Changes in Glial Cell Line-derived Neurotrophic Factor Expression in the Rostral and Caudal Stumps of the Transected Adult Rat Spinal Cord

Limited information is available regarding the role of endogenous Glial cell line-derived neurotrophic factor (GDNF) in the spinal cord following transection injury. The present study investigated the possible role of GDNF in injured spinal cords following transection injury (T9–T10) in adult rats. The locomotor function recovery of animals by the BBB (Basso, Beattie, Bresnahan) scale score showed that hindlimb support and stepping function increased gradually from 7 days post operation (dpo) to 21 dpo. However, the locomotion function in the hindlimbs decreased effectively in GDNF-antibody treated rats. GDNF immunoreactivty in neurons in the ventral horn of the rostral stump was stained strongly at 3 and 7 dpo, and in the caudal stump at 14 dpo, while immunostaining in astrocytes was also seen at all time-points after transection injury. Western blot showed that the level of GDNF protein underwent a rapid decrease at 7 dpo in both stumps, and was followed by a partial recovery at a later time-point, when compared with the sham-operated group. GDNF mRNA-positive signals were detected in neurons of the ventral horn, especially in lamina IX. No regenerative fibers from corticospinal tract can be seen in the caudal segment near the injury site using BDA tracing technique. No somatosensory evoked potentials (SEP) could be recorded throughout the experimental period as well. These findings suggested that intrinsic GDNF in the spinal cord could play an essential role in neuroplasticity. The mechanism may be that GDNF is involved in the regulation of local circuitry in transected spinal cords of adult rats.
Introduction
Spinal cord injury (SCI), a common result of car accidents, high-altitude falls and crashes, and other violent injuries is increasing yearly. It has been reported that there are about 15–40 traumatic SCI cases per million persons annually population worldwide [1]. Once the spinal cord is damaged, it usually results in severe neurologic dysfunction and disability. Over the past several years, many extensive experiments have been done in order to find a way to promote the repair of the injured spinal cord. However, no good therapeutic approaches have yet been found. Recently, it has been shown that delivery of exogenous neurotrophic factors (NTFs) to the injured spinal cord has beneficial effects in improving functional recovery [2], indicating the potential value of NTFs in the treatment of SCI.
Glial cell line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor-β (TGF-β) family, was originally purified using an assay based on its ability to maintain the survival and function of embryonic ventral midbrain dopaminergic neurons in vitro [3]. GDNF supports neural survival in several neuronal populations, including midbrain dopamine neurons and motoneurons in the central nervous system. It promotes the survival and the differentiation of many peripheral neurons, such as sympathetic, parasympathetic, and sensory neurons [3–10]. Following injury, GDNF showed a benefit effect to rescue motoneurons degeneration after spinal root avulsion and distal nerve axotomy [11], promotion of axonal regeneration [12] and enhancing regeneration of dorsal roots (DR) into the adult rat spinal cord [13]. In addition, the transplantation of GDNF-producing cells greatly enhances the survival of spinal cord motorneurons [11, 14, 15] and regeneration of several spinal systems, including dorsal column sensory, and regionally projecting propriospinal pathways [16, 17] after SCI. The biological action of GDNF is mediated by a two-component receptor GFRα-1 and protein tyrosine kinase Ret [18–21]. The GFRα-1 receptors expressed on a variety of neurons that project into the spinal cord, including supraspinal neurons, dorsal root ganglia, and local neurons. GDNF mRNAs are widely distributed in a variety of neuronal and non-neuronal tissues of embryos and adults [4, 22–25].
Although exogenous GDNF is available partially to promote propriospinal axonal regeneration and locomotion functional recovery, the role of intrinsic GDNF in injured spinal cords is largely unknown. Therefore, the present study investigated the possible role of GDNF at various time intervals following spinal cord transection injury in adult rats, so as to gain insights into the contribution of endogenous GDNF in spinal neuroplasticity.
Materials and methods
Characterization of antibodies
To localize GDNF protein in the spinal cord, an affinity-purified rabbit polyclonal antibody (D-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used in this study. The specificity of antibody for GDNF was confirmed by Western blots using rat spinal cord homogenates.
Preparation of probe for in situ hybridization
For detection of GDNF cellular expression in situ, we used Digoxigenin-labeled oligonucleotide probe designed by Primer premier 5.0 package which was complementary to the rats GDNF gene sequence (33 mer, 5′-GCCCTACTTTGTCAC–TCACCAGCCTTCTATTTC-3′). This antisense DNA single-stranded oligonucleotide probe was synthesized by Takara Biotechnology Company.
Animal grouping and surgery
Sixty-five adult Sprague-Dawley rats of either sex (weighing 200–220 g) were obtained from the Animal Experiment Center of Sichuan University of Medical Sciences. Fifty rats among all animals, designated as sham-operated group and spinal cord transection group that animals were allowed to survive 3, 7, 14 and 21 days post operation (dpo), were used for immunohistochemistry and in situ hybridization (n = 5 in each group), and Western blot analysis (n = 5 in each group) respectively. Another five rats were intraperitoneally injected anti-GDNF solution (2.5 ml/kg) once every 2 days until 21 dpo, designed as antibody neutralization group. Five animals, subjected to cord transaction, were injected with distilled water as the distilled water group. The last five operated rats were used to investigate corticospinal tract sprouting. Every effort was taken to reduce the number of animals and suffering during the experiments. All the experiments using animals were carried out according to the guidelines of the NIH Guide for Care and Use of Laboratory Animal. For the cord transection operation, the rats were anesthetized with 3.6% Chloral Hydrate (1 ml/100 g). A midline incision was made in the back, then the T7–T8 spinous processes and the vertebral laminae were removed to expose the spinal cord at T9–T10 level. The cord was transected between T9 and T10 segments with a pair of microscissors. After surgery, the superficial back muscles and the skin were sutured along the midline.
Behavior tests
All rats in each group were evaluated using the BBB (Basso, Beattie, Bresnahan) scale score [26]. The BBB score for evaluation of hindlimbs function after transected injury ranges from zero, which corresponds to flaccid paralysis, to 21, which is normal gait. Animals were allowed to walk around freely in a circular field (1.2 m in diameter) for 4 min while movements of the hindlimbs were closely observed. Ranking according to the scoring system described by Basso et al. [26] includes frequency and quality of hindlimb movement as well as forelimb/hindlimb coordination.
Measurement of somatosensory evoked potentials (SEP)
The right peroneal nerve of the rats was dissected and a stimulation electrode was placed on it while a hole (3–4 mm in diameter) was made on the skull (2 mm to the left of the midline and 2 mm in front of the fonticulus posterior). An electrode was placed on the dura of brain cortex to record the SEP. The reference electrode was inserted at the nose epithelium. A ground electrode was inserted at the tail. The stimulus intensity was set high enough to produce a marked muscle twitch in the hind limb, about 1.1 mA, the amplitude was 0.2 ms and the frequency 3 Hz. The SEP Tracings represented the average of 200 responses.
Immunohistochemistry
After anesthesia with 3.6% Chloral Hydrate (1 ml/100 g), rats were perfused with 500 ml of cold phosphate-buffered saline (PBS) for 5 min and 500 ml cold 4% paraformaldehyde solution for 30 min. The T9 and T10 spinal cord segments in the sham-operated group, and the cord stumps rostral and caudal to the injury site in the experimental group were harvested. This was followed by a postfix for 6–12 h, then immersed in 0.1 M PBS containing 20% sucrose overnight. Sections of 20 μm thickness were cut on a freezing microtome and used for freefloating GDNF immunostaining, then rinsed with 0.01 M PBS and soaked in PBS containing 3% H2O2 for 30 min at room temperature to quench the endogenous peroxidase activity. They were immersed in PBS containing 5% goat serum and 0.3% TritonX-100 solution at room temperature for 2 h, then incubated at 4°C for 24 h with anti-GDNF (1:1,000, Chemicon) rabbit polyclonal antibody. Sections were incubated with biotinylated goat anti-rabbit IgG antibody (1:100 dilution) for 1.5 h at room temperature, and by reaction with avidin-biotinylated peroxidase complexes (1:250, ABC Elite, Vector Labs). GDNF immunoreactivity was visualized with brown staining using diaminobenzidine (DAB) and H2O2 as substrate for 5 min, and observed with a light microscope. Negative control experiments in which PBS was substituted for the primary antibody were performed to ascertain the specificity of antibody staining.
In situ hybridization
Sections from sham-operated rats of 25 μm thickness were also cut on a freezing microtome and used for in situ hybridization. Sections were fixed in 4% paraformaldehyde in 0.1 M PBS, pH 7.2 (all treatments were performed at room temperature unless otherwise indicated), and further treated with 0.3% TritonX-100 solution for 10 min and proteinase K (5 μg/ml) at 37°C for 25 min, refixed with 4% paraformaldehyde for 5 min, repeatedly immersed in 0.1 M PBS, then acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) to prevent non-specific binding of the probes. The sections were washed with 2× SSC (pH 7.0) and then prehybridized in a hybridization solution (50% formamide, 10% dextran sulfate, 1× Denhardt’s solution, 0.2 mg/ml Herring sperm DNA, and 10 mM dithiothreitol) without probes at 37°C for 2 h before hybridization, then hybridized in 100 μl hybridization solution containing 1 μl probes at 37°C for 12–16 h in a moist chamber. This was followed by washing in decreasing concentrations of SSC, from 4× SSC (pH 7.0) at 37°C for 20 min, 2× SSC (pH 7.0) at 42°C for 20 min, 1× SSC (pH 7.0) at 48°C for 20 min and ending with 0.5× SSC (pH 7.0) at 50°C for 20 min. Then sections were incubated at 37°C in 1% blocking buffer (Roche) for 1 h, subsequently reacted in 1:1,000 sheep anti-digoxygenin-alkaline phosphatase (AP) antibody in 1% blocking buffer at 4°C overnight. Lastly, AP activity was detected using nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) substrate (Roche). The sections were visualized with blue and purple sedimentation then observed with a light microscope.
GFAP/GDNF double-label immunohistochemistry
Two-color immunohistochemical staining for simultaneous detection of glial fibrillary acidic protein (GFAP)/GDNF expression was performed as described above. Briefly, sections were stained with anti-GDNF rabbit polyclonal antibody (1:1,000), and using DAB solutions as a substrate. This was followed by second incubation with anti-GFAP rabbit polyclonal antibody (1:1,000, AB5804, Chemicon), and finally, reacted with TMB solutions. Double labeling showed a combination of brown and blue product.
Western blotting
The spinal cord from the rostral and caudal stumps was obtained. After carefully removing the spinal meninges, the cords were homogenized on ice in a Lysis Buffer containing 0.05 M Tris–HCl (pH 7.4, Amresco), 0.5 M EDTA (Amresco), 30% TritonX-100 (Amresco), NaCl (Amresco), 10% SDS (Sigma) and 1 mM PMSF (Amresco), then centrifuged at 12,000g for 30 min. The supernatant was obtained and stored at −80°C for late use. Protein concentration was assayed with BCA reagent (Sigma, St. Louis, MO, USA). A 20 μl aliquot of the samples was loaded on to each lane and electrophoresed on 12% SDS-polyacrylamide gel (SDS-PAGE) for 2.5 h at a constant voltage of 120 V. Proteins were transferred from the gel to a nitrocellulose membrane for 435 min at 24 V. The membrane was blocked with phosphate-buffered saline containing 0.05% Tween-20 (PBST) with 10% non-fat dry milk overnight at 4°C. The membrane was rinsed with PBST and incubated with the primary antibody for GDNF (1:1,000) at 4°C. The membrane was incubated with a HRP-conjugated goat anti-rabbit IgG (1:5,000; Vector Laboratories, CA) for 2 h at room temperature. The membrane was developed in ECM kit and exposed against X-ray film in a darkroom. Densitometry analysis for the level of GDNF protein was performed by Bio-Gel Imagining system equipped with Genius synaptic gene tool software. β-actin (the primary antibody, 1:1,000, the secondary antibody, 1:2,000; Santa Cruz Biotechnology) was used as an internal control.
BDA anterograde tracing
At 14 dpo, the animals for this part were anesthetized and fixed in a David Kopf Instruments (Tujunga, CA) stereotaxic head-holder device. Burr holes were made in the dorsal cranium, and biotinylated dextran amine (BDA) (10% BDA solution, Molecular Probes) was microinjected into eight sites at a depth of 0.7 mm from the cortical surface (0.5 μl/site) to cover the hindlimb region. Animals were then sacrificed 2 weeks later to allow sufficient time for axonal transport of BDA in corticospinal tract. The spinal cords were removed and postfixed at 3 days in cold 4% paraformaldehyde in 0.1 M PBS (pH 7.2). Transverse sections (30 μm) of spinal cord at the injury site and neighboring rostral and caudal parts to the injury site were processed for the presence of BDA-labeled axons by incubation in avidin-HRP (Molecular Probes). Lastly, DAB stain was performed to visualize the positive fiber, as brown color staining.
GDNF antibody neutralization
After 14 dpo, each rat was intraperitoneally injected with 0.5 ml (30 mg/ml, 30 mg of anti-GDNF diluted in 1 ml of distilled water) anti-GDNF solution once every 2 days until 21 dpo. GDNF antibody was the distilled water replace as control in another five rats. The locomotion in hindlimbs by BBB score was evaluated at 3, 7, 14, and 21 dpo.
Statistical analysis
All data were expressed as the mean ± S.E.M. They were analyzed using One-way ANOVA and LSD-q test by SPSS software package. The statistical significance was defined as P < 0.05.
Results
Behavior tests
The BBB score for locomotor function in sham-operated rats hindlimbs was 21. Compared with the sham-operation group, the BBB score of animals in the group with only the cord transection and distilled water group increased gradually from 7 to 21 dpo. A significant decrease in the BBB score for the GDNF-antibody treated group was observed (P < 0.05) (Table 1).
Table 1Mean values of BBB scores in cord transected rats (mean ± S.E.M)Group3 Days7 Days14 Days21 DaysSham-operation group5 ± 0.621 ± 021 ± 021 ± 0Transection group0 ± 00.8 ± 0.32.4 ± 0.73.6 ± 0.5Distilled water group0 ± 00.6 ± 0.72.0 ± 0.43.3 ± 0.3GDNF-antibody treated group0 ± 00.2 ± 0.40.6 ± 0.30.9 ± 0.5BBB score in rats subjected to the sham operation was constant. Following transection injury, the BBB score of animals increased significantly from 7 to 21 dpo (P < 0.05). There had no statistical significance in BBB score between cord transection group and distilled water group (P > 0.05), while a significant decrease of BBB score in GDNF-antibody treated group was observed (P < 0.05)
Somatosensory evoked potentials (SEP)
Evoked potentials are the electrical signals generated by the nervous system in response to sensory stimuli. Somatosensory evoked potentials consist of a series of waves that reflect sequential activation of neural structures along the somatosensory pathways following electrical stimulation of peripheral nerves. SEP (P1N1P2 type) was recorded in the control rats and shown in Fig. 1. Throughout the experimental period, no SEP could be recorded in rats subjected to cord transection.
Fig. 1Somatosensory evoked potential was recorded in normal adult rats (P1N1P2 type). The latency of SEP: P1 (10.3 ± 0.26) ms, N1 (8.81 ± 0.34) ms, P2 (15.5 ± 0.43) ms, amplitude: P1 (2.34 ± 0.02), N1 (16.3 ± 0.14), P2 (−5.06 ± 0.05)
The GDNF immunostained intensity changes
In the control group, GDNF immunoreactivity was observed in the cytoplasm and nuclei of the neurons from the ventral horn in the gray matter (Fig. 3a). Most of the small and medium-sized neurons in the intermediate zone and dorsal horn were stained faintly. GDNF positive profiles (Fig. 3b) simultaneously labeled with GFAP, were present in the white matter (Fig. 2b). In the negative control group, no specific immunopositive staining was detected (Fig. 2a).
Fig. 2In the negative control group, no specific immunopositive staining was detected (a). GDNF positive profiles, simultaneously label-GFAP, were present in the white matter (b)
In the rostral stump, strong immunostaining for GDNF was observed in neurons from the ventral horn at 3 and 7 dpo (Fig. 3c, d; Table 2). Moderate to intense immunostained neurons were seen at 14 and 21 dpo, as shown in Figs. 3g, 4a and Table 2. In the white matter, astrocytes showed strong immunoreactivity of GDNF from 3 to 21 dpo (Figs. 3d–f, 4b; Table 2).
Fig. 3Under higher magnification (400×), GDNF immunoreactivity (IR) was observed in neurons from the ventral horn of the gray matter in the normal spinal cord (a, arrow). GDNF (IR) in astrocytes was also seen in white matter (b, arrow). In the rostral stump, strong GDNF IR was detected in neurons from the ventral horn at 3 and 7 dpo (c, e, 400×, arrows). Moderate to intense immunostained neurons were labeled at 14 dpo (g, 400×, arrow). Intensely immunostained IR in astrocytes of the white matter were observed from 3 to 14 dpo (d, f, h, 400×, arrows)Table 2GDNF immunostaining intensity in neurons and astrocytesNormal 3 Days7 Days14 Days21  DaysNeurons+++++++++∼+++++∼+++Rostral part Astrocytes ++++++++++++++Caudal part Neurons++∼+++++++++Astrocytes++++++++++++Intensity: + weak, ++ moderate, +++ strongNeurons in the ventral horn of the rostral stump was strongly stained for GDNF at 3 and 7 dpo, and in the caudal stump at 14 dpo. The GDNF positive staining in astrocytes was also stronger at all time-points after transection injury than seen in control groupFig. 4In the rostral stump, moderate to intense immunostained neurons were labeled at 21 dpo (a, 400×, arrow). Intensely immunostained IR in astrocytes of the white matter were also observed at 21 dpo (b, 400×, arrow). In the caudal stump, immunostained products were detected in neurons of the gray matter at 3 and 7 dpo (c, d, 400×, arrows). Strong immunostained IR for GDNF in neurons was observed at 14 dpo (e, 400×, arrow). Under higher magnification, immunoreactive structures of GDNF were localized in both cytoplasm and nuclei at 21 dpo (g, 400×, arrow), GDNF IR in astrocytes of the white matter was intensely immunostained at 14 and 21 dpo (f, h, 400×, arrows)
In the gray matter of the caudal stump, moderately immunostained neurons were detected at 3, 7 and 21 dpo, and more so at 14 dpo (Fig. 4c–e, g; Table 2). The subcellular localization for GDNF was observed both in cytoplasm and nuclei at 21 dpo (Fig. 4g). At both 14 and 21 dpo, astrocytes in the white matter strongly stain with GDNF immunoreactivity as shown in Fig. 4f, h and Table 2.
Localization of GDNF mRNA in spinal cord of rats
Signals of hybridization for GDNF mRNA were markedly detected in the Central canal and lamina III, IV and V, in which small neurons were moderately NBT/BCIP stained (Fig. 5a). Medium-sized neurons were seen in the field of the intermediomedial nucleus and nucleus dorsalis (Fig. 5a). In the ventral horn, GDNF mRNA-positive large neurons were also observed in lamina IX (Fig. 5b). A few scattered glial cells were probe-labeled with light density in white matter (Fig. 5c).
Fig. 5In the spinal cord of adult rats, GDNF mRNA-positive product were markedly detected in neurons from the lamina III, IV and V and the central canal (a). Medium-sized neurons were seen in the field of intermediomedial nucleus and Nucleus dorsalis. In the ventral horn, GDNF mRNA-positive large neurons were also observed in lamina IX. Under higher magnification (200×), positive products with blue and purple staining were localized in the cytoplasm (b, arrows), also in white matter scattered glial cells were probe-labeled with light density (c, arrows)
Western blotting
A single positive band was observed at a molecular weight of about 34,000, which corresponds to the molecular weight of GDNF.
In the rostral stump, the level of GDNF protein increased at 3 dpo, when compared with the control group (P < 0.05), then decreased quickly to its lowest level at 7 dpo (P < 0.05) which was less than that of the control level, and then began to increase again at 14 dpo. However, GDNF protein was higher than what was seen in the control group at 21 dpo (Figs. 6a, 7). In the caudal stump, GDNF protein immediately decreased at 3 dpo, reached its lowest level at 7 dpo (P < 0.05), then increased from 14 to 21 dpo, but it did not go back to the control level (Figs. 6b, 7).
Fig. 6In the rostral stump, GDNF protein level increased at 3 dpo (P < 0.05), then reached its lowest point at 7 dpo which was below the control group (P < 0.05), then began to recover at 14 dpo, but it was less than that of normal level. This was followed by a continuous increase higher than the control group at 21 dpo (a). In the caudal stump, GDNF protein decreased at 3 dpo, reached its lowest point at 7 dpo (P < 0.05), then started to increase at 14 dpo, but it was lower than the normal level at 21 dpo (b)Fig. 7GDNF levels were significantly different from each other (P < 0.05), except for the comparison between the 21 dpo and normal group, as well as the 21 and 3 dpo group in the rostral stump. The groups were significantly different from each other (P < 0.05), except for the comparison between the 3 and 14 dpo group in the caudal stump
BDA tracing
BDA-positive axons which stained as brown silkiness were detected in rostral cord sections of the injury site (Fig. 8a), but not in the field of scar tissue (Fig. 8b), and in the caudal segment near the injury site (Fig. 8c).
Fig. 8BDA-positive axons staining as brown silkiness were detected in rostral cord sections near the injury site (a), but not in the field of scar tissue (b), and caudal cord sections near the injury site (c)
Discussion
In the present study, GDNF immunoreactive products in neurons from the gray matter and in astrocytes from the white matter of the spinal cord were observed in the sham-operation group. This indicates that GDNF could be involved in either the physiological function or the maintenance of survival for all kinds of neurons and astrocytes. It has been shown that GDNF mRNAs widely distributed in a variety of neuronal and non-neuronal tissues of embryos and adults [4, 23–25]. In addition, GDNF mRNA positive signals were found in the ventral horns, the field of nucleus dorsalis, and the dorsal horns in the spinal cords of adult rats [2, 16, 27] and in the dorsal root ganglia in newborn rats [23]. Therefore, it raised the possibility that GDNF was synthesized by cells of the spinal cord and the GDNF expression may be changed after lesion, which implies that GDNF plays a potential role in both physiological and pathological conditions.
Following cord transection, rats exhibited spinal cord dysfunction throughout the experimental period. There was an absence of SEP in surface recordings, indicating a loss of sensory function in the hindlimbs following transection injury. Comparatively, locomotion function recovery was observed from 7 to 21 dpo, which was parallel to the improvement in BBB score. It showed that locomotor functional plasticity in the hindlimbs of rats did occur following SCI. As corticospinal tract regeneration was not seen in the caudal part near the injury site and scar tissues, it appears impossible to reestablish connectivity between the areas rostral and caudal of the injury site. The possible reason for locomotor function improvement may be related to the modulation of spinal circuit activity from endogenous neurotrophins. In the present study, BBB scores in the GDNF-antibody treated rats was inferior to that of the group not receiving the antibody treatment, indicating intrinsic GDNF may be available for improving locomotor function recovery to some extent in the hindlimbs of rats [15, 28]. It has been reported that GDNF can promote the repair or survival of spinal motoneurons [4, 7, 29–32]. Several other studies have shown that the administration of GDNF could promote axonal regeneration, and enhance locomotion functional recovery [2, 15, 30, 33–36].
GDNF immunoreactivity products in neurons from the ventral horn of the rostral stump were strongly stained at 3 and 7 dpo, and in the caudal stump at 14 dpo, suggesting that the GDNF expression in neurons was up-regulated following injury. This may be related to the locomotion functional recovery of hindlimbs. Glazner et al. [37] showed that strong labeling for GDNFR-α and c-ret mRNA was observed in large neurons from the ventral horn. Our results also supported the mRNA expression for GDNF in neurons of the ventral horn, as well as astrocytes in white matter using in situ hybridization. RET expression was unregulated following peripheral axotomy in alpha-motoneurons [38], and topical application of GDNF 30 min after SCI significantly improved motor function and reduced blood–spinal cord barrier (BSCB) breakdown, edema formation, and cell injury at 5 h has been reported [39]. Therefore, GDNF is available to assist in locomotion functional plasticity by exerting behavioral and anatomic neuroprotection following SCI.
In the present study, the expression patterns of endogenous GDNF indicated its possible role in maintaining motorneuron survival because GDNF mRNA-positive products were detected in neurons from the ventral horn by in situ hybridization. Of course, GDNF retrograde axonal transport from a target tissue to neuronal cell bodies [40, 41] is not absolutely excluded. It has been reported that GDNF produced by skeletal muscle is taken up at the nerve terminals and retrogradely transported to motoneurons of the ventral horn by axons [30].
Comparing the results of immunostaining, the GDNF level varied throughout the spinal cord at 3 dpo. In the caudal stump, GDNF showed a significant decrease. This was probably due to the transection injury that triggers a process destructive to ascending and descending tracts conduction and that extends tissue loss. Differently, a rapid up-regulation of GDNF was observed in the rostral stump. This suggested that GDNF had accumulated around the transection site due to interruption of its transport within the axons. Satake et al. [42] reported GDNF transcription began to increase within 30 min after injury and peaked within 3 h after spinal cord contusion injury. Interestingly, the level of GDNF protein reached its lowest point at 7 dpo in both stumps, probably due to the consequences of cell death or apoptosis and/or interruption of the spinal cord pathways. Subsequently, GDNF protein levels gradually started to recover and surpassed the control level in the rostral stumps at 21 dpo, which may be explained by (1) GDNF transport within axons was strengthened, (2) inflammation leads to the increase of GDNF synthesis [36], and (3) induction of de novo synthesis of GDNF by neurons and/or non-neuronal cells.
Noticeably, the GDNF positive cells in the white matter were labeled by GFAP simultaneously, which identified the labeled cells as astrocytes. Indeed, an association between reactive astrocytes and regenerating nerve fibers in the white matter of the central nervous system in vivo has been reported [43, 44]. Also, increased GFR-1 in astrocytes of degenerating white matter in adult rat spinal cords after mechanical injury has been shown [2]. These results indicated that GDNF expression in astrocytes may be involved in neuroplasticity following spinal cord transection injury.
The present study provides new evidence to understand the role of intrinsic GDNF in the rostral and caudal stumps of injured spinal cords. It suggests that GDNF plays an essential role in the neuroplasticity of the local circuitry, especially in the caudal stump of the transected spinal cords.

rats
gdnf
antibody
western blot
spinal cord transection
immunohistochemistry
in situ hybridization


Bilateral pedicle stress fracture in the lumbar spine of a sedentary office worker

A case of bilateral pedicle fracture in the lumbar spine of a sedentary office worker is being presented. No such case has been reported in the literature previously. Bilateral pedicle fracture is a rare entity. Few cases have been reported in literature. All the reported cases had some underlying causative factors like degenerative spine disease, previous spinal surgery or stress-related activities, e.g. athletes. Our case is a 36-year-old sedentary office worker with none of the factors mentioned. We present a case of a 36-year-old sedentary worker with long-standing low backache. There were no root tension signs. Plain radiographs were inconclusive. The patient had a CT scan. The CT scan revealed long-standing defects in the pedicles of L2 vertebra with pseudoarthrosis. Infiltration with anaesthetic relieved the symptoms. Our patient was managed conservatively with spine rehabilitation physiotherapy program. Pedicle fracture can develop due to abnormal stresses in the pedicle either because of previous spinal surgery or spondylitic changes in the spine. Bilateral pedicle fracture in the absence of these conditions is extremely rare.
Introduction
Isolated pedicle fracture in the spine is uncommon. It has been reported in association with previous spine surgery, or in highly active athletic individuals. We report a case of bilateral pedicle fracture in a patient with chronic backache without the history of trauma or any of the above-mentioned conditions. The patient was a sedentary worker with minimal physical demands.
Case report
We present a case of a 36-year-old female with a history of chronic backache. Her symptoms were of mild to moderate in intensity. Discomfort was mainly related to activity but she was performing her normal duties as an office manager without significant problems. On examination there were no neurological deficit or root tension signs. Plain radiographs were inconclusive. Clinical examination revealed localized deep tenderness at L2; therefore, a CT scan was performed. The CT scan showed bilateral long-standing defects through the pedicles of 2nd lumbar vertebra with pseudoarthrosis and sclerosis (Fig. 1a, b). Infiltration with local anaesthetic, under the X-ray guidance, in the involved area relieved the symptoms. Surgical management was discussed with the patient, which was declined. The patient underwent spine rehabilitation physiotherapy program, which resulted in the improvement of the symptoms.
Fig. 1a CT scan of the vertebra showing bilateral pedicle fracture. b CT scan showing bilateral pedicle fracture with pseudoarthrosis
Discussion
Bilateral pedicle stress fracture in the spine is a rare finding. Traughber and Havlina [10] reported the first case of a bilateral pedicle stress fracture. Cyron et al. [2] demonstrated that the parsintereticularis is thought to be the weakest site in the neural arch, followed by the pedicle. In cases with established unilateral stress fracture, there is a redistribution of forces in the neural arch, which leads to compensatory sclerosis of contralateral bony structures. A compensatory hypertrophy of the contralateral pedicle has been documented [1, 8]. This entity was first described by Wilkinson and Hall [11] in 1974. These authors reported on seven patients whose radiographs were suspicious for a neoplastic process occurring in a pedicle (e.g. osteoid osteoma or osteoblastoma). Further investigation revealed unilateral spondylolysis with sclerosis of the contralateral posterior elements. The incidence of bilateral pedicle stress fracture is unknown in an otherwise normal spine. Gunzburg and Fraser [3] introduced the term pediculolysis in 1991 in a patient with multilevel facet joint osteoarthritis and minimal spondylolisthesis at L4–L5. Pedicle fracture has been reported in cases following posterolateral instrumented spinal fusion [4, 5, 7]. Bilateral pedicle stress fractures or pediculolysis have been documented in an athlete of high physical demand [6]. Stanley and Smith [9] reported a case of pedicle fracture following laminectomy. In our patient the fractures appear to be old with evidence of sclerosis at the fracture margins along with pseudoarthrosis. This type of bilateral pedicle stress fracture has not been documented in a normal spine of a sedentary office worker without a history of major trauma or surgery.
Conclusion
Pedicle fracture is uncommon but has been reported in association with degenerative spondylolisthesis, unilateral spondylolysis, following spinal surgery but not in an otherwise normal spine. Pedicle stress fracture may show either as a recent fracture or may have signs of established pseudoarthrosis. So far there is no case of bilateral pedicle stress fracture in an otherwise normal spine.

bilateral
stress fracture
pedicle fracture
backache


Pediatric DXA: clinical applications

Normal bone mineral accrual requires adequate dietary intake of calcium, vitamin D and other nutrients; hepatic and renal activation of vitamin D; normal hormone levels (thyroid, parathyroid, reproductive and growth hormones); and neuromuscular functioning with sufficient stress upon the skeleton to induce bone deposition. The presence of genetic or acquired diseases and the therapies that are used to treat them can also impact bone health. Since the introduction of clinical DXA in pediatrics in the early 1990s, there has been considerable investigation into the causes of low bone mineral density (BMD) in children. Pediatricians have also become aware of the role adequate bone mass accrual in childhood has in preventing osteoporotic fractures in late adulthood. Additionally, the availability of medications to improve BMD has increased with the development of bisphosphonates. These factors have led to the increased utilization of DXA in pediatrics. This review summarizes much of the previous research regarding BMD in children and is meant to assist radiologists and clinicians with DXA utilization and interpretation.
Introduction
Peak bone mineral accrual occurs during early puberty and peak bone mass is achieved in young adulthood. Low BMD can result from a wide variety of childhood diseases or be due to the effects of their treatment. Increasingly, pediatric specialists are requesting evaluation of BMD and radiologists are expected to be knowledgeable in the technique, interpretation and clinical applications of DXA. A review of the technical and interpretive aspects of DXA has been recently presented [1]. The clinical uses of DXA in pediatrics are exceedingly broad. This paper reviews the clinical data regarding pediatric DXA and can be used to assist radiologists and clinicians in DXA utilization and interpretation.
Gastrointestinal disorders
Gastrointestinal diseases may impact bone health in several ways. Poor calcium intake, as in patients with a milk allergy, and reduced calcium absorption, as in patients with untreated celiac disease, result in low BMD. Early correction of the underlying deficiency allows normal bone mineralization to occur. In addition to poor calcium absorption, inflammatory bowel disease likely impacts bone health through other factors including chronic diarrhea, decreased lean tissue mass, reduced physical activity, increased inflammatory cytokines, and CS therapy. As in all chronic conditions, correction of short stature and delayed maturation will allow better identification of those patients with abnormal DXA findings who will have significant bone mineral deficits.
Milk allergy
Henderson and Hayes [2] reported a positive correlation between LS and hip BMD and calcium intake in children with significant milk allergy. They found a 5–8% increase in LS Z-scores and a 9–13% increase in hip Z-scores in patients with daily calcium intake above the recommended dietary allowances. These findings were confirmed in children with reduced dairy intake due to a variety of other causes [3, 4]. There was a positive correlation between BMD and calcium intake with normal values in the subgroup with normal dietary calcium intake and decreasing BMD as the daily intake of calcium decreased. LS BMD was maintained, even with severe dairy restrictions when calcium intake was maintained through diet or supplementation.
Inflammatory bowel disease
Boot et al. [5] found decreased LS and TBBMD in children with Crohn disease or ulcerative colitis. The decreases were statistically significant even when they accounted for the delayed bone age in their patients. Similarly, Ahmed et al. [6] found reduced LS and TBBMD Z-scores in nearly 70% of children with Crohn disease and, to a lesser extent, ulcerative colitis, when compared to healthy age-matched controls. However, when height was taken into account, only 22% of their patients had low BMD. Burnham et al. [7] found decreased height, lean tissue mass and TBBMC in children with Crohn disease when compared to normal controls. However, with correction for height, age, race, Tanner stage and lean tissue mass, no statistically significant differences were found between the study and control groups. The authors also found no correlation between CS treatment and height-corrected BMC. Similar findings were reported by Walther et al. [8]. Thus, in patients with inflammatory bowel disease, the apparent low LS BMD and TBBMC is largely a result of short stature, delayed maturation and decreased lean tissue mass.
Celiac disease
Kalayci et al. [9] found lower LS BMD in untreated celiac disease patients compared to treated patients. They found that nearly all children with celiac disease whose treatment began before the age of 4 years reached normal BMD compared to only 50% of children whose treatment began after the age of 4 years. They also found that all patients who initially presented with gastrointestinal symptoms had normal BMD after 1 year of a gluten-free diet. In prepubertal children with celiac disease, Barera et al. [10], Tau et al. [11] and Szathmari et al. [12] found an increase in height, weight, BMC and BMD after 1.5 years of a gluten-free diet when compared to baseline measurements. These reports indicate that in patients with celiac disease, normalization of DXA findings can be expected with early diagnosis and treatment.
Liver disease
The effects of liver dysfunction on bone health are complex and may involve vitamin malabsorption, failure of vitamin D activation, calcium malabsorption, bile salt deficiency and chronic malnutrition. Argao et al. [13] evaluated children with a variety of chronic cholestatic liver diseases and found distal radial BMC to fall quickly after birth and in infancy. The values remained low throughout childhood and reflected the severity of the underlying hepatic dysfunction. In a study of prepubertal children with Alagille syndrome, Olsen et al. [14] found reduced height, weight and TBBMC compared to healthy controls matched for age, gender, ethnicity and physical maturity. Malabsorption of fat-soluble vitamins, particularly vitamin D, significantly reduces LS and TBBMC. D’Antiga et al. [15] found normal LS BMC and BMD in patients with childhood liver failure who were at least 1 year out from orthotopic liver transplantation. Return to normal values appeared to be unaffected by the severity of bone disease or cholestasis prior to transplantation. Thus, liver dysfunction with disordered vitamin D metabolism results in early decreased bone mineral accrual. With successful liver transplantation, normalization of DXA findings can be expected.
Renal diseases
Chronic kidney disease results in abnormal bone metabolism via disturbances in calcium and phosphate handling, altered vitamin D and parathyroid hormone levels and function, and altered renal clearance of other metabolites. Additional factors that affect the BMD in these patients include malnutrition, metabolic acidosis, anemia and growth hormone abnormalities resulting in growth retardation. As with many chronic medical conditions, short stature will greatly affect BMD and needs to be accounted for in DXA interpretation.
Bakr [16] found reduced LS BMD in 60% of pre- and postdialysis patients. The degree of osteopenia correlated with biochemical markers of secondary hyperparathyroidism. However, the DXA BMD values were not corrected for height. Similar results were reported by Pluskiewicz et al. [17] following a 2-year longitudinal study of adolescents with chronic kidney disease. They found decreased or stable LS BMD (uncorrected for height) during a time when an increase would be expected. The most severe decreases in BMD were found in patients who were treated with CS. In contrast, Ahmed et al. [18] found normal TBBMD and TBBMC as well as normal LS BMD in children with chronic kidney disease when the DXA values were corrected for height. Similar findings were reported in children before transplantation [19]. Boot et al. [20] reported normal LS and TBBMD in patients with kidney disease of relatively short duration and in the setting of adequate vitamin D replacement. Two-year follow-up of these patients demonstrated continued maintenance of normal BMD [21]. Several authors found that the height-corrected LS BMD was reduced for up to 2 years after transplantation; this was felt to be due to high-dose CS treatment required to suppress transplant rejection. However, by 3 years after transplantation, LS BMD had recovered to normal values [18, 22–24].
Idiopathic hypercalciuria results in excessive urinary excretion of calcium and has increasingly been identified as a cause of decreased BMD in children [25–28]. The degree of BMD reduction does not appear to correlate with the presence of urolithiasis or urinary uric acid concentration. Studies of children and their mothers with idiopathic hypercalciuria indicate that low LS and hip BMD persist into adulthood [25, 26].
In summary, the various metabolic disturbances resulting from altered renal physiology may compromise normal bone mineralization and remodeling, but children with chronic kidney disease often maintain normal BMD if calcium and vitamin D metabolism are normal. CS treatment, either before transplantation or when used to suppress transplant rejection, appears to contribute to low BMD but short-term follow-up indicates eventual normalization of LS BMD. Regardless of the nature of renal disease, correcting for height is essential for accurate interpretation of DXA results in these patients.
Endocrinological diseases
Skeletal development is significantly influenced by multiple hormone systems that are essential to normal bone mineral accrual. Both insufficient and excessive hormone levels can have deleterious effects, resulting in inadequate bone formation or disproportionate bone resorption. Pubertal hormones play a critical role in bone mass acquisition with major differences occurring based on sex and skeletal site [29]. Patients with early or delayed puberty will show early or delayed peak bone mass accrual, respectively [30]. Treatment of precocious puberty includes gonadotropin-releasing hormone analogs that could theoretically interfere with normal bone mineral acquisition. Short-term treatment does not interfere with normal BMD increases in these patients [31].
Disorders of growth hormone, insulin and glucocorticoids
Accurate DXA evaluation of children with growth hormone deficiency or idiopathic short stature must account for their smaller bones [1]. Children with growth hormone deficiency will present with low areal [32] and volumetric LS [33] BMD when first diagnosed. Children with idiopathic short stature have also been found to have low areal LS and hip BMD prior to therapy [34]. With sustained growth hormone replacement these values have been reported to remain stable [33] or normalize rapidly [34]. Children with growth hormone deficiency have a robust response to replacement therapy with increases in height, lean tissue mass and TBBMC [33]. Increases in BMD occur even after peak height is achieved, and this indicates the need to continue hormone therapy until peak BMD is achieved [35, 36].
Type 1 diabetes mellitus is a known risk factor for osteoporosis, but the underlying mechanisms are not fully determined. Adolescent patients have lower bone mass and bone size despite normal growth and maturation [37]. Studies suggest that children and adolescents with diabetes have reduced bone formation and increased bone resorption resulting in low bone mass and BMD [38, 39]. Hemoglobin A1C level, a measure of glycemic control, negatively correlates with BMD [40] and TBBMC [41].
The negative effects of chronic high-dose CS therapy on bone have been well documented. CS replacement therapy, as in congenital adrenal hyperplasia, has not been found to result in reduced BMD [42, 43]. However, young women with congenital adrenal hyperplasia are at risk of reduced BMD if there is biochemical evidence of androgen suppression due to CS therapy [44]. Patients with either childhood-onset or adult-onset Cushing disease often have severe reductions in BMD due to increased osteoclastic activity and bone resorption with normal or reduced bone deposition [45]. Additionally, concomitant reductions in sex and growth hormones contribute to their low BMD. Adolescents appear to be particularly vulnerable to reduced trabecular bone density as measured at the LS. BMD deficits and biochemical measures of bone turnover showed marked improvement after 2 years remission from hypercortisolism when compared to baseline values, but low LS BMD persisted in the majority of patients [46].
Disorders of reproductive hormones
Reproductive hormones play an important role in the acquisition of bone mineral; estrogen is known to have a protective effect against the development of osteoporosis in postmenopausal women. The effects of hormonal birth control use on BMD in adolescent girls have been investigated by several researchers. The trend toward lower estrogen levels in oral contraceptives has been associated with decreased BMD in adolescent girls who use oral contraceptives [47, 48]. Progesterone-mediated contraceptives (Depo-Provera) have been shown to inhibit bone mineral acquisition in the LS and hip in adolescent girls [49–51]. The extent of this reduction appears to correlate with the duration of treatment and can be lessened with supplemental estrogen [51]. Partial or full BMD recovery can occur after cessation of treatment and can be facilitated with increased physical activity and adequate calcium and vitamin D intake.
Girls with anorexia nervosa have been found to have decreased BMD at multiple skeletal sites [52–54]. These reductions are thought to be due to a combination of nutritional (decreased calcium and caloric intake), hormonal (decreased estrogen levels, delayed puberty), and mechanical (decreased lean tissue mass) factors. With resumption of normal caloric intake and return of menses, there is biochemical evidence of increased bone turnover; however, BMD levels remain low and Z-scores may continue to fall for at least 1 year [53]. It is not known if full BMD recovery will occur by early adulthood, but adult women who had adolescent-onset anorexia nervosa have more dramatic reductions in BMD than those with onset during adulthood [55, 56]. Estrogen replacement with oral contraceptives and vigorous exercise can protect against BMD loss in anorexia nervosa [57].
Untreated congenital hypothyroidism can result in markedly reduced BMD. Children with adequate long-term thyroid replacement therapy show normal LS and hip BMD and BMC [58, 59] and normal findings continue into young adulthood [60]. Later in childhood, hypothyroidism is usually caused by Hashimoto thyroiditis. LS and distal radial BMD are expected to be normal at diagnosis and to be maintained with thyroid hormone therapy, and normal peak bone mass is to be expected in these patients [61]. Hyperthyroidism in Graves disease markedly decreases LS and hip BMD, but these normalize within 1 year following thyroid ablation and hormone therapy [62].
In summary, hormonal imbalances from any of a large number of endocrine disorders are associated with abnormal DXA findings and reduced BMD. With hormone replacement therapy, improved bone mineral accrual is to be expected in most of the endocrine deficiencies. However, with anorexia nervosa, especially with onset in adolescence, there appears to be a sustained reduction in BMD even after resumption of normal caloric intake and menses.
Respiratory diseases
Cystic fibrosis
In cystic fibrosis (CF), low BMD may be caused by reduced gastrointestinal absorption of calcium and vitamin D, reduced testosterone levels, chronic hypoxia, chronic CS use, and reduced lean tissue mass. Because of the multifactorial nature of reduced BMD in CF, BMD Z-scores often reflect the severity of illness. Clinically stable children with mild CF have been shown to have normal BMD [63–65]. However, adolescents and adults with CF have accelerated bone loss over time reflecting disease progression [66, 67]. Henderson and Madsen [68] and Buntain et al. [66] found progressively decreasing BMD Z-scores with increasing age in CF patients. LS, hip and TBBMD in the prepubertal group were normal. TBBMD was reduced in adolescents; however, all other sites remained normal. In adult patients, BMD was reduced at all sites. In a follow-up study, Haworth et al. [69] reported that patients older than 25 years showed accelerated LS and hip BMD losses. Bhudhikanok et al. [70] found that LS, hip, and TBBMD were low at baseline and documented further bone loss at follow-up. CS use was identified as the most modifiable factor accounting for this bone loss and the authors suggested that bisphosphonates may be beneficial in these patients. The benefits of bisphosphonates in CF were confirmed by Aris et al. [71, 72] and Haworth et al. [73] who found increases in LS and hip BMD following treatment with intravenous or oral bisphosphonates, respectively.
Asthma
Asthma may inhibit normal bone metabolism due to chronic hypoxia and decreased physical activity. Additionally, asthmatic patients are treated with both oral and inhaled CS to reduce airway inflammation. Patients treated with low or moderate levels of inhaled CS have been shown to have normal BMD Z-scores when compared to CS-naive asthmatic controls but reduced BMD Z-scores when compared to normal controls when height is not taken into account [74–77]. Allen et al. [78] and Harris et al. [79] found significant reductions in BMD in asthmatic children receiving high doses of inhaled CS.
Hematological diseases
Anemia and hemophilia
Chronic anemias can affect bone structure and density through bone marrow hyperplasia (resulting in expansion of the medullary space, trabecular coarsening and cortical thinning), and vasoocclusion (resulting in medullary and diaphyseal infarction). Additional factors for reduced BMD include reduced lean tissue mass, decreased physical activity and hypogonadism, and in the case of thalassemia, endocrine dysfunction. Brinker et al. [80] found low BMD in children with sickle cell anemia. These findings confirmed those of Soliman et al. [81] who reported low LS BMD in prepubertal children with sickle cell anemia. Lal et al. [82] and Buison et al. [83] noted markedly reduced BMD and BMC, which correlated with disease severity in these children. These changes were found to persist into adulthood [84]. Additionally, low calcium intake and low vitamin D levels may contribute to decreased BMD in these patients. Vogiatzi et al. [85] and Benigno et al. [86] evaluated children with thalassemia major who were fully treated with transfusion and chelation therapy and found reduced BMD at diagnosis and showed further reductions at follow-up.
Patients with hemophilia are at risk for osteoporosis due to reduced levels of physical activity and sports, particularly those involving running, jumping and axial loading of the skeleton. The severity of hemophilic arthropathy can be quantitated using a clinically derived joint score, and this has been found to correlate with BMD [87]. Low BMD is independent of height and weight but correlates with disease severity and extent. These changes can be expected to persist into adulthood [88].
Oncological diseases
Acute lymphocytic leukemia
ALL is the most common childhood malignancy, but because of its excellent prognosis, most children with ALL survive into adulthood. Arikoski et al. [89] found reduced BMD in ALL survivors up to 20 years after treatment. Of their 29 patients, 20 had received cranial irradiation and four males had received testicular irradiation. The use of high-dose methotrexate and cranial irradiation were found to be significantly correlated with low BMD. Differing results were presented by Brennan et al. [90] who also reported significantly reduced LS, hip and distal radial BMD in long-term survivors of ALL but found no statistically significant correlation of BMD with cranial irradiation, growth hormone status or height Z-scores. They suggested that the low BMD found in their patients was related to the effects of chemotherapy rather than cranial irradiation. Van der Sluis et al. [91] found normal LS and TBBMD a mean of 10 years after ALL treatment that included high-dose methotrexate and CS but not cranial irradiation. Their patients had all been prepubertal at the time of initial diagnosis and none had signs of significant gonadal or growth hormone dysfunction at the time of follow-up. The authors concluded that the deleterious effects of ALL and its treatment in childhood on BMD may be due to cranial irradiation but, in its absence, BMD will normalize by early adulthood. This conclusion is supported by other researchers [92, 93]. Jarfelt et al. [94] examined bone turnover and growth hormone status with respect to physical activity levels and BMD in adult survivors of childhood ALL. The patients were all prepubertal at the end of treatment and had been treated with high doses of CS and methotrexate. No patient had evidence for gonadal or endocrine dysfunction. The BMD values were normal for the group. Only the level of physical fitness at follow-up correlated positively with BMD. The authors stressed the importance of physical activity in restoring and maintaining normal BMD in survivors of childhood ALL.
In summary, children with ALL will have reduced BMD during treatment and shortly thereafter. The recuperative capacity in young children is high and normal BMD should be expected even after high-dose methotrexate and CS therapy. The role of adequate physical activity is being increasingly stressed as an important factor in normal BMD recovery. Children who have survived ALL may be at risk of persistently low BMD in adulthood if they have confounding factors such as gonadal dysfunction or if the course of their disease coincided with the period of expected rapid accrual of bone mineral that normally occurs during puberty.
Other malignancies
There are only limited data regarding the effects of other childhood malignancies on BMD. Aisenberg et al. [95] and Vassilopoulou-Sellin et al. [96] found reduced femoral neck and TBBMD in young adult survivors of various childhood cancers. Gonadal dysfunction due to pelvic or cranial irradiation was the factor most strongly correlated with reduced BMD. CS treatment did not correlate with low BMD. Nysom et al. [97] reported normal size-adjusted TBBMC in adult survivors of childhood lymphoma and found no relationship between TBBMC and cumulative methotrexate or CS doses. Kelly et al. [98] evaluated adult survivors of various pediatric solid tumors and found reduced BMD in at least one site in half of their patients. Only the total number of chemotherapeutic agents correlated with reduced BMD. Five of six extremities involved with a bone sarcoma showed reduced BMD. Similar results were found in a group of sarcoma survivors reported by Ruza et al. [99]. Interestingly, they found that those diagnosed prior to puberty had more severe BMD reductions later in life. This may be due to the extensive physical disabilities associated with amputations and limb salvage procedures in these patients. Odame et al. [100] found that patients with childhood brain tumors treated with cranial irradiation had reduced LS and TBBMD and these were correlated with reduced physical activity and poorer quality of life. The authors postulated that the higher cranial radiation doses used for the treatment of brain tumors had a profound effect on long-term BMD compared to the relatively lower doses used in the treatment of ALL.
In summary, there are multiple factors that affect short- and long-term BMD in these patients/survivors. These include pubertal status at diagnosis, type of malignancy, local (sarcomas, central nervous system tumors) versus systemic (leukemia) disease, initial (malnutrition, immobilization) versus prolonged (amputation) disease-related disability, types of chemotherapy, and radiation ports and dosages that may cause gonadal or growth hormonal dysfunction.
Neurological diseases
Cerebral palsy
Because of decreased muscle development and ambulation, children with cerebral palsy are at increased risk of osteoporosis-associated fractures. Frequently, assessment of the LS and hips with DXA is impossible due to difficulties in positioning patients with contraction deformities, muscular spasm-induced motion artifacts or orthopedic hardware-related artifacts. Because of these limitations, Harcke et al. [101] suggested scanning the distal femur with these patients positioned on their side. Distal femoral BMD was found to be highly correlated with hip BMD and the technique yielded highly reproducible BMD data [102].
Henderson et al. [103] demonstrated reduced LS and hip BMD in children with cerebral palsy. Measures of nutritional and ambulatory status were found to be the best predictors of BMD. In children with moderate to severe disease, Henderson et al. [104] found distal femur and LS BMD to be markedly reduced and both closely correlated with disease severity. Distal femoral BMD was negatively correlated with age, indicating progressive bone mineral deficits as these children grew older. The lack of a strong correlation between LS BMD and fracture risk in the lower extremities emphasizes the need for direct assessment of the distal femur with DXA in these patients. In a prospective longitudinal study of patients with cerebral palsy, Henderson et al. [105] found that low initial LS and distal femur BMD Z-scores were associated with poor growth, nutritional status and motor function, and these contributed to reduced bone mineralization at follow-up. Treatment with bisphosphonates for 1 year resulted in a substantial increase in distal femoral BMD in children with quadriplegic cerebral palsy [106]. The improvement was sustained for at least 6 months after the last treatment. However, Bachrach et al. [107] found that LS BMD returned to baseline values 2 years after bisphosphonate therapy was terminated. Importantly, despite the lack of sustained BMD improvement, no patient had a fracture during the treatment or follow-up periods.
In summary, children with CP show decreased bone mineral status that reflects the duration and severity of their disease. Distal femoral measurements better reflect the bone status at the sites more at risk of fracture. Bisphosphonate therapy increases BMD during treatment and it appears to have sustained benefits with reduced fracture rates even after treatment is terminated.
Meningomyelocele
Quan et al. [108] found reduced forearm BMD in children with meningomyelocele. Patients with a history of fracture had substantially lower forearm BMD than those without a fracture history. The reduced upper extremity BMD in these patients with preserved upper extremity function may indicate that both systemic and local factors affect bone mineralization. Hypercalciuria in nonambulatory patients with meningomyelocele may be such a factor. Quan et al. [109] later evaluated the effect of thiazide treatment for hypercalciuria in these patients and found no change after 1 year in forearm BMD when compared to placebo-treated controls. Valtonen et al. [110] found normal forearm and LS BMD in adult patients with meningomyelocele, but reduced hip BMD in one-third of the patients. There was a trend for low hip BMD in the nonambulatory compared to ambulatory patients but not for the LS. This dissociation of LS and hip BMD values was thought to be due to relatively preserved axial loading on the LS in upright patients with meningomyelocele. The normalization of forearm BMD in these adult patients was attributed to increased upper extremity muscular stress with the use of crutches and manual wheelchairs.
Connective tissue diseases
Juvenile rheumatoid arthritis and systemic lupus erythematosus
Rheumatoid diseases have long been known to affect bone health negatively. Disease extent, severity, subsequent disability and CS therapy negatively impact bone mineralization. Pepmueller et al. [111] and Pereira et al. [112] found decreased regional, LS, and TBBMD in patients with JRA. The decrease was more severe in children with longer disease duration and was similar for oligoarthritis and polyarthritis. Lien et al. [113] found decreased TBBMC and TBBMD in patients with juvenile idiopathic arthritis. Disease duration and severity correlated with TBBM but not CS therapy. Henderson et al. [114] found no statistically significant difference in TBBMD in prepubertal children with mild-to-moderate JRA and no history of CS treatment when compared to normal controls. When a similar study was performed on CS-naive older girls with mild-to-moderate JRA, Henderson et al. [115] found decreased TBBMC. As with other chronic diseases of childhood, it appears that persistent disease activity through puberty results in decreased BMC. Mul et al. [116] found marked reductions in LS BMD and BMC in children with rheumatic diseases treated for at least 1 year with high-dose CS. Bianchi et al. [117] found that long-term methotrexate for JRA did not result in reduced LS or TBBMD.
In summary, prepubertal children with JRA of mild or moderate severity without a history of CS treatment will have TBBMD similar to healthy children. With increasing disease severity and duration, especially through puberty, TBBMC will decrease when compared to normal children.
These changes are in contrast to the normal DXA findings reported in patients with juvenile systemic lupus erythematosus [118, 119]. Unlike patients with JRA, these patients typically do not have bone and joint involvement and thus are more likely to have preserved BMD. However, factors that may negatively impact bone health in these patients include immobility, limited exposure to sunlight, and CS therapy.
Musculoskeletal diseases
For normal mineralization to occur, adequate muscle-induced mechanical stresses are required to induce bony remodeling. Insufficient muscle mass and activity result in poor bone accrual and low BMD. Additionally, therapy of primary muscle disorders with CS also inhibits normal bone mineralization.
Duchenne muscular dystrophy and dermatomyositis
In a longitudinal study of boys with Duchenne muscular dystrophy, Larson and Henderson [120] found that LS and hip BMD correlated with functional mobility level with ambulatory boys having higher LS BMD than nonambulatory boys. Hip BMD was decreased before loss of ambulation and showed progressive reduction over time. Fractures occurred in nearly half of patients, most typically involving the lower extremity and frequently resulting in loss of ambulation. Bianchi et al. [121] found decreased LS and TBBMD in ambulatory boys with Duchenne muscular dystrophy when compared to healthy boys. LS BMD was more severely reduced in the subset of Duchenne muscular dystrophy patients treated with CS. The authors noted greater lower extremity than upper extremity bone mineral loss using regional DXA analysis, especially in the CS-treated patients, and they attributed this to reduced mechanical stress on the lower extremities. Hawker et al. [122] found an increase in LS and TBBMD after 2 years of bisphosphonate treatment, supplemental calcium, and vitamin D. Improvement in TBBMD was inversely related to age at baseline.
In summary, patients with Duchenne muscular dystrophy have reduced LS BMD, especially in the setting of CS treatment. The low LS and hip BMD worsen with loss of ambulatory status and are frequently associated with fractures of the lower extremities.
Studies of a small number of children with dermatomyositis have found low LS BMD that worsens with ongoing CS therapy [123, 124]. Patients treated with bisphosphonates for osteoporosis-related compression fractures show increases in LS BMD [123].
Osteogenesis imperfecta
Osteogenesis imperfecta includes a spectrum of genetic disorders of collagen synthesis resulting in abnormal skeletal and connective tissues. Reduced BMD and other factors result in fragile bones and multiple fractures. Fracture occurrence and DXA findings in these children vary with subtype with few fractures and normal or near-normal DXA findings in type I osteogenesis imperfecta and more frequent fractures and marked reductions in BMD in types III and IV [125, 126]. (Type II typically results in perinatal demise.) Since as many as 40% of patients with osteogenesis imperfecta will have normal BMD and BMC, a normal DXA study does not preclude this diagnosis or distinguish these cases from non-accidental injuries [125]. In recent years, clinical trials using bisphosphonates in osteogenesis imperfecta have yielded impressive results with multiple studies demonstrating increases in LS, hip and TBBMD and improved quality of life [127–130]. Patients with the lowest baseline bone mass experienced the most significant gains with treatment. Improved mobility, ambulation, muscle force and reduced chronic pain and fatigue lead to improved quality of life, and the stimulus of physical activity is known to be beneficial to bone. Increased bone mass in the skull following bisphosphonate therapy in these patients implies that the drug also has a direct effect on bone accrual irrespective of physical activity [129]. The theoretical concern that this therapy might negatively impact linear growth in these children has not been shown to be true [131].

pediatrics
dxa
bone mineral density
osteoporosis


Why do house-hunting ants recruit in both directions?

To perform tasks, organisms often use multiple procedures. Explaining the breadth of such behavioural repertoires is not always straightforward. During house hunting, colonies of Temnothorax albipennis ants use a range of behaviours to organise their emigrations. In particular, the ants use tandem running to recruit naïve ants to potential nest sites. Initially, they use forward tandem runs (FTRs) in which one leader takes a single follower along the route from the old nest to the new one. Later, they use reverse tandem runs (RTRs) in the opposite direction. Tandem runs are used to teach active ants the route between the nests, so that they can be involved quickly in nest evaluation and subsequent recruitment. When a quorum of decision-makers at the new nest is reached, they switch to carrying nestmates. This is three times faster than tandem running. As a rule, having more FTRs early should thus mean faster emigrations, thereby reducing the colony’s vulnerability. So why do ants use RTRs, which are both slow and late? It would seem quicker and simpler for the ants to use more FTRs (and higher quorums) to have enough knowledgeable ants to do all the carrying. In this study, we present the first testable theoretical explanation for the role of RTRs. We set out to find the theoretically fastest emigration strategy for a set of emigration conditions. We conclude that RTRs can have a positive effect on emigration speed if FTRs are limited. In these cases, low quorums together with lots of reverse tandem running give the fastest emigration.
Introduction
Organisms often employ more than one mechanism to accomplish a task. For instance, animals typically navigate with multiple ‘input channels’. The classic example is homing by the rock dove Columba livia, for which magnetic fields, the sun, landmarks and geophysical processes have all been shown to be used (Wiltschko and Wiltschko 2003).
The range of behaviours or mechanisms organisms employ may be puzzling. At times, an apparent simplicity is observed. Defence strategies against predators are a well-studied example here. Acacia trees (Acacia spp.) either have chemical defences to ward off herbivores or have symbiotic relationships with protective ants (Rehr et al. 1973). Hosts parasitised by Eurasian cuckoos Cuculus canorus famously reject cuckoo eggs, but never reject cuckoo chicks (Davies 2000). This simplicity may be the result of evolutionary lag but, more interestingly, may also be caused by one strategy making another maladaptive by reducing predator abundance (Planqué et al. 2002; Britton et al. 2007).
Conversely, the portfolio of behaviours may be varied and complex. Different mechanisms may complement one another, and true redundancy is often hard to show (Able and Bingman 1987). Indeed, the existence of a suite of mechanisms against a broad ensemble of predators is readily understandable. One exemplar is the vertebrate immune system (Perelson 2002).
Another striking example of a system in which different mechanisms augment and complement one another, but now at a collective level, is house hunting in social insects. This has become one of the model systems to study distributed decision making in animals. When the nest is destroyed, the colony has to decide collectively where to settle next during a time of crisis (Franks et al. 2003a). Individual ants or bees have been shown to combine sophisticated assessments of potential nest sites (Seeley 1977; Seeley and Morse 1978; Mallon and Franks 2000; Franks et al. 2003b) with various recruitment mechanisms to collate information, and thus make collective decisions (Mallon et al. 2001; Pratt et al. 2002; Pratt 2005; Seeley and Visscher 2003, 2004; Visscher 2007).
A typical emigration by a colony of Temnothorax albipennis may be described as follows. When the old nest is destroyed, a fraction of ants goes out scouting to find a new home. Upon finding a nest, the nest is assessed (Mallon and Franks 2000) and ants start recruiting other ants to it with a latency that is inversely proportional to the perceived nest quality (Mallon et al. 2001), using a process called forward tandem running (Möglich et al. 1974). During a forward tandem run, a knowledgeable ant teams up with a naïve ant. The leader slowly progresses towards the new nest, each time waiting for the follower to catch up, thereby teaching her the way (Franks and Richardson 2006). Through this slow recruitment process, information on the location of the new nest spreads, and recruiter numbers increase. Once a nest population reaches a certain quorum threshold, the recruiters switch from slow tandem running to much faster social carrying, and transport the remaining passive ants and brood to the new nests (Pratt et al. 2002; Pratt 2005).
This description has been the basis of several models (Pratt et al. 2002; Pratt et al. 2005; Marshall et al. 2006; Planqué et al. 2006). However, a behaviour commonly employed by these ants is usually not included (but see Pratt et al. 2005) and has never been analysed. After the quorum, recruiter ants are not only engaged in social carrying, but also regularly perform tandem runs from the new back to the old nest. These so-called reverse tandem runs (Möglich 1978) are often more common than forward tandem runs (Mallon et al. 2001; Pratt et al. 2002), but their function is much less well understood.
To maximise fitness, the colony should emigrate as quickly as possible to avoid predation and other hazards. Therefore, during house hunting, a fast build up of recruiters is essential. Why then do ants mix fast carrying with slow reverse tandem running, when they already have forward tandem running at their disposal?
In this paper, the role of reverse tandem running is theoretically investigated. In particular, through the use of mathematical models, we explore under what circumstances reverse tandem running can have a positive influence on emigration speed.
Materials and methods
We present two mathematical models to investigate the possible role of reverse tandem runs in ant colony emigrations. Reverse tandem running does not contribute to the decision-making process of which new nest to choose (Pratt et al. 2002; Franks et al. 2003a). We, therefore, restrict ourselves to emigrations to one new nest only.
We model the emigration as follows (Fig. 1). Only a small fraction of the ants in a colony are actively involved during emigrations (Pratt et al. 2002; Langridge 2006). In this paper, we thus divide the colony’s N ants into fractions FN of active ants A, and (1 − F)N passive ants P. This assumption to divide ants into active and passive is a crucial one, without which the models collapse. We will come back to this in “Discussion”. Numbers of scouts and recruiters are denoted S and R, respectively. The rates at which active ants leave the nest and become scouts, and scouts recruiters, are given by μ and k, respectively. Forward tandem running occurs at a rate λ until the quorum Q is met, after which recruiters carry passive ants and brood at a rate φ. To incorporate reverse tandem runs, we need to model which ants follow these tandem runs. Available data from nest-choice experiments (Mallon et al. 2001) suggest two possibilities: Either the reverse tandem runs are followed by ants that have not found the new nest yet, or by ants that have been carried to the new nest. These two options are not necessarily mutually exclusive: the carried ants could have been scouts. We modelled both these two possible interpretations. Model 1 assumes that reverse tandem runs are followed by uncommitted scouts in the arena, and model 2 assumes they are followed by passive ants that were carried to the new nest.
Fig. 1Diagrams of the two different models for which reverse tandem runs are hypothesised to increase speed. FTR Recruitment through forward tandem running; RTR recruitment through reverse tandem running. The parameters are explained in Table 1
To capture in detail the influence of reverse tandem runs on emigration dynamics, we need to consider the following points:
Both tandem running and social carrying involve a pair of ants from two different classes. Hence, recruitment can only occur if ants of both participating classes are available;Once the quorum has been met, recruiters cannot carry and perform reverse tandem runs simultaneously (we also assume recruiters are not involved in other activities than these two).
The interaction between different classes of ants has been modelled using simple interaction terms. We assume that ants of both classes are well mixed in the part of the arena (or nest) where they meet. With populations of ants of size X and Y meeting, the number of ants that on average meet is then proportional to XY/(X + Y). Importantly, the smallest class limits the interaction rate, as is to be expected.
We thus also have to specify how much post-quorum time recruiters spend on carrying or reverse tandem running (they are assumed not to spend any time on other behaviours). Before the quorum is met, the rate at which active ants at the old nest, A, become recruiters, R, through tandem running is given by λRA/(A + R). Now let f be the fraction of post-quorum time spent on reverse tandem runs, and the remainder 1 − f spent on social carrying. Then the mean number of scouts becoming recruiters through reverse tandem runs is
For carried ants becoming recruiters through reverse tandem running, we have by analogy
Similarly, the mean number of passive ants P that become carried ants is given by (1 − f) φRP/(P + R). Recruiter ants should not perform reverse tandem runs when there are no scouts or carried ants left. Therefore, we replace f by  in Eqs. 1 and 2, where  and min {Cf, f}, respectively. The min operation is for computational reasons only and ensures that  decreases continuously but rapidly to zero as S or C decreases, respectively. It has no influence on the models’ predictions. We drop the bar on  in the rest of the paper.
Forward tandem running only occurs before the quorum is met and carrying and reverse tandem running only after. These are modelled with functions l, c and r, respectively, as follows.
and, setting B to A for model 1, and to P for model 2,
To aid the reader, we state the full equations for both models. The equations for model 1 are given by
with initial conditions (A,S,R,P,C)(0) = (FN − 2ε, ε,ε,(1 − F)N,0). Model 2 is specified by
with the same initial conditions as model 1. Models 1 and 2 only differ in the placement of the term fr(λ,R,Q,C). In both models, the ε in the initial conditions is necessary to avoid singularities in the denominators of the interaction functions l, r and c. We have used ε = 0.01 throughout.
We also explored a number of other models in which some assumptions were relaxed. These are briefly discussed in the final section of this paper.
The main hypothesis we tested on both these models was: Reverse tandem runs speed up the emigration if recruiter numbers failed to increase early in the emigration; this occurs through a combination of the new nest being hard to find and forward tandem running being prohibited. We tested this hypothesis by finding the fastest emigration strategy for given parameter settings and determining whether reverse tandem runs formed part of this optimal strategy. We first fixed the scouting parameter, μ, the fraction of active ants at the old nest, F, and k, the rate at which scouts become recruiters. Then quorum size Q and fraction of post-quorum reverse tandem running time f were varied to optimise emigration speed. Individual runs were performed in Matlab using a standard ode45 solver. Emigrations were termed completed when all passive ants and active ants at the old nest had disappeared. In particular, we set the threshold for emigration completeness at P + A = 0.01. Optimal strategies were found using the Nelder–Mead simplex method (Nelder and Mead 1965). Parameters μ and F were varied on an equidistant 20×20 grid spanning [0.01,0.2] ×[0.05,0.5]. Recruitment latency k was set at either 0.0001 or 0.001. The ranges of these parameters are inspired by experimental estimates in (Pratt et al. 2002; Pratt 2005). Parameter values are summerised in Table 1.
Table 1Values or ranges, where applicable, for the parameters used in models 1 and 2 depicted in Fig. 1ParametersDescriptionValue/rangeNColony size250FFraction of active ants[0.05,0.5]QQuorum thresholdn.a.fFraction of post-quorum reverse tandem running timen.a.μRate at which active ants at old nest become scouts (ant − 1 min − 1)[0.01,0.2]λRate at which ants following tandem runs become recruiters (ant − 1 min − 1)0.1φRate at which passive ants are carried to new nest (ant − 1 min − 1)0.2kRate at which scouts independently become recruiters (ant − 1 min − 1){0.0001,0.001}Parameter choices for λ, φ and N were taken from the ranges in Pratt et al. (2005).
Results
For both models, the optimal emigration strategy included reverse tandem runs for a wide range of parameters, together with low quorum thresholds (Fig. 2). Fixing k whilst varying μ and F, the optimal strategy often contained more reverse than forward tandem runs for a large part of the parameter range (Fig. 2). The fraction of time spent on reverse tandem running f and the quorum threshold Q were negatively correlated. When either the fraction of active ants F decreased or the scouting parameter μ increased, fraction f increased, and the quorum Q decreased. Choosing a higher recruitment latency by decreasing k gave more reverse tandem running and lower quorum thresholds (Fig. 2).
Fig. 2Optimal fractions of post-quorum time spent on reverse tandem runs (top figures), and optimal quorum thresholds (bottom figures) for models 1 (left two columns) and 2 (right two columns) for varying fractions of active ants F and scouting probabilities μ and for two values of recruitment latencies k. Other parameter values used are given in Table 1. See text for simulation details. For both values of k, reverse tandem runs are part of the optimal emigration strategy when scouting probability is high or when fraction of active ants is low. Lowering k enhances the use of reverse tandem running
Note that, although models 1 and 2 broadly give similar predictions, they differ in the amount of post-quorum time spent on reverse tandem runs. In model 1, this reaches a full 100% in model 1, but never so in model 2.
Overall, the models predict that reverse tandem running should be used more than forward tandem running, and the quorum threshold lowered, if the recruitment latency increases by decreasing k (scouting ants wait longer before starting their first recruitment act), in combination with either a decreasing fraction of active ants F, or an increasing scouting parameter μ. For all but very large F, the optimal quorum threshold corresponded to the time when all active ants have left the old nest to go scouting. In the absence of multiple new nests, the decision when to switch from forward tandem running to social carrying is thus best made at the old nest. Recruiters should thus apply the following rule: Continue forward tandem running until there are no ants left to perform them with and then switch to social carrying; if few forward tandem runs have been performed (by the recruiters), combine carrying with reverse tandem runs; otherwise, do not.
The numbers of forward tandem runs, reverse tandem runs, the numbers of carried ants in the new nest and total emigration time were computed for each of the optimal strategies of models 1 and 2 (Fig. 3). We note four points. First, for a large parameter range, there are more reverse tandem runs than forward tandem runs. This is broadly consistent with the experimental data from nest choice experiments in Mallon et al. (2001). Numbers there range between 3 and 17 forward tandems, and between 9 and 25 reverse tandems, and reverse tandems were always performed more often than forward tandems. Second, as a validation of our optimisation method, note that the optimal emigration time varies smoothly under parameter changes, as is to be expected for this type of model. Third, for model 1, despite a clear drop in the post-quorum time spent on reverse tandem runs with increasing F (see Fig. 2, top left), the number of reverse tandem runs in fact varies smoothly. Fourth, the number of carried ants that remained in the nest at the end of the emigration is clearly different between models 1 and 2. In model 1, this number is just (1 − F)N, the number of passive ants in the colony. In model 2, however, over half of the colony may end up being recruiters by drawing recruits from the carried class using reverse tandem runs.
Fig. 3Numbers of forward and reverse tandem runs, number of carried ants and emigration time, computed for each of the optimal strategies for models 1 (top row) and 2 (bottom row), illustrated in Fig. 2. See that figure for details and parameter choices. Here we have only illustrated k = 0.001
Discussion
To maximise their fitness, ants should try to achieve the fastest emigrations to minimise vulnerability (Franks et al. 2003a; Franks et al. 2003b). Therefore, the active ants either have to become scouts, discover a new site and then become recruiters or wait at the old nest until a recruiter leads them to the new nest. Both of these processes may be hampered: When all the active ants go out scouting, recruiter numbers slowly increase if the new nest is hard to find or if those few cannot find any active ant back at the old nest to tandem run with. In terms of the models, this could occur if scouts slowly become recruiters (low value for k), in combination with either a small class of active ants at the old nest (F is small) or all active ants having gone scouting (high value for scouting rate μ). Under either or both of these circumstances, the model predicts that ants should not waste time trying to recruit by forward tandem runs but should do the next best thing and use a low quorum threshold to quickly switch to carrying. The recruiters should then invest a fraction of their time to recruit scouts or carried ants using reverse tandem runs, thus boosting the recruiter population and speeding up the emigration.
Conversely, the model also predicts that reverse tandem runs should not be used if either the new nest is easy to find (recruiter numbers then build quickly anyway), or when there are many ants to follow a forward tandem run.
These predictions fit quite well with previous experimental work. Ants have been shown to leave their intact old nest if the new nest is sufficiently better, but have lower standards when their nest is destroyed (Dornhaus et al. 2004). In these experiments, reverse tandem runs were mainly observed when the old nest was destroyed, combined with few forward tandem runs. The model offers a simple explanation for this: The greater panic might have caused fewer scouts to remain at the old nest, thereby obstructing early recruitment.
Whilst investigating speed–accuracy trade-offs, Franks et al. (2003a) found lower quorums under harsh than under mild conditions. This again fits with the models. On the other hand, the models also predict higher numbers of reverse tandem runs. In the experiments, this difference in reverse tandem running activity between mild and harsher conditions was found to be non-significant (Franks et al. 2003a).
Critique on model 2
Although evidence for which ants follow reverse tandem runs is scarce, both models give the same qualitative predictions. Note, however, that the dynamics of the different ant classes during a simulated emigration in model 2 poorly match observed experimental dynamics (see, e.g., Planqué et al. 2006). In many cases, the final number of ants active in an emigration is much greater than the original number of active ants. An example is given in Fig. 4. These recruiter numbers far exceed observed numbers of active ants (available in Table 3 in Pratt et al. 2005). Indeed, simulated emigrations often end with half the colony being recruiters and less than half carried into the new nest (Fig. 3, bottom row, third from left).
Fig. 4Examples of temporal dynamics for models 1 and 2. At μ = 0.05, F = 0.1447, we have taken parameters optimal for models 1 and 2, respectively. Notice that, in model 1, the number of recruiters rises to about 35, but in model 2, there are no less than 100 recruiters at the end of the emigration, indicating that recruitment from the carried class (model 2) may give rise to very unrealistic emigration dynamics
Reverse tandem activity
Figure 2 (top left) shows that recruiters in model 1 should use a sequential strategy if F is small (and μ is large): When the quorum is met, they first spend all of their time on reverse tandem runs until all scouts have become recruiters, and then switch to carrying. In contrast, when F is large, recruiters mix tandem running and carrying. This qualitative difference may be understood as follows.
The total number of recruiters is bounded by FN, the number of active ants in the colony. As F decreases, the remaining recruiters take longer to carry all the passive ants. Hence, the time costs for not having the recruiters increases and the time to recruit the remaining scouts decreases (as there are fewer scouts too). Hence, in this situation, recruiters should devote their post-quorum time, first, all on reverse tandem running before starting carrying.
When F is large, the reverse argument applies. With less passive ants, there should be less emphasis on additional recruitment by reverse tandem running. One does not have to make many hands if the work was light to start with.
Note that this behaviour for F →0 is different for model 2. In this paper, we never observe sequential strategies (Fig. 2, top right), as there is no end to building recruiter numbers but by completing the entire emigration.
This argument also explains another difference between these models: The number of reverse tandem runs during an emigration. In model 1, there is a clear maximum for intermediate F, whereas in model 2 the number of reverse tandems strictly decreases with F (Fig. 3, second from left, top and bottom).
Nonlinearities in the models and divisions between active and passive ants
Contrary to the models in this paper, two previous models of house hunting by T. albipennis ants (Pratt et al. 2002; Planqué et al. 2006) assumed linear terms for tandem running and social carrying. There, these processes occurred at rates only proportional to the number of recruiters. The predictions of the current models proved to be strongly dependent on the assumption of non-linearity of these terms. The corresponding linear models predicted that reverse tandem running should not be used for practically any parameter choices in F and μ or k.
Another ingredient in this model shared with the previous (Pratt et al. 2002; Planqué et al. 2006), models of this collective decision-making system is the division between active and passive ants. There is as yet little experimental evidence suggesting this division really exists. All we know at present is that a limited fraction of ants is actively engaged during an emigration. We have thus also explored models in which this division was absent, using both linear and non-linear interaction terms such as those in models 1 and 2 presented in this paper. In none of these models did reverse tandem running contribute to the optimal emigration speed. The division between active and passive ants is thus a crucial ingredient for reverse tandem running to have a positive impact on emigration speed, which should be experimentally validated.
Hypothesised explanations
Several hypotheses on the potential role of reverse tandem running have been put forward (Pratt et al. 2002). First, the ants might have a “home nest”, which changes during the emigration, thereby reversing the direction of any recruitment events from “home” to another nest (Pratt et al. 2002). If true, this would predict a change of direction when about half of the colony had been displaced. This is not in agreement with the available data (Mallon et al. 2001). Moreover, this hypothesis does not offer a suggestion why tandem running often occurs early in the emigration. In other words, it might explain the direction, but not the occurrence itself.
Second, it has been suggested that reverse tandem running may re-allocate recruitment (Pratt et al. 2002). Again, this does not fit the available data from Mallon et al. (2001). Reverse tandem runs were nearly always observed between the best nest and the old nest. The models in this paper do not incorporate choice between nest sites, but we conjecture that early flexible commitment (Planqué et al. 2006) will be more efficient in redirecting ants to better nests than late recruitment. Other experimental results also corroborate that reverse tandem running does not influence the decision-making process (Franks et al. 2003a).
The first models in which reverse tandem runs have been explicitly incorporated to analyse their role have yielded clear predictions: under a range of conditions, we expect a negative correlation between levels of early and late recruitment. This finding lends itself well to simple experiments, and we aim to present those in the near future.
The build up of recruiter numbers serves two purposes: to decide on a nest and to increase the number of ants actively involved in transport. The decision-making process and the implementation of this decision are thus conflated. This in itself is a side-effect of the distributed nature of this system. Reverse tandem running may thus be a logical extension to overcome this inherent problem. This suggests that such additional backup behaviours could be a common feature of decentralised collective decision-making systems.

temnothorax albipennis
tandem running
social insects
recruitment methods
collective behaviour