Impaired adult olfactory bulb neurogenesis in the R6/2 mouse model of Huntington's disease

Zacharias Kohl
Martin Regensburger
Robert Aigner
Mahesh Kandasamy
Beate Winner
Ludwig Aigner
Jürgen Winkler
Impaired adult olfactory bulb neurogenesis in the R6/2 mouse model of Huntington's disease
R E S E A R C H A R T I C L E Open Access
Background: Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder linked to expanded CAG-triplet nucleotide repeats within the huntingtin gene. Intracellular huntingtin aggregates are present in neurons of distinct brain areas, among them regions of adult neurogenesis including the hippocampus and the subventricular zone/olfactory bulb system. Previously, reduced hippocampal neurogenesis has been detected in transgenic rodent models of HD. Therefore, we hypothesized that mutant huntingtin also affects newly generated neurons derived from the subventricular zone of adult R6/2 HD mice. Results: We observed a redirection of immature neuroblasts towards the striatum, however failed to detect new mature neurons. We further analyzed adult neurogenesis in the granular cell layer and the glomerular layer of the olfactory bulb, the physiological target region of subventricular zone-derived neuroblasts. Using bromodeoxyuridine to label proliferating cells, we observed in both neurogenic regions of the olfactory bulb a reduction in newly generated neurons. Conclusion: These findings suggest that the striatal environment, severely affected in R6/2 mice, is capable of attracting neuroblasts, however this region fails to provide sufficient signals for neuronal maturation. Moreover, in transgenic R6/2 animals, the hostile huntingtin-associated microenvironment in the olfactory bulb interferes with the survival and integration of new mature neurons. Taken together, endogenous cell repair strategies in HD may require additional factors for the differentiation and survival of newly generated neurons both in neurogenic and non-neurogenic regions.

Background

Huntington's disease (HD) is a devastating autosomal dominant hereditary neurodegenerative disease caused by a CAG trinucleotide repeat expansion within the huntingtin gene encoding an extended polyglutamine tract in the huntingtin (htt) protein [1]. The progressive clinical phenotype consists of involuntary choreic movements, cognitive decline and psychiatric symptoms (reviewed in [2]). Impaired olfactory function was also observed in patients as well as presymptomatic gene carriers [3,4]. Emerging evidence suggests that mutant htt leads to selective neuronal damage by a gain of toxic function inducing neuronal loss and gliosis predominantly in the neostriatum and the cortex (reviewed in [5]).

The presence of neural stem cells in the adult central nervous system (CNS) has been widely described both in rodents and in humans (reviewed in [6]). In adult mammals these cells are present in neurogenic regions of the CNS, the subventricular zone (SVZ) of the lateral ventricle wall and the dentate gyrus (DG) of the hippocampus. New cells are continuously generated in the SVZ [7,8] and physiologically migrate via the rostral migratory stream (RMS) to the olfactory bulb (OB) [9,10]. Here they mainly differentiate into GABA-ergic interneurons in the granular cell layer (GCL) or to a lesser extent in the glomerular layer (GLOM), and functionally integrate [11,12]. New GLOM neurons are further subdivided based on their immunoreactivity against tyrosine hydroxylase (TH), calbindin or calretinin [13]. Neurogenesis in the OB is a life long phenomenon in the mammalian brain, leading to an increase in the total number of neurons in the GLOM, while the number of neurons in the GCL appears to be counterbalanced by continuous cell death [14]. Importantly, neurogenesis in the adult CNS can be modulated by various molecular and environmental factors, in part acting differentially on both neurogenic regions [6]. The stimulation of this endogenous pool of immature neurons may be a useful tool for a cell-based therapeutic approach in neurodegenerative disorders [15].

In transgenic animal models of HD impaired adult neurogenesis has been described in the DG of the hippocampus [16,17], which could be partially restored by environmental stimuli or antidepressants [18,19]. In contrast, the proliferation of neural stem cells in the SVZ is not affected in transgenic HD mouse models [20,21]. The R6/2 HD mouse model carries exon 1 of the human HD gene with an expanded CAG trinucleotide repeat strand (130-150 CAG repeats; [22]) and exhibits decreased striatal and total brain size, ubiquitinated nuclear and cytoplasmic inclusion bodies [23], and progressive motor and cognitive deficits [24]. These mice have an early onset of symptoms associated with a fast progression and a limited life span of approximately 12 to 17 weeks, depending on the colony (reviewed in [25]).

To further rule out the impact of mutant htt on the endogenous generation of new neurons we investigated the SVZ/OB system in the R6/2 mouse model and analyzed the survival and differentiation of SVZ-derived new neurons migrating towards their physiological target region, the OB, and to the adjacent severely affected striatum.

Results

Aggregates of mutant huntingtin appear in mature neurons, but not in neuroblasts in R6/2 mice To determine whether aggregates of mutant htt were present in the SVZ/OB (Fig. 1E), we used an α-huntingtin antibody specific for the N-terminus of human mutant htt with a repeat expansion > 82 repeats (MAB5374; [26]). We detected intranuclear immunoreactivity in some mature calretinin-expressing neurons of both the GCL and the GLOM (Fig. 1I,J) and in THexpressing neurons of the GLOM (Fig. 1K). In contrast, mutant htt was not detected in doublecortin (DCX)expressing neuroblasts of R6/2 animals in the GCL and the GLOM of the OB (Fig. 1G, H), or in migrating DCX positive neuroblasts in the RMS (Fig. 1F) and the SVZ (Fig. 1C). Besides the DCX expressing SVZ neuroblasts (or A cells) neither glial fibrillary acidic protein (GFAP) expressing B cells (Fig. 1 ) nor epidermal growth factor (EGF)-receptor expressing C cells (Fig. 1) showed intranuclear immunoreactivity for htt aggregates. In contrast, mature neurons of the adjacent striatum expressing dopamine-and cAMP-regulated phosphoprotein-32 (DARPP-32) contained aggregates of htt (Fig. 1D). No immunoreactivity of mutant human htt was observed in the SVZ/OB of WT mice (data not shown).

Increased migration of neuroblasts towards the striatum in R6/2 mice

We observed an increased number of neuroblasts in the striatum adjacent to the SVZ. Quantification of DCXpositive cells in the striatum revealed significantly higher numbers in transgenic animals compared to WT (44.0 ± 28.1 in WT vs. 348.0 ± 237.7 in R6/2, p < 0.05, Fig. 2D). These cells were predominantly located in close proximity to the SVZ with a decline of cell number away from the SVZ ( Fig. 2A-C). This may be due to neuroblasts migrating into the striatum. At distances greater than 300 μm from the SVZ no DCX-immunopositive neuroblasts were observed. To investigate if any new cells obtained a mature neuronal phenotype in the striatum, we investigated possible colocalization of bromodeoxyuridine (BrdU) with the mature neuronal marker NeuN. In contrast to a previous study [20], we did not observe BrdU/NeuN positive cells 4 weeks after BrdU treatment. Moreover, as none of the DCX-positive striatal cells colabeled with BrdU, we excluded that the lack of striatal neurogenesis was due to a delayed maturation longer than 4 weeks. These results in line with previous studies [21,27] suggest that neuronal maturation of newly generated cells is not present within the R6/2 striatum of 9-week old mice.

SVZ cell proliferation not affected in R6/2 mice Proliferation in the SVZ was assessed by quantification of cells labeled for proliferating cellular nuclear antigen (PCNA). In accordance with previous data [21,28] the number of proliferating cells in the SVZ of R6/2 animals was unchanged compared to WT (8.49 ± 2.16 vs. 7.42 ± 3.11 (×10 3 ); Table 1, Fig. 3).

Decreased olfactory neurogenesis in HD mice

To study the amount of newly generated cells in the OB which showed no structural abnormalities we analyzed the number of BrdU-labeled cells in the GCL and in the GLOM separately. For further investigation, the neuronal identity of these newly generated cells was determined by double-labeling with BrdU and NeuN. The total number of newly generated BrdU/NeuN double-labeled cells was determined to evaluate the effect of mutant htt on OB neurogenesis. In the GCL we observed a significant 33% reduction of BrdU-positive cells in R6/2 animals compared to the WT group (46.8 ± 11.3 vs. 69.9 ± 15.1 (×10 3 ); p < 0.05; Table 1, Fig. 4A-C). In the GCL no significant difference in neuronal differentiation was detected (87% vs. 88%, p > 0.05; Table 1). Due to the considerable reduction in newly generated cells this resulted in a significant decrease in total BrdU/NeuN cell  (35)). Aggregates of mutant huntingtin (green) are absent in GFAP expressing SVZ stem cells (red, A), EGF-R expressing transit-amplifying cells (red, B) and neuroblasts with DCX immunoreactivity (red, C), but appear in DARPP-32 positive mature neurons of the adjacent striatum (red, D) of R6/2 mice. Moreover, neuroblasts of the RMS (F), the GCL (G) and the GLOM (H) do not show immunoreactivity for mutant huntingtin. In contrast, aggregates of mutant huntingtin (green) were detected in mature, calretinin-expressing neurons (red) of the GCL (I) and the GLOM (J) and in TH expressing neurons of the GLOM (red, K) of the olfactory bulb. Topro-3 (blue) was used as counterstain for cell nuclei. Scale bar represents 20 μm. numbers by 33% (40.7 ± 11.2 vs. 61.5 ± 15.1 (×10 3 ); p < 0.05; Table 1; Fig. 5A-C).

In the GLOM, an even more pronounced 52% reduction of BrdU positive cells was measured in the R6/2 group compared to WT (4.8 ± 1.7 vs. 10.1 ± 3.9 (×10 3 ); p < 0.01; Table 1, Fig. 4E-G). As in the GCL, differentiation into a neuronal phenotype remained unchanged (91% vs. 90%, p > 0.05; Table 1). The resulting number of newly generated neurons was significantly reduced in R6/2 mice compared to WT mice (4.4 ± 1.7 vs. 9.1 ± 3.5 (×10 3 ); p < 0.05; Table 1; Fig. 5D-F). Interestingly, the fraction of BrdU-labeled newborn cells that also expressed TH as a marker of a dopaminergic phenotype was elevated in the R6/2 group (14 vs. 8%; p < 0.05; Table 1; Fig. 6A, B). This led to the finding that the total number of newly generated dopaminergic GLOM neurons was unchanged comparing R6/2 with WT animals (656 ± 270 vs. 775 301; p > 0.05; Table 1).

Increased apoptosis in the OB of R6/2 mice

An increase of cell death has been described in the cortex and the striatum of R6/2 mice [29]. To examine if increased cell death is, at least in part, attributable to the reduction in adult neurogenesis in the OB of these HD animals, we performed immunohistochemical staining for terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the  OB. We found a substantial increase of TUNEL labelled cells in the GCL of R6/2 mice compared to WT mice (Fig. 7A, B). This indicates that there is increased cell death in the presence of mutant htt in the OB.

Discussion

Our data provide evidence that the expression of mutant human htt with a CAG repeat expansion in transgenic R6/2 mice is associated with a significant impairment in the survival of newly generated neurons in the OB. We demonstrated that both neurogenic regions of the OBthe GCL and the GLOM -are severely affected in this HD model. Interestingly, mutant htt could not be detected in migrating neuroblasts of the SVZ/OB system or in cells of earlier stages of neural development in the SVZ (B and C cells). In contrast, numerous mature neurons in the striatum and OB contain aggregates of mutant htt. Thus, impaired adult neurogenesis in the OB may be caused by non cell-autonomous mechanisms. Furthermore, it is noteworthy that it remains unclear whether htt aggregates have a potential neuroprotective or toxic function [30]. While there are recent studies on aggregation kinetics of mutant htt and its different fragments [31,32], htt aggregration in developing neurons remains elusive. Adult neurogenesis in the SVZ/OB system is a highly regulated process consisting of proliferation of neural precursor cells in the SVZ [33] and the subsequent maturation into neuroblasts that migrate along the RMS to the OB. While hippocampal neurogenesis is reduced in the R6/2 mouse model [16,21,28], we confirmed previous studies that the proliferation of neural precursors in the SVZ of R6/2 animals is unchanged [20,21]. Since mutant htt severely affects the adjacent striatum, one potential repair strategy to repopulate this region is to redirect SVZderived neuroblasts to the striatum. Redirection of migrating neuroblasts to a lesion site was first described in an animal model of cerebral ischemia [34]. In a model of Parkinson's disease, the combined application of growth factors such as epidermal growth factor and fibroblast   growth factor 2 was sufficient to enhance migration of neuroblasts to the striatum; however it did not result in neuronal maturation [35]. In the present study, we observed significantly more cells expressing the neuroblast marker DCX in the striatum of R6/2 mice. These neuroblasts are localized in close proximity to the SVZ, indicating that these precursor cells may migrate as neuroblasts into the diseased R6/2 striatum. Using colocalization of BrdU with NeuN and of BrdU with DCX, we showed that these neuroblasts do not acquire a mature neuronal phenotype. Striatal neurogenesis in HD animal models was previously assessed with conflicting findings: The number of DCX-expressing striatal cells was increased in quinolonic acid-lesioned animals [36], and was even more pronounced when neural and mesenchymal stem cells were transplanted into the lesioned striatum [37]. On the other hand, no alterations in DCX-expressing cells were observed in untreated transgenic R6/2 mice compared to WT controls [21]. Moreover, another study revealed the presence of BrdU/NeuN double-positive cells in the striatum of R6/2 animals [20], which we could not confirm with our present findings. Furthermore, the intraventricular application of combined brain-derived neurotrophic factor and Noggin was effective to generate significant numbers of BrdU-labeled mature striatal neurons expressing DARPP-32 and glutamic acid dehydrogenase-67 (GAD67) in R6/2 mice [27] indicating that additional neurotrophic factors may be required for neuronal maturation and survival in the striatum.

The main focus of this study was the analysis of the survival of newborn neurons in the OB, the physiological target region of SVZ-derived neural precursors. In healthy animals, the first newly generated neuroblasts arrive in the OB after two days: by 14 days the vast majority reaches either their final destination or undergo apoptosis [10]. Previously, a reduced number of BrdUlabeled cells was shown in the RMS or the OB of R6/2 mice three or seven days after injection [20,38]. The period of 28 days after the first BrdU application comprises nearly all labeled cells arriving in the OB. These newly generated mature neurons usually integrate into the existing network of the OB. We observed that numbers of newly generated OB neurons in the GCL and GLOM were reduced by 33% and 50%, respectively, in 9-week old R6/2 animals. There are different potential explanations for the reduced number of new neurons in the OB. A decreased migration speed was proposed by a recent study of RMS neuroblasts in R6/2 mice, because of a shift in the number of BrdU-labeled cells from the rostral to the caudal portion of the RMS [38]. Alternatively, the reduction in OB neurogenesis may be related to an increased cell death in HD mice. Previously, we estimated a 50% cell survival rate of SVZ-derived newly generated neurons in the adult OB [12]. Using TUNEL staining in the GCL of R6/2 mice, we conclude that increased cell death plays an important role in the reduction of newborn OB neurons, comparable to other brain areas of this HD animal model like the cortex and the striatum, where increased cell death occurs [29]. We hypothesize that progressive dysfunction is caused by mutant htt rather in mature OB neurons and not in DCX-positive neuroblasts. Moreover, mutant htt may interfere with the integration of newly generated neurons into the preexisting neural networks in the GCL and GLOM, leading to increased cell death of developing OB neurons. Nevertheless, we can not rule out an early toxic effect of mutant htt in monomeric or oligomeric form, as the used htt antibody mainly binds to htt aggregates and is not able to distinguish different forms of transgenic mutant htt.

w?>Further analysis of newly generated mature neurons in the GLOM revealed a shift to a dopaminergic phenotype. While most of the GLOM neurons are GABAergic, a small portion of these cells co-expresses TH [39]. Our finding that the percentage of TH-positive new neurons was increased in HD animals, was unexpected, but may indicate that the survival of these cells is less affected by mutant htt in comparison to GABAergic neurons. This further supports that mutant htt differentially affects various neuronal phenotypes differentially in the HD brain [40].

Conclusion

Taken together, OB neurogenesis was severely affected in the R6/2 animal model of HD. Unchanged SVZ proliferation and the absence of aggregates of mutant htt in migrating neuroblasts suggest that the impact on new neurons occurs at later stages of neural maturation. While there are some indications of olfactory deficits in HD patients [4,41], it remains unclear whether the reduced OB neurogenesis may be related to this deficit. Moreover, despite the supposed increase of neuroblast migration from the SVZ into the striatum of R6/2 HD mice, the hostile microenvironment of this region lacks the appropriate signals to support mature neuronal differentiation.

Methods

Animals

Female B6CBAF1/J mice transplanted with ovaries from female B6CBATg(Hdexon1) 62Gpb/1J mice (R6/2, [22]) were bred to male B6CBAF1/J mice. All animals were obtained from Jackson Laboratories (Bar Harbor, USA). Tail DNA was used for determining the CAG repeat length in transgenic animals using a polymerase chain reaction assay [22]. Female mice wild type (WT; n = 6) and transgenic (R6/2; n = 6) mice were kept in normal light-dark cycle of 12 hours and had free access to food and water. Labeling of replicating cells was performed by intraperitoneal injection of the thymidine analogue BrdU (Sigma, Steinheim, Germany) at 50 mg/kg of body weight using a sterile solution of 10 mg/ml of BrdU dissolved in a 0.9% (w/v) NaCl solution. The BrdU injections were performed daily for 5 consecutive days at 5 weeks of age. Animals at 9 weeks of age were deeply anaesthetized using a mixture of ketamine (20.38 mg/ ml), xylazine (5.38 mg/ml) and acepromazine (0.29 mg/ ml). Transcardial perfusion was performed with 0.9% (w/v) NaCl solution, followed by a 4% paraformaldehyde, 0.1 M sodium phosphate solution (pH 7.4). Brains were removed and post-fixed in the paraformaldehyde solution overnight at 4°C. Tissues were then cryoprotected in a solution containing 30% (w/v) sucrose and 0.1 M sodium phosphate (pH 7.4). Brains were cut into 40 μm-thick sagittal sections using a sliding microtome on dry ice. Sections were stored at -20°C in cryoprotectant solution (ethylene glycol, glycerol, 0.1 M phosphate buffer pH 7.4, 1:1:2 by volume). All experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/ EEC) and were approved by the local governmental commission for animal health.

Immunostaining Immunostainings were conducted as described previously [16]. For BrdU and PCNA labeling, tissue was pretreated with formamide and HCl to denature DNA. Free-floating sections in Tris-buffered saline (TBS; 0.15 M NaCl, 0.1 M Tris-HCl, pH 7.5) were treated with 0.6% H 2 O 2 . Following extensive washes in TBS, sections were blocked with a solution composed of TBS, 0.1% Triton X-100 and 3% donkey serum (Sigma). This buffer was also used during the incubation with antibodies. Primary antibodies were applied overnight at 4°C. For chromogenic immunodetection, sections were incubated with biotin-conjugated species-specific secondary antibodies followed by a peroxidase-avidin complex solution from the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, USA). The peroxidase activity of immune complexes was detected with a solution of TBS containing 0.25 mg/ml 3,3'-diaminobenzidine (Vector Laboratories), 0.01% (v/v) H 2 O 2 , and 0.04% (w/v) NiCl 2 . Sections were put on Superfrost Plus slides (Menzel, Braunschweig, Germany) and mounted in Neo Mount (Merck, Darmstadt, Germany). For fluorescence immunodetection, sections were washed extensively and incubated with fluorochrome-conjugated species-specific secondary antibodies for 2 h. Sections were put on slides and mounted with Prolong Antifade reagent (Molecular Probes, Eugene, USA). Light microscopy images were obtained using a Leica microscope (Leica, Wetzlar, Germany) equipped with a Spot™ digital camera (Diagnostic Instrument Inc, Sterling Heights, USA) and fluorescent images were obtained using a confocal scanning laser microscope (Leica TCS-NT).

The To determine cell death in the OB, the TUNEL assay was performed using the Apoptag In Situ Cell Death Detection Kit (Intergene, Purchase, NY, USA). The modified protocol for free floating sections was administered which was previously described in detail [42]. For chromogenic immunodetection, the identical protocol using peroxidase activity as above was used.

Counting procedures and microscopy

All morphological analyses were conducted on blindcoded slides. Every sixth section (240-μm interval) of one hemisphere was selected from each animal and processed for immunohistochemistry. To analyze cell survival after 4 weeks in the GCL and GLOM, the number of BrdU-immunopositive cells was determined using the counting procedure described by Williams and Rakic [43]. Results were multiplied by 6 to obtain an estimate of the total immunopositive cell numbers. Total numbers of labeled cells within the GLOM were counted after determining the region of interest. BrdU-positive cells within the GCL were estimated with the optical dissector method [44] using a virtual counting frame of 50 × 50 μm spaced in a 300 × 300 μm counting grid. Labeled cells which intersected the uppermost focal plane (exclusion plane) or the lateral exclusion boundaries of the counting frame were not counted. The stereology system finally calculated a precise estimation of the total number of BrdU labeled cells within the whole thickness of the OB as decribed previously [45]. To quantify striatal neuroblasts, DCX-positive cells were exhaustively counted within the whole caudate/putamen region bordered by the corpus callosum, the RMS, the ventricle and the SVZ as well as the accumbens nucleus [45]. Total numbers of PCNA labeled cells within the SVZ were counted after determining the region of interest. All counting procedures and measurements of reference volumes were conducted on a light microscope (Leica) equipped with a semi-automatic stereology system (Stereoinvestigator, MicroBrightField, Colchester, USA) as previously described [45]. All extrapolations were calculated for one hemisphere and should be doubled to represent the total brain values.

To determine the frequency of neuronal differentiation of newborn cells, every sixth brain section (240-μm interval) was stained for BrdU and NeuN by immunofluorescence and examined using a confocal laser microscope (Leica TCS-NT) equipped with a 20× PL FLUOTAR oil objective (0.75 numeric aperture) and a 40× PL APO oil objective (1.25 numeric aperture) and a pinhole setting that corresponded to a focal plane of 2 μm. In the GCL, 100 BrdU-labeled cells per animal were analyzed for neuronal differentiation. BrdU-positive cells were characterized as solely BrdU-positive (newborn cells) and BrdU+/NeuN+ double-positive cells (newborn neurons). In the GLOM, 50 BrdU-positive cells per animal were counted for neuronal differentiation, identifying BrdU+/NeuN+ newborn neurons and newly generated neurons with a dopaminergic phenotype (BrdU+/TH+). Further fluorescence images labeling htt in combination with various other markers were also obtained on the confocal scanning laser microscope (40× PL APO oil objective, pinhole corresponding to a focal plane of 2 μm).

Statistical analysis

The data are presented as mean values ± standard deviations (SD). Student's t-test was used to test for differences in BrdU and DCX cell numbers, BrdU/NeuN and BrdU/TH percentages, total number of BrdU labeled neurons and total number of BrdU-labeled dopaminergic neurons. Statistical analysis was performed using Prism (Prism Graph Pad Software, San Diego, USA). Differences were considered significant at p < 0.05, unless otherwise indicated.

Abbreviations

Figure 1
1Expression pattern of mutant huntingtin in developing and mature neurons of the subventricular zone and the olfactory bulb in R6/2 mice. SVZ derived neurons originate from dividing GFAP expressing cells ("stem cells", B cells) that generate transit-amplifying cells (C cells) which in turn give rise to neuroblasts (A cells). DCX-expressing neuroblasts migrate along the RMS to the olfactory bulb and differentiate into different neuronal cell types in the GCL and the GLOM. Schematic representations of regions of adult neurogenesis in the SVZ/ olfactory bulb (E, adapted from

Figure 2
2R6/2 mice show increased migration of neuroblasts to the affected striatum compared to controls, without signs of substantial striatal neurogenesis. Representative images of DCX-expressing neuroblasts in wild-type (WT; A) and R6/2 mice (R6/2; B), higher magnification (from insert in B) of cells exhibiting heterogeneous morphologies (C): Some cells possess only short or no processes, some display multiple well-developed processes characteristic of migrating cells. Arrows indicate migrating DCX positive cells leaving the SVZ. Quantification of DCX-positive cells in the striatum of WT and R6/2 mice, predominantly in close proximity to the SVZ (D). Error bars represent SD, * indicates p < 0.05. Scale bars represent 50 μm.

Figure 3
3Cell proliferation in the subventricular zone of the R6/2 HD animals is unchanged compared to WT controls. Staining for dividing cells with the cell cycle marker PCNA revealed no difference in the amount of stem and precursor cell proliferation in the SVZ of WT (A) and R6/2 animals (B). Scale bar represents 20 μm.

Figure 4
4Impaired survival of newly generated cells in the two neurogenic regions of the olfactory bulb in HD mice compared to controls. Significant reduction of BrdU-labeled newly generated cells in the GCL and the GLOM 4 weeks after BrdU administration in R6/2 mice compared to controls (C, G). Representative images of immunostainings for BrdU in the GCL (A, B) and the GLOM (E, F) of both groups. Schematic representation of the OB areas analyzed (D). Error bars represent SD, * indicates p < 0.05. Scale bars represent 50 μm.

Figure 5
5The generation of new neurons in the neurogenic regions of the olfactory bulb is impaired in R6/2 animals compared to controls. In the GCL the percentage of newly generated cells that differentiate into a neuronal phenotype is unchanged comparing WT and R6/2 animals, as determined by double-labeling for BrdU and NeuN (A, B). Calculation of absolute numbers of surviving new neurons in the GCL revealed significantly decreased neurogenesis in HD mice compared to controls (C). Analysis of differentiation in the GLOM demonstrated unchanged neuronal differentiation in this region as well. Calculation of the total number of newly generated neurons in the GLOM showed a reduction of more than 50% in R6/2 mice compared to WT controls (F). D and E show representative immunostainings for BrdU and NeuN in the GLOM. Error bars represent SD, * indicates p < 0.05. Scale bars represent 20 μm.

Figure 6
6Generation of dopaminergic neurons in the glomerular layer is less severely affected in HD mice compared to WT aminals. Further analysis of the phenotype of newly generated neurons in the GLOM of the olfactory bulb revealed that the proportion of new dopaminergic neurons (labeled for BrdU, NeuN and TH, A) was significantly increased in HD mice compared to controls (B). Scale bar represents 20 μm.

Figure 7
7Increased cell death in the olfactory bulb of R6/2 mice. TUNEL staining of the GCL in the OB of WT and R6/2 animals shows an increase of TUNEL-positive profiles in transgenic animals carrying the mutant huntingtin (B) compared to WT mice (A). Scale bar represents 50 μm.

following antibodies and final dilutions were used. Primary antibodies: rat anti-BrdU (1:500; Oxford Biotechnology, Oxford, UK), mouse anti-PCNA (1:500), goat anti-DCX (1:200), goat anti-DARPP-32 (1:250; all from Santa Cruz Biotechnology, Santa Cruz, USA), rabbit anti-GFAP (1:1000; Molecular Probes, Eugene, USA), sheep anti-EGF-receptor (1:100, Upstate, Lake Placid, USA), rabbit anti-calretinin (1:500; Swant, Bellinzona, Switzerland), sheep anti-tyrosine hydroxylase (TH; 1:500), mouse anti-NeuN (1:500) and mouse antimutant htt (1:500; all from Chemicon, Temecula, USA). The mouse anti-huntingtin antibody MAB5374 detects N-terminal mutant human htt with a repeat length > 82, in particular as htt aggregates, and much weaker soluble forms, respectively. Secondary antibodies were donkey-derived, species-specific and conjugated with Alexa 488, Alexa 568, Alexa 660 (1:1000, Molecular Probes) or biotin (1:500; Jackson Immuno Research, West Grove, USA). For labeling cell nuclei, ToPro-3 (1:2000; Molecular Probes) was diluted in TBS and applied directly to the sections for 10 min.

Table 1
1Quantification of adult neurogenesis in the olfactory bulb of wildtype and R6/2 mice Morphological quantification of neurogenesis in the olfactory bulb of WT and R6/2 mice comprises the following measures: total number of proliferating cells in the subventricular zone (PCNA), total number of surviving newly generated cells (BrdU), percentage of newly generated neurons (% BrdU/ NeuN) and total number of new neurons (BrdU × % BrdU/NeuN) in the granular cell layer. For the glomerular layer, total number of surviving newly generated cells (BrdU), percentage of newly generated neurons (% BrdU/ NeuN), percentage of newly generated dopaminergic neurons (% BrdU/TH), total number of new neurons (BrdU × % BrdU/NeuN) and total number of new dopaminergic neurons (BrdU × % BrdU/TH) are indicated. All measures are expressed as mean ± SD. # indicates significance comparing WT vs. R6/2 (p < 0.05).WT
R6/2

Proliferation in the SVZ

PCNA (×10 3 )
7.42 ± 3.11 8.49 ± 2.16

Neurogenesis in the OB granular cell layer

BrdU (×10 3 )
69. 9 ± 15.1 46.8 ± 11.3 #

% BrdU/NeuN
88 ± 4
87 ± 3

BrdU × % BrdU/NeuN (×10 3 )
61.5 ± 15.1 40.7 ± 11.2 #

Neurogenesis in the OB glomerular layer

BrdU (×10 3 )
10.1 ± 3.9
4.8 ± 1.7 #

% BrdU/NeuN
90 ± 3
90 ± 6
BrdU × % BrdU/NeuN (×10 3 )
9.1 ± 3.5
4.4 ± 1.7 #

% BrdU/TH
8 ± 2
14 ± 3 #

BrdU × % BrdU/TH
775 ± 301
656 ± 270

AcknowledgementsThe authors would like to thank Jacqueline Schneider for excellent technical support, Leslie Crews for editing the manuscript and J. Ecke, Munich for the artwork. The study was supported by the School of Medicine at the University of Regensburg, Germany (ReForM program to ZK) and the Bavarian State Ministry of Sciences, Research and the Arts (ForNeuroCell grant to BW, JW). MK was supported by a fellowship sponsored by the "Bayerische Forschungsstiftung", Munich, Germany. BW is a Lynen-fellow of the Alexander von Humboldt Foundation.Authors' contributions ZK carried out the experiments, participated in the design of the study and drafted the manuscript. MR carried out experiments and drafted the manuscript. RA carried out animal and immunohistochemical experiments. MK participated in the design of the study and carried out experiments. BW participated in the design of the study and drafted the manuscript. LA conceived of the study, and participated in its design and coordination and helped to draft the manuscript. JW conceived of the study and its design and drafted the manuscript.
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Impaired adult olfactory bulb neurogenesis in the R6/2 mouse model of Huntington's disease. Kohl, BMC Neuroscience. 11114Cite this article asCite this article as: Kohl et al.: Impaired adult olfactory bulb neurogenesis in the R6/2 mouse model of Huntington's disease. BMC Neuroscience 2010 11:114.


Clinical registry of dental outcomes in head and neck cancer patients (OraRad): rationale, methods, and recruitment considerations

Rajesh V Lalla
for the OraRad Study Group

Leslie Long-Simpson
for the OraRad Study Group

James S Hodges
for the OraRad Study Group

Nathaniel Treister
for the OraRad Study Group

Thomas Sollecito
for the OraRad Study Group

Brian Schmidt
for the OraRad Study Group

Lauren L Patton
for the OraRad Study Group

Michael T Brennan
for the OraRad Study Group

Clinical registry of dental outcomes in head and neck cancer patients (OraRad): rationale, methods, and recruitment considerations
10.1186/s12903-017-0344-yS T U D Y P R O T O C O L Open Access
Background: Most head and neck (H&N) cancer patients receive high-dose external beam radiation therapy (RT), often in combination with surgery and/or chemotherapy. Unfortunately, high-dose RT has significant adverse effects on the oral and maxillofacial tissues, some of which persist for the life of the patient. However, dental management of these patients is based largely on individual and expert opinion, as few studies have followed patients prospectively to determine factors that predict adverse oral sequelae. In addition, many previous studies were conducted before widespread adoption of intensity-modulated radiation therapy (IMRT) and concurrent chemotherapy. The objective of this multi-center study is to systematically evaluate the oral health of subjects for 2 years after commencement of RT, with the goal of identifying risk factors that predict adverse oral outcomes post-RT.Methods: This is a prospective multi-center longitudinal cohort study of H&N cancer patients who receive high-dose RT with curative intent. Planned enrollment is 756 subjects at 6 primary clinical sites (and their affiliated sites) in the USA. A baseline visit is conducted prior to the beginning of RT. Follow-up visits are conducted at 6, 12, 18 and 24 months from the start of RT. The primary outcome measure is the 2-year rate of tooth loss in patients who have received at least one session of external beam RT for H&N cancer. Secondary outcome measures include the incidence of exposed intraoral bone; incidence of post-extraction complications; change in Decayed Missing and Filled Surfaces (DMFS); change in periodontal measures; change in stimulated whole salivary flow rates; change in mouth opening; topical fluoride utilization; chronic oral mucositis incidence; changes in RT-specific quality of life measures; and change in oral pain scores.Discussion: This study will contribute to a better understanding of the dental complications experienced by these patients. It will also enable identification of risk factors associated with adverse outcomes such as tooth loss and osteoradionecrosis. These findings will support the development of evidence-based guidelines and inform the planning of future interventional studies, with the goal of advancing improvements in patient care and outcomes. Trial registration: ClinicalTrials.gov Identifier NCT02057510, registered 5 February 2014.

Background

Most head and neck (H&N) cancer patients receive high-dose external beam radiation therapy (RT), often in combination with surgery and/or chemotherapy [1]. Unfortunately, high-dose RT has significant adverse effects on the oral and maxillofacial tissues, some of which persist for the life of the patient [2]. The salivary glands are often included in the fields of radiation, which frequently leads to irreversible hyposalivation [3], resulting in increased rates of dental caries and tooth loss postradiation [4]. Radiation-induced soft tissue fibrosis can lead to restricted mouth opening (trismus), which reduces the ability to maintain oral hygiene [5]. Furthermore, radiation impairs bone homeostasis and ability to heal, leading to a life-long risk of jaw osteoradionecrosis (ORN), which is often precipitated by dental extractions [6]. Consequently, these patients are caught in a vicious circle, being at higher risk of needing dental extractions, but with extractions to be avoided post-RT due to the risk for ORN.

To attempt to reduce the frequency of such complications, pre-RT dental evaluation and management is considered a best practice and standard of care in the USA and many other countries. However, pre-RT dental management practices vary widely among centers, due to a lack of reliable data on dental morbidity in these patients and on outcomes of dental management strategies. Although some studies indicate that these patients have significant dental morbidity post-RT, definitive data to document the extent, severity and risk factors for these complications do not exist. Consequently, pre-RT dental management is largely based on expert opinion [7]. For example, an international survey of dentists/oral surgeons experienced in providing pre-RT care found that approximately 50% would extract a mandibular tooth in the radiation field with a small periapical lesion and pain on percussion, while 50% would treat the tooth endodontically [8]. Clinicians mentioned that the lack of adequate data reduced their confidence in their treatment decisions. Such disparate decision-making can result in sub-optimal dental and medical outcomes for these complex patients.

The objective of this prospective observational cohort study is to collect data on dental outcomes in patients who have received RT to the H&N region. Systematic collection of this data in a large multi-center cohort will lead to a better understanding of the dental complications experienced by these patients. In addition, it will enable the identification of risk factors associated with negative outcomes such as tooth loss and ORN. These findings will support the development of evidence-based guidelines and should lead to improvements in patient care and outcomes. The purpose of this manuscript is to describe the study protocol (version 4, 29 October 2015) for this important ongoing study and provide insight based on our experience related to subject recruitment and complexity of study procedures.

Methods

Study design

This is a prospective multi-center longitudinal cohort study of H&N cancer patients who receive high-dose RT.

Outcome measures

The primary outcome measure is the 2-year rate of tooth loss in patients who have received at least one session of external beam RT with curative intent for H&N cancer.

"Tooth loss" is defined as a dental extraction that has been performed or recommended. Teeth that are lost without the involvement of a healthcare professional are also included in this definition. Since dental extractions are often avoided in this population because of the increased risk of ORN, tooth loss also includes teeth having a dental procedure to avoid extraction of a tooth that would have been extracted if the individual had not received RT. The following categories constitute teeth that would otherwise be recommended for extraction: non-restorable because of fracture or extent of caries; amputated crown with root remaining; uncontrolled or persistent periodontal or odontogenic infection. Such teeth are classified as "hopeless teeth" for the purposes of this study.

Secondary outcome measures include the following additional oral health outcomes associated with RT in H&N cancer patients and potential risk factors for negative outcomes: Incidence of exposed intraoral bone, suggestive of ORN. This will be defined as exposed maxillary or mandibular bone with an avascular appearance in a quadrant that has received RT; Incidence of post-extraction complications; Change in Decayed, Missing, and Filled Surfaces (DMFS); Change in periodontal measures; Change in stimulated whole salivary flow rates; Change in mouth opening in mm; Use of fluoride to prevent new caries; Incidence of chronic oral mucositis; Change in RT-specific quality of life measures; and Change in pain scores as measured with the UCSF Oral Cancer Pain Scale.

Study organization and funding

Subjects are enrolled at six primary clinical study sites, which are (in alphabetical order): Brigham and Women's Hospital, Carolinas Medical Center, New York University, University of Connecticut Health, University of North Carolina, and University of Pennsylvania. Personnel at each site include the site Principal Investigator (PI), study coordinator(s), and clinical examiner(s). Some of the primary clinical sites have also established local affiliated sites. The Data Coordinating Center (DCC) for the study is located at the University of Minnesota (Division of Biostatistics) and includes the site PI/study statistician, study manager, and database administrator. The study is funded by the US National Institute for Dental and Craniofacial Research (NIDCR) through a cooperative agreement (U01) mechanism. NIDCR staff are closely involved in the development and oversight of the study.

Safety of human subjects and data integrity

Approval from the Institutional Review Board (IRB) at each clinical study site was obtained before beginning enrollment at that site. Any changes are also communicated to IRB and other stakeholders as appropriate. In addition, each site was individually activated by the NIDCR after passing all relevant requirements for human subjects safety and personnel training. All subjects participate in an informed consent process conducted by the study coordinator, including the signing of a written informed consent document. An independent Data and Safety Monitoring Board (DSMB) has been constituted by the NIDCR to ensure subject safety. It has 3 members, all of whom are free from competing interests. It meets annually and additional meetings can be called as needed. The DSMB reviews any adverse events as well as other study data. No safety concerns have been identified to date.

Study data is entered into a secure database, hosted at the DCC, using web-based data-entry screens created for this study. DCC staff monitor study data to identify missing data or forms and communicate with the clinical sites to resolve these issues. Formal monitoring of study records is conducted by an independent contract research organization. Study Monitors visit each clinical site annually for formal study monitoring.

Training and calibration

All study personnel receive training on the activities they conduct in the study. This includes training on carrying out clinical assessments as well as training on completing study forms, data entry, and all non-clinical procedures. In addition, annual in-person calibration on healthy volunteers is conducted for all clinical examiners for DMFS and periodontal measurements. Clinical examiners must meet minimum standards for intra-and inter-rater consistency annually for these measurements. Specifically, examiners must achieve at least 95% intraexaminer reproducibility within ± 2 mm for both clinical attachment loss and pocket depth; at least 75% interexaminer agreement for pocket depth within ± 1 mm; and at least 60% inter-examiner agreement for clinical attachment loss within ± 1 mm. Examiners also receive hands-on training on measuring furcation involvement and tooth mobility.

Subject selection criteria Inclusion criteria

To be eligible to participate in this study, a subject must meet all of the following criteria:

Diagnosed with H&N squamous cell carcinoma (SCC) or a salivary gland cancer (SGC), and intends to receive external beam RT with curative intent (tumor eradication), with or without concomitant chemotherapy; OR Diagnosed with a non-SCC, non-SGC malignancy of the H&N region, and intends to receive RT, with or without concomitant chemotherapy. The subject must be expected to receive at least 4500 cGy to at least 1 of 26 specified sites in the H&N region; Aged 18 years and older; Willing and able to provide signed and dated consent form; At least 1 natural tooth remaining or expected to remain in the mouth after completion of the pre-RT dental extractions, if any; Willing to comply with all study procedures; and Willing to participate for the duration of the study.

After the completion of the baseline study visit, it must be verified that the subject has received at least one session of RT, to confirm eligibility for continued follow-up in the study.

Exclusion criteria

A potential subject who meets any of the following criteria will be excluded from participation in this study:

Receiving palliative RT; History of prior curative RT to the H&N region to eradicate a malignancy; Incarcerated at time of screening; or Anything that would place the subject at increased risk or preclude the subject's full compliance with or completion of the study. Figure 1 depicts the sequence of events in a subject's participation. A baseline visit is conducted before the beginning of RT but after completing any recommended pre-RT extractions and other invasive oral procedures. Follow-up visits are conducted at 6, 12, 18, and 24 months (+/-30 days) from the start of RT. Table 1 summarizes the procedures conducted at each study visit.

Study assessments

Details of study assessments, including instruments/ scales used, are as follows:

A panoramic radiograph taken within 180 days before the baseline study visit is evaluated to assess the presence of intrabony devices, periapical lesions and impacted teeth. Periodontal measures are collected at baseline, 12, and 24 month visits. Probing depths and the distance from the cemento-enamel junction to the gingival margin are measured using a UNC 15 probe at six sites on each tooth. The clinical attachment level is calculated by the DCC using these two recorded measurements. The presence of bleeding upon probing is documented at the six periodontal probing sites. These assessments are obtained on all teeth other than third molars. Additionally, at the baseline visit, a plaque score is recorded for the Ramfjord teeth, furcation involvement is measured using a Naber's probe, and tooth mobility is assessed [9]. The invasive periodontal measures are not performed on patients needing antibiotic prophylaxis prior to invasive dental procedures. Decayed, missing, filled surfaces (DMFS) are recorded at baseline, 6, 12, 18, and 24 month visits. The dentition is examined for active decay, missing teeth because of extraction and/or spontaneous exfoliation, and teeth that have restorations. A No. 23 explorer or a No. 2A explorer is used to detect decay. Five tooth surfaces on the posterior teeth and four tooth surfaces on the anterior teeth are scored for DMFS. The DMFS is also used to reconcile newly missing teeth between study visits.   Salivary flow rate is collected at baseline, 6, and 18 month visits. Participants are provided with unflavored paraffin (gum base) and two 50 ml test tubes. They are instructed to chew the gum base for 2 min, meanwhile expectorating saliva into one of two test tubes. This is done as practice to standardize the technique and stabilize the flow rate. The same chewing/expectorating method is used for 5 min for the final flow rate assessment, timed using a digital timer. The saliva collected in 5 min is weighed and recorded.

Additional assessments completed at all visits include clinical examination of the subject's oral cavity for "hopeless teeth" (using criteria described above), grading of oral mucositis using the World Health Organization (WHO) mucositis scale [10], inspection of both the mandible and maxilla for exposed intraoral bone, and measurement of maximal mouth opening using a Therabite® Range of Motion Scale in millimeters. Patient-reported questionnaires are administered at all study visits. These questionnaires collect information on the participant's daily oral hygiene habits, oral pain using the UCSF Oral Cancer Pain Scale [11], and quality of life related to their oral health using items from the EORTC QLQ-H&N35 scale [12]. Additional "Event visits" are conducted if a subject has any of the following "events": has a dental extraction, reports spontaneous exfoliation of a tooth, or has an area of exposed intraoral bone. Any signs and symptoms the subject may present with at the visit are recorded, including those related to infection and wound healing. Additional data, related to the event, is collected from the medical and dental records, including radiation doses to affected locations and the condition of the teeth before extraction.

Statistical considerations Sample size and power considerations

Total planned enrollment is 756 subjects across all clinical sites. The primary outcome used to determine sample size is the rate of tooth loss after the end of RT. Although ORN is an important outcome in these patients, the relative infrequency of ORN [13] makes it unfeasible to conduct a study with ORN as a primary outcome measure. Tooth loss is an ideal outcome measure because it is more common than ORN, it is easily measured, and ORN in this population often results from post-RT dental extractions. Previous studies found that 20 of 168 patients either had or were recommended to have a dental extraction after median follow-up of 7.6 months after end of H&N RT [14], and that 10 of 50 patients (20%) reported having had an extraction or a procedure to avoid an extraction at a median follow-up of 728 days after the end of RT, with median time of 442 days from the end of RT to the procedure (Unpublished data, University of Connecticut). We also estimated that up to 30% of enrolled participants would withdraw from study participation, be lost to follow-up, or become deceased during 2 years of follow-up. Based on the foregoing, we assumed 20% of subjects would have an extraction or a procedure to avoid an extraction by 2 years after RT, so that 756 enrolled subjects would yield 529 participants with 2-year follow-up data on tooth loss and an expected 95% confidence interval (around 20%) of 16.7%-23.7%, which we deemed adequately narrow. If the actual rate of tooth loss is lower than 20%, a 95% confidence interval will be narrower; if the actual rate of tooth loss is higher than 20%, a 95% confidence interval will be wider. Under these assumptions, 88-125 subjects will have a toothloss event. Based on common rules of thumb, this suffices to consider 8-12 predictors (risk factors) for tooth loss [15].

Planned analyses

The study's primary objective is descriptive so the analyses are mainly descriptive. The primary objective can be interpreted in two ways: What fraction of subjects had a tooth-loss event in the two years after RT? And how many teeth were lost per year? Analyses of the former will use time-to-event (survival) methods, where the event is the first tooth loss. This type of analysis accounts for partial follow-up. The 2-year fraction with tooth-loss events will be estimated using the survivor function and associated confidence interval. To estimate the rate of teeth lost per year, each subject's outcome is the number of teeth they lost, analyzed using a negative binomial model with log link and offset log follow-up time (in years) and no predictors. This model's intercept, raised to the power e (the base of the natural logarithm), estimates the rate of teeth lost per year. (The negative binomial model allows analysis of a count outcome with a flexible error variance. Obviously this model is inappropriate if many participants lose large numbers of teeth but this seems unlikely.) Secondary analyses will cull potential risk factors for tooth loss events and for increased rate of tooth loss by considering them as predictors in the regression analyses described above. Other secondary analyses will consider the secondary outcomes, many of which are measured at multiple follow-up visits, so their analyses will be longitudinal, accounting for the presumed correlation of a subject's multiple measurements.

One complication of these analyses is how a subject's initial number of teeth affects tooth loss. We will examine this question in preliminary analyses that include in the regression a flexible function of the initial number of teeth (e.g., a 4°-of-freedom spline). A further complication arises from subjects who have incomplete follow-up due to death. We account for this by defining the outcome being estimated as "rate [or chance] of tooth loss assuming survival to 2 years", which can be estimated by various methods that accommodate participants with incomplete follow-up, e.g., inhomogeneous Poisson processes or logistic regression including a flexible function of follow-up time as an adjuster.

Discussion

The results of this ongoing study are expected to be valuable for several reasons. First, this study will definitively document the oral and dental morbidity resulting from high-dose RT to the H&N region. This includes the impact on salivary flow, mouth opening, caries, periodontal disease, tooth loss, and development of ORN. A better understanding of these downstream effects of RT will inform the pre-RT dental evaluation and reinforce its importance to all stakeholders including patients, medical and dental providers, and third-party payers. Second, this study should allow the identification of risk factors for adverse oral and dental outcomes. As mentioned previously, the pre-RT dental management of these patients varies widely and is largely based on expert opinion. Identification of specific risk factors will allow for evidence-based decision making and better clinical outcomes. Thus, our findings have the potential to lead to significant changes in clinical practice. Third, this study provides a unique opportunity to compare pre-RT dental management practices across a number of different sites. An assessment of important factors, such as number of pre-RT dental extractions, duration between extractions and start of RT, and subsequent development of ORN, in this large cohort can be highly informative.

There are some limitations of this study design. Two years is a relatively short follow-up period as tooth loss may occur after this time. While a longer follow-up period would be desirable, funding constraints did not allow for this and it can also be challenging to follow such medically complex patients for longer periods. Important assessments such as periodontal measurements and salivary flow are not conducted at each follow-up visit. These are completed at alternating follow-up visits to reduce the length of the follow-up visits and burden on the subject. Although the study Manual of Procedures recommends that each subject should be examined by the same clinical examiner at all visits, in practice this has been difficult to achieve due to scheduling issues and staff turnover. However, all clinical examiners are calibrated to strict standards for inter-and intra-rater consistency.

Recruitment presents a challenge in most clinical studies and especially in the oncology population. It has been estimated that only about 3% of patients with cancer participate in a clinical trial and even at some tertiary academic centers, the rate may be below 10% [16]. A barrier to recruitment for the present study is that in order for the baseline visit to be conducted prior to the start of RT, patients may need to be approached soon after their cancer treatment plan has been determined. At this time, patients are often emotionally fragile and overwhelmed with details from various providers on planned surgery, chemotherapy, and RT. In the context of a life-threatening diagnosis, patients can be bewildered about being approached for participation in an observational dental study that does not offer any direct impact on their cancer prognosis. Indeed, as of December 2015, the most common reason (60%) for an approached patient not being formally screened is that they are "not interested/too busy". Careful and patient explanation of the dental and oral complications of RT, and possible benefits of study participation, can be helpful, particularly if reinforced by the radiation oncologist. It is highly desirable to have a team-based approach, where all parties recognize and reinforce the importance of the study and benefits of participation, to the subject and to society. Potential benefits to the subject include the regular and detailed monitoring of their oral and dental status at no charge, which can facilitate the earlier diagnosis and management of potential oral complications. Appropriate monetary compensation is also provided to subjects for their time and effort in attending study visits. However, the study does not directly provide or support clinical care, which may have a negative effect on some patients' interest in study participation.

An important aspect of recruitment is access to the patient population. It is standard practice in the USA for these patients to be referred to a dentist for a pre-RT dental assessment [1]. This dental assessment may occur at the cancer treatment site itself or at a community dental practice. Our experience at the different clinical sites has shown that recruitment is enhanced when the study team works closely with the clinical dental providers receiving the clinical referral and/or the oncology team managing the medical care of these patients. However, the pre-RT clinical dental evaluation can also be a reason patients decline study participation. Specifically, when the pre-RT dental evaluation by regular clinical providers includes periodontal probing, patients have sometimes been unwilling to then undergo the additional detailed periodontal examinations conducted at the baseline study visit by the calibrated study examiner.

The number of eligible patients also varies across sites, with sites located in large urban centers or tertiary care centers usually having a larger patient population.

Indeed, such higher profile referral centers have been the strongest enrolling sites. However, a proportion of patients at such magnet referral centers travel from a distance for their cancer care but may receive follow-up closer to home. This can lead to sub-optimal compliance with the subsequent follow-up visits after treatment. Thus, appropriate selection of patients is important. The main barrier to recruitment at smaller centers is access to an adequate number of eligible patients. The smaller sites, however, also tend to have better attendance at follow-up visits. Strategies we have adopted at sites with lower enrollment include the establishment of a satellite study location at a nearby healthcare facility, where enrollment activities and/or study visits can take place. While this has had a modest positive impact on enrollment, it does create new challenges in terms of obtaining medical records from the satellite site.

An additional factor affecting recruitment is adequate staffing, given the ongoing demands related to scheduling and conducting follow-up visits as well as data management. To better optimize use of the study staff's time, the amount of medications data being collected at follow-up visits was significantly reduced after about 250 subjects had been enrolled. An analysis of the medications data collected to that point revealed that, beyond the 6 month visit, very few subjects were using the medications being collected. Therefore, collection of detailed medications data from medical records after that point was determined as not being critical to achieve the main study objectives.

Notwithstanding all of the above-mentioned recruitment considerations, over 450 subjects have been enrolled to the study so far. Thus, this landmark study is expected to meet its planned enrollment target and result in significant advances in the care of patients receiving RT to the H&N region.

Fig. 1
1Sequence of a subject's participation

at baseline include clinical attachment loss, bleeding on probing, pocket depth, gingival recession, plaque index, tooth mobility and furcation involvement; at follow-up visits, plaque index, tooth mobility and furcation involvement are not recorded b Findings from clinical radiograph are recorded; radiograph is taken for study only if a current (<180 days) radiograph from routine clinical care is not available c Measured at early withdrawal visit only if it would have been done at next scheduled visit

Table 1
1Study Procedures at each time-point (per SPIRIT guidelines)Procedures

Screening Baseline Visit

(0
-42 days
before RT begins)

Baseline records
review after RT

Follow-up Visit 1

(182 ± 30 days
after RT begins)

Follow-up Visit 2

(364 ± 30 days
after RT begins)

Follow-up Visit 3

(546 ± 30 days
after RT begins)

Follow-up Visit 4

(728 ± 30 days
after RT begins)

Early Withdrawal
Visit

Unscheduled

Visit

Consent for screening, assess eligibility,
record basic demographic data

X

Obtain signed study consent
form, confirm eligibility

AcknowledgementsThe authors gratefully acknowledge the significant contributions of Dr. Jane C. Atkinson of the NIDCR to the development of the study protocol, Ms. Mary Cutting of the NIDCR for editing the study protocol, and the contributions of Ms. Helen Voelker of the Data Coordinating Center in establishing the study database.FundingAuthors' contributions RVL prepared the first draft of this manuscript and the study protocol, made revisions to this manuscript and the study protocol for important intellectual content, incorporated comments from other authors into this manuscript, and finalized the manuscript. LLS made revisions to this manuscript and the study protocol for important intellectual content. JSH made revisions to this manuscript and the study protocol for important intellectual content. NT made revisions to this manuscript and the study protocol for important intellectual content. TS made revisions to this manuscript and the study protocol for important intellectual content. BS made revisions to this manuscript and the study protocol for important intellectual content. LLP made revisions to this manuscript and the study protocol for important intellectual content. MTB prepared the first draft of the study protocol, and made revisions to this manuscript and the study protocol for important intellectual content. All authors have read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Consent for publicationNot applicable. This manuscript does not contain any individual person's data in any form.Ethics approval and consent to participate• We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal Submit your next manuscript to BioMed Central and we will help you at every step:
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