The present research extended their observations. Our observations confirm that single cell-derived MAPCs transcribe Oct4 and genes of the three germ layers simultaneously, and could differentiate into multiple lineages. It may be concluded that under appropriate circumstances, single cell-derived MAPCs can maintain not only characteristics of stem cells but multi-lineage differentiation potential which has expressed corresponding regulating genes, and single cell-derived MAPCs could differentiate into multiple lineages through quantitative modulation of regulating gene expression, rather than switching on expression of specific genes.

multipotent adult progenitor cell
differentiation
pluripotency
oct 4


Neck pain in chronic whiplash syndrome treated with botulinum toxin. A double-blind, placebo-controlled clinical trial

Objectives Neck pain in chronic whiplash syndrome is a major burden for patients, healthcare providers and insurance companies. Randomized data on treatment of botulinum toxin in chronic whiplash syndrome are scarce. We conducted a randomized, placebo-controlled clinical trial to prove efficacy of botulinum toxin for neck pain in chronic whiplash syndrome.
Introduction
Intractable chronic neck pain after a whiplash trauma is a major burden for patients, healthcare providers and insurance companies. It is estimated that 14–42% of patients develop chronic symptoms after an acute whiplash trauma [2].
These patients often have reduced mobility of the neck with tender muscles and increased muscle activity [25]. Pathophysiology is still unknown, but myofascial triggerpoints can develop in cervical muscles from excessive neck and shoulder muscle contraction due to an overt injury such as acceleration of the neck in a motor vehicle accident [5]. Botulinum toxin is used to treat a variety of neurological disorders associated with pathologically increased muscle tone.
Two previous studies have reported that botulinum toxin might be successful in patients with chronic neck pain following whiplash injuries [6, 12]. These studies were uncontrolled and the follow-up period was relatively short. In a randomized placebo- controlled trial involving 26 patients with chronic neck complaints after whiplash injury a significant improvement on total pain was suggested [7, 8].This study however has some methodological problems, such as a relatively short follow-up period of 4 weeks and a small sample size [7, 8]. Based upon the present evidence botulinum toxin cannot be recommended as standard treatment for chronic neck pain after whiplash injury.
We therefore conducted a double blind placebo-controlled randomized trial to confirm if botulinum toxin in treatment of neck pain is indeed effective in chronic whiplash syndrome.
Methods
Patients
Patients were eligible if they had a whiplash type neck distortion defined as a soft tissue injury of the neck following a vehicle collision, with symptoms lasting longer than 6 months. They also met criteria of the Quebec Task Force grade 1 or 2 whiplash-associated disorders (WAD). WAD grade 1 means only neck pain and stiffness and grade 2 requires also musculoskeletal signs, e.g. a decreased range of motion [24]. Exclusion criteria were: age < 18; pregnancy; neuromuscular disorders; previous use of other investigational new drugs in the past 30 days prior to the screening visit; previous use of botulinum toxin; traumatic abnormalities on radiological examinations. The institutional ethics committee approved the study protocol, and patients gave informed consent before inclusion.
Study design
The trial had a double-blind, placebo-controlled parallel design. After patient’s consent was obtained, we recorded demographics (age, gender). During a baseline-period of 4 weeks, patients used a diary to record the presence of neck pain, number of neck pain hours per day, number of days on which medication was taken, and number of tablets taken per day. At the end of this baseline-period (week 0) patients recorded their mean neck pain intensity scored on 100 mm Visual Analogue Scale of the previous 4 weeks (week –4 to 0, VAS-pre-treatment). At week 0, patients were randomly assigned to receive 100 units botulinum toxin (Botox®) or placebo (saline) in 2 cc syringes. At 12 weeks follow-up they recorded again their mean neck pain intensity of the previous 12 weeks (week 0 to 12, VAS-post-treatment). The local trial pharmacist prepared the drug, coded the syringes, and kept treatment codes. The pharmacist had no further participation in the trial. An experienced clinical neurophysiologist (D.T.) performed all injections. Injection sites were selected individually (i.e. ‘follow the pain’ approach) in muscles with clinically increased muscle tone or muscle tenderness, and included m. occipitofrontalis, m.temporalis, m. masseter, m. sternocleidomastoideus, m. splenius capitis, m. trapezius and m. semispinalis.
Outcomes
After patients were injected at week 0 in one single treatment-session, clinical characteristics were recorded using diaries during the 12 weeks post-treatment. The primary outcome measure was the intensity of the neck pain of the total study period, week 0–12, scored on the VAS at week 12 (one single measurement) compared with baseline (VAS week –4 to 0). Patients were asked to rate (in a single value) severity of neck pain which at times may be mild and perhaps at others severe, by “integrating severity over time”.
Secondary outcome measures were average number of neck pain days, average number of neck pain hours per day, average number of days on which symptomatic treatment was taken, mean number of symptomatic tablets per day at 12 weeks compared with baseline. Additionally, a blinded investigator asked patients whether there was any improvement in their condition after 4, 8 and 12 weeks in the two treatment groups (five-point scale: great worsening, any worsening, no improvement, any improvement and great improvement). Range of cervical motion was measured with a cervical range of motion (CROM) device to measure the six conventional movements of the spine at week 12 compared with baseline. For measurements of CROM, the patients were seated in a chair with their shoulders supported on a backrest. Patients were instructed to assume a comfortable position and then to perform each required movement and return to the start position. The movements were assessed in this order: right rotation, left rotation, right flexion, left flexion, extension, flexion and then summated to determine the mean total range of motion.
We also divided all patients in responders and non-responders at week 12. We considered at least 45% reduction in the VAS compared with baseline to be clinically relevant (e.g. responder). All adverse events during the 3 months follow-up were recorded by the investigators.
Sample size determination
At baseline we expected mean neck pain intensity on the VAS of 40/100 mm. The estimated placebo effect was a 8 mm reduction to a VAS intensity of 32/100 at 12 weeks. We hypothesized a 18 mm reduction in the treatment group to a VAS intensity of 22/100, although there are no earlier data on treatment effect of BTX in these patients. To detect this clinically relevant effect (8 versus 18 mm improvement) at a 5% level of statistical significance (two-tailed), with a power of 80%, a total of 40 patients was needed.
Statistical analysis
We calculated mean differences between baseline VAS data and VAS data after 12 weeks, using the t-test; statistical uncertainty was expressed in 95% confidence intervals. The responders at week 12 in each treatment group and the differences in improvement scores were compared by use of the Chi-square test. All these analysis were done with SPSS (version 10.0).
Results
Between November 1999 and March 2003 40 patients were included at Leyenburg hospital The Hague. Of the 59 patients assessed for eligibility, 19 patients were excluded. Four patients did not meet the inclusion criteria, eight patients refused to participate and seven patients withdrew in the baseline period. No patient was lost to follow-up.
In 1 patient the neck pain diary was incomplete during the whole study period (missing data on neck pain duration, days on which medication was taken and mean number of tablets per day). In 7 patients the neck pain diary for the post-treatment period was partly incomplete (missing data on neck pain duration, days on which medication was taken and mean number of tablets per day). There were no differences in baseline characteristics between the two treatment groups. Neck pain intensity, VAS at baseline was 64.5 mm (SD = 14.8) in the botulinum group and 62.1 (SD = 20.3) in the placebo group (Table 1).
Table 1Baseline characteristicsCharacteristicBotulinum toxin (n = 20)Placebo (n = 20)Mean age, years (range)39 (20–58)34 (20–49)Female sex (%)14 (70%)13 (65%)Ongoing claims15 (75%)18 (90%)Mean duration of pain, months (SD)40 (71)37 (20)Neck pain severity, mean VAS (SD)64.5 (14.8)62.1 (20.3)Mean number of neck pain days, % (SD)97% (11) *196% (12)Mean neck pain duration per day, hours (SD)12.7 (4.0) *311.7 (4.5)Mean number of medication days, % (SD)50% (41) *136% (40)Mean number of analgesics per day, tablets (SD)1.4 (1.9)1.3 (2.1)Mean total cervical range of motion, degrees (SD)266 (73) *1266 (60) *1*1 = one missing data; *3 = three missing data
At 12 weeks the VAS scores were 52.0 mm (SD = 29.2) in the botulinum group and 56.7 mm (SD = 29.6) in the placebo group. Mean differences between VAS pre- and post-treatment was 12.5 in the botulinum group and 5.5 in the placebo group. The therapeutic gain ( = difference in improvement between the two treatment groups measured in mm) was –7.0 (p = 0.31; 95% CI (–20.7 to + 6.7)). Six patients in the botulinum group were responders as defined, versus four in the placebo group (NS, p = 0.5).
Patients’ assessment of improvement/worsening showed no significant difference between the two groups at week 4 (p = 0.9), week 8 (p = 0.4) and 12 (p = 0.4) (Table 2).
Table 2Improvement scaleImprovementAt 4 weeksAt 8 weeksAt 12 weeksBotulinum toxin (n = 20)Placebo (n = 20)Botulinum toxin (n = 20)Placebo (n = 20)Botulinum toxin (n = 20)Placebo (n = 20)Great worsening400212Any worsening241423No improvement688768Any improvement555454Great improvement236363Total19*12020202020*1 = one missing data
There were no significant differences between the two treatment groups in mean number of neck pain days, mean number of neck pain hours per day, mean number of days on which medication was taken, and mean number of tablets taken per day pre- and post-treatment (Table 3).
Table 3Secondary outcome measuresOutcomeBotulinum toxin (n = 20)Placebo (n = 20)Therapeutic gain (95% CI)Neck pain days (% ± SD)  Baseline (4 weeks)97% (11) *196% (12)  Post-treatment (12 weeks)89% (23) *289% (25) *2  Mean difference8% (18)7% (22)−1% (−15% to + 13%)Neck pain duration (hours/day ± SD)  Baseline (4 weeks)12.7 (4.0) *311.7 (4.5)  Post-treatment (12 weeks)12.2 (4.6) *711.0 (5.1) *2  Mean difference0.86 (2.2)0.72 (4.7)−0.14 (−3.0 to + 2.7)Days on which analgesics were taken (% ± SD)  Baseline (4 weeks)49% (42%)35% (40%)  Post-treatment (12 weeks)42% (38%) *324% (32%) *2  Mean difference6.4% (16%)5.7% (24%)0.7% (−15% to + 13%)Number of analgesics taken per day (tablets/day ± SD)  Baseline (4 weeks)1.4 (1.9) *11.3 (2.1)  Post-treatment (12 weeks)1.2 (1.8) *20.95 (1.9) *1  Mean difference0.32 (0.8)0.18 (0.6)−0.14 (−0.6 to + 0.4)Total cervical range of motion (degrees ± SD)  Baseline266 (73) *1266 (60) *1  Post-treatment (week 12)293 (59) *2277 (62) *3  Mean difference22 (43)11 (36)−11 (−40 to + 17)*1 = one missing data; *2 = two missing data; *3 = three missing data *7 = seven missing data
At baseline all patients showed a reduced cervical range of motion (CROM) in all directions [9]. There was a slight improvement in total CROM in favour of the botulinum group. However, CROM was not significantly different between BTX group and placebo, nor between responders and non-responders.
Twenty-seven patients reported minor side-effects, 15 in the botulinum toxin group and 12 in the placebo group. The main complaint was “short-lasting pain at the injection sites”.
Discussion
Our study could not demonstrate botulinum toxin to be more effective than placebo in treatment of neck pain in patients with chronic whiplash syndrome.
Previous open label studies with botulinum toxin in patients with chronic whiplash syndrome suggested some beneficial effect on neck pain [12]. Our study is the second published randomized-controlled trial to determine efficacy of botulinum toxin in patients with chronic whiplash syndrome. The other randomized placebo-controlled trial in 26 patients with chronic whiplash syndrome reported a beneficial effect of botulinum toxin [7, 8].
This study, however, has some methodological shortcomings. Primary outcome was improvement of headache intensity scored on the VAS after 4 weeks compared with baseline. The post treatment difference between the two treatment groups was reported to be significant. However, baseline-characteristics were significantly different, with a significant higher headache intensity in the botulinum group at baseline. (VAS 6.5 (range 2–9) versus 3.0 (0–8) in the placebo group (p < 0.01)). The reported improvement may mainly have been caused by the unequal distribution of the patients at baseline. In their second publication ( on the same trial) the mean total pain score was reported, ranging from 0–30, expressing the total pain experience for neck pain, headache and shoulder pain. After 4 weeks the mean total pain was 10.0 (SE 1.3) in the botulinum group and 14.1(SE 21) in the placebo group, which was also reported to be significant. These data are difficult to interpret without individual scores. Calculation of the standard deviation could suggest that some patients even had a negative mean total pain score. In any case, the distribution was truncated and skew to the right.
There are some points of discussion in our study. We used injections at trigger points on an individual basis, instead of following a standardized protocol. This might seem subjective and arbitrary, but resembles clinical practice and is usually recommended for botulinum toxin (BTX) treatment in pain syndromes [10, 16]. We used doses of 100 U Botox®, which should be sufficient for a single treatment session. There was only slight, not significant, improvement of cervical range of motion in the botulinum group, suggesting some, if any, effect on muscle tenderness. Also, none of our patients reported muscular weakness as a side effect. Therefore, further increase of the dosage of botulinum toxin might be possible. There is some evidence that multiple treatment sessions are needed for sufficient clinical benefit [20]. In our study there was not even a trend for a positive treatment effect after this single treatment session of BTX. Still, a positive effect of multiple treatment injections cannot be entirely ruled out [16].
Pathophysiology of chronic pain in whiplash syndrome is still a matter of debate. There is evidence that myofascial pain is a major factor in chronic pain syndromes like chronic neck pain and chronic tension-type headache [14]. The hypothesis of BTX being effective in chronic whiplash syndrome could be pain reduction by decreasing muscle tenderness and/or activity. If increased muscle tenderness is a key feature in chronic neck pain after a whiplash injury, a more beneficial effect of BTX would have been expected. However, in other chronic pain syndromes where muscle tenderness is thought to play a role BTX was also proven ineffective [17, 23]. BTX might only be effective in neck pain in combination with cervical dystonia [26]. In our study were no patients with cervical dystonia.
Patients with chronic whiplash syndrome also form a very heterogeneous group, with a wide spectrum of symptoms, varying from neck pain to headache, with pain of different degrees. Many different factors seem to play a role in the development of acute and chronic pain after a whiplash injury.
There is ample evidence that central mechanisms might play a more important role in the development of chronic neck pain in whiplash patients [4], as in other chronic pain syndromes. Damaged peripheral tissue releases inflammatory mediators (like bradykinine and prostaglandins) that changes the sensitivity of high-threshold nociceptors (peripheral sensitization). This, in turn, sets up changes in the responsiveness of neurons in the central nervous system (CNS), ultimately increasing the excitability of neurons within the CNS, representing central sensitization [21]. Patients with chronic whiplash syndrome display exaggerated pain after sensory stimulation as a consequence of this state of hyperexcitability of the central nociceptive system [1, 13]. In other words, an alteration in function of the central nervous system, the way it responds to normal inputs, seems present in these patients.
This central hypersensivity is probably independent of the nociceptive input arising from the painful and tender muscles [4], and possibly a consequence of deficient pain inhibitory systems.
Many somatic, psychological or cultural factors may influence central sensitization. Different studies show that prognosis of chronic whiplash syndrome is also dependent on cultural factors [15, 18, 22]. It has even been shown that presence or absence of compensation claims is one of the most important predictors of bad outcome in patients after whiplash injury (also present in our group in 75–90% of the patients) [11, 19]. The etiological contribution of all different psychological factors, like for example coping style [3], to central sensitization is not yet clarified. It is not to be predicted that BTX will improve all different factors, especially psychological and cultural factors, which may contribute to development of this chronic whiplash syndrome.
Based on present evidence BTX cannot be recommended as treatment for neck pain in chronic whiplash patients. Future studies directed on possible central mechanisms of this complicated chronic pain syndrome are warranted.
Contributors
The trial was designed by S.F.T.M. de Bruijn, D.L.J. Tavy and M. Padberg. Statistical analysis was performed by M. Padberg.
Conflict of interest statement
Non declared.

neck pain
chronic whiplash syndrome
botulinum toxin


Re-irradiation of a second localization of idiopathic midline destructive disease in the head and neck area

Idiopathic midline destructive disease is a rare disease, characterized by a progressive ulceration and destruction of midline facial structures. We report a case with localization on the palate for which she received radiotherapy. Later she developed a second localization on the posterior pharyngeal wall for which she was re-irradiated, without severe sequels. Twice a complete regression was observed.
Introduction
Idiopathic midline destructive disease is a rare, which is characterized by a progressive ulceration and destruction of midline facial structures, including the nasal cavity, the paranasal sinuses and the palate. In most cases, radiotherapy is an effective treatment [1]. We describe a woman with twice a localization of idiopathic midline destructive disease, which were both treated with irradiation.
Case report
A woman, 46 years of age, was referred with complaints of nasal discharge while drinking. She was otherwise healthy and there was no history of substance use. Physical examination revealed an almost complete destruction of the membranous nasal septum and perforations of the soft and hard palate of 3 and 5 mm, respectively. Further examination of the head and neck region and general examination showed no abnormalities. Computed tomography showed extensive destruction of the hard palate. Biopsy showed a non-specific inflammation and fibrosis (Fig. 1). There were no signs of Wegener’s granulomatosis, malignant lymphoma, tuberculosis or Epstein-Barr virus infection. Serum tests showed negative results for anti-neutrophil cytoplasmic antibodies (ANCA) and syphilis. A diagnosis of idiopathic midline destructive disease was made.
Fig. 1Histological picture of idiopathic midline destructive disease detail (40× objective, Hematoxilin & Eosin stain) showing predominantly small irregular lymphocytes admixed with histiocytes and some granulocytes. The lymphoid cells do not appear obviously malignant. Low power view of biopsy (5× objective, Hematoxilin & Eosin stain) show an ulcerative surface (left) and the infiltrate (right) with prominent vessels with swollen endothelial cells
The patient was then treated with radiation therapy to a total dose of 52 Gy in 26 fractions. At the end of the radiation course, a grade 1 mucositis and grade 1 erythema were observed. A complete regression of the disease occurred. After 4 years, the defect in the palate was closed surgically using a temporalis muscle flap.
Four years later she developed a sore throat and swallowing complaints. On examination, a tumor-like lesion with a central ulcer was seen on the posterior pharyngeal wall. A CT-scan of the head and neck region showed a soft tissue mass on the posterior pharyngeal wall with extension from the level of the soft palate and nasopharynx to the level of the epiglottis. Biopsy showed the same histologic appearance as previously. The site of the original lesion was without any signs of abnormalities.
It was decided to treat this new localization with re-irradiation. During treatment, the patient experienced grade 1 erythema and grade 2 mucositis. She experienced swallowing complaints for 4 months. Complete regression occurred again.
Four years after re-irradiation the patient presented again with progressive swallowing complaints and nasal speech. On physical examination, an ulcer was seen on the posterior pharyngeal wall with a diameter of 1-cm, which had a different clinical appearance than the former two lesions. Therefore, the differential diagnosis included a radiation-induced ulcer or recurrence midline destructive disease. Biopsy was not taken because it was most likely that pathological examination of this lesion could not distinguish between these two entities and because of an increased probability of wound-healing problems after biopsy in a highly irradiated area. We decided to treat this lesion as a radiation ulcer and the patient was proposed to undergo hyperbaric oxygen therapy, which she refused. Therefore, as an alternative, we decided to treat her with pentoxyfilline and high dose vitamin E, which she has used for one year. In the subsequent 12 months, the ulcer as well as her complaints gradually decreased.
During the last follow-up visit (more than 5 years after re-irradiation), there were no signs of recurrent tumor or other severe radiation sequels, while a small ulcer still persists. Besides a mild xerostomia, she had no other complaints.
Discussion
Idiopathic midline destructive disease (IMDD), also known as lethal midline granuloma (LMG), is a rare disease which is clinically characterized by a progressive, unrelenting ulceration and necrosis of the nasal cavity or the midline facial structures. This disorder has to be distinguished from other progressive, ulcerative disorders of the midline facial tissues, including cocaine use, sarcoidosis, infections like tuberculosis and syphilis, various neoplasms like vestibular or septal carcinoma, Wegener’s granulomatosis, and polymorphic reticulosis (PMR) [2]. In contrast to all the other diseases, which have distinct histopathological features that distinguish them from IMDD, the histopathological findings of IMDD are nonspecific. It is essentially a diagnosis per exclusionem.
Some authors believe that IMDD is an early phase of PMR [2]. Others state that all cases referred to as IMDD or PMR are in fact nasal T-cell (natural killer cell) lymphomas (NTL) [1, 3], with a possible association with the Epstein-Barr virus [4]. However, Barker and Hosni recently reported a case of IMDD in which no other cause could be diagnosed by current techniques and they proposed that IMDD remains a valid description in a small number of cases [5]. In our case the clinical course (with only localized disease) and histology (no atypia or Epstein-Barr virus in the tumor) argue strongly against lymphoma and for IMDD.
Untreated IMDD is eventually fatal. No more than approximately 100 cases have been reported, of which only part is characterized as IMDD and the other cases as PMR or NTL. Therefore outcome parameters, such as survival and local control, are difficult to interpret. The preferred treatment of IMDD is radiation therapy to a dose of at least 50 Gy [1, 3]. A 5-year overall survival after radiation therapy has been reported from 15 to 75%. Local control varies between 63 and 77% [1–4]. Cases reported as NTL do worse compared to IMDD [1]. Some authors administer chemotherapy in case of NTL, although no difference in survival in patients with or without the addition of chemotherapy could be established [3].
In our case, a total dose of 52 Gy in 26 fractions was used which is generally considered to be sufficient. The margins taken during the first treatment were adequate according to the clinical tumor extension. Also because of the long time-interval, the second lesion can be considered to be a new localization of the disease.
It was decided to treat this new localization with re-irradiation for the following reasons. The largest part of the new localization was outside the initial treatment field implying that only part of the previously irradiated area would be treated again with the second course of radiotherapy. Second, the interval between the initial course of radiotherapy and relapse was more than 8 years and the total dose administered was 52 Gy, which may imply that a substantial amount of initial radiation damage to the normal tissues could have recovered [6–8].
Data on re-treatment of IMDD after local failure are scarce. Both Chen et al. [1] and Sakata et al. [3] mention a case with local relapse with successful re-irradiation without giving any detailed information. One patient with a local relapse of IMDD benefited from salvage chemotherapy [3], whereas two other patients, who were diagnosed as NTL, failed on a combination of re-irradiation and chemotherapy [1]; also on these patients no detailed information regarding radiotherapy was provided.
In conclusion, radiation therapy is the treatment of choice for idiopathic midline destructive disease. In case of a second localization in the irradiated area re-irradiation should be considered.

idiopathic midline destructive disease
radiotherapy
midline granuloma