Cellular and Behavioral Effects of Cranial Irradiation of the Subventricular Zone in Adult Mice

Cellular and Behavioral Effects of Cranial Irradiation of the Subventricular Zone in Adult Mice

Françoise Lazarini1,2, Marc-André Mouthon3, Gilles Gheusi1,2, Fabrice de

Chaumont4, Jean-Christophe Olivo-Marin4, Stéphanie Lamarque5,6, Djoher Nora

Abrous5,6, François D. Boussin3, Pierre-Marie Lledo1,2*

1 Institut Pasteur, Laboratory for Perception and Memory, Paris, France, 2 Centre National de la Recherche

Scientifique (CNRS) Unité de Recherche Associée (URA), Paris, France, 3 CEA, DSV, iRCM, SCSR, Laboratoire de

RadioPathologie, INSERM U967, Fontenay-aux-Roses, France, 4 Institut Pasteur, Unité Analyse d'Images

Quantitative, CNRS (URA 2582), Paris, France, 5 INSERM U862, Neurocentre Magendie, Neurogenesis and

Pathophysiology group, Bordeaux, France, 6 Université de Bordeaux, Bordeaux, France

Abstract Top

Background

In mammals, new neurons are added to the olfactory bulb (OB) throughout life. Most of these

new neurons, granule and periglomerular cells originate from the subventricular zone (SVZ)

lining the lateral ventricles and migrate via the rostral migratory stream toward the OB.

Thousands of new neurons appear each day, but the function of this ongoing neurogenesis

remains unclear.

Methodology/Principal Findings

In this study, we irradiated adult mice to impair constitutive OB neurogenesis, and explored the

functional impacts of this irradiation on the sense of smell. We found that focal irradiation of the

SVZ greatly decreased the rate of production of new OB neurons, leaving other brain areas

intact. This effect persisted for up to seven months after exposure to 15 Gray. Despite this robust

impairment, the thresholds for detecting pure odorant molecules and short-term olfactory

memory were not affected by irradiation. Similarly, the ability to distinguish between odorant

molecules and the odorant-guided social behavior of irradiated mice were not affected by the

decrease in the number of new neurons. Only long-term olfactory memory was found to be

sensitive to SVZ irradiation.

Conclusion/Significance

These findings suggest that the continuous production of adult-generated neurons is involved in

consolidating or restituting long-lasting olfactory traces.

Citation: Lazarini F, Mouthon M-A, Gheusi G, de Chaumont F, Olivo-Marin J-C, et al. (2009) Cellular and

Behavioral Effects of Cranial Irradiation of the Subventricular Zone in Adult Mice. PLoS ONE 4(9): e7017.

doi:10.1371/journal.pone.0007017

Editor: Kenji Hashimoto, Chiba University Center for Forensic Mental Health, Japan

Received: May 26, 2009; Accepted: July 13, 2009; Published: September 15, 2009

Copyright: © 2009 Lazarini et al. This is an open-access article distributed under the terms of the Creative

Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium,

provided the original author and source are credited.

Funding: This study was supported by the Agence Nationale de la Recherche (ANR-2007 SEST-01411) (P-ML

and FB) and Electricité de France (FB). PML is also supported by the Fondation pour la Recherche Médicale

(“Equipe FRM”), the Groupe Arpège, and the Ecole des Neurosciences de Paris. DNA is supported by INSERM

and University of Bordeaux. PML's laboratory is a member of the Network of European Neuroscience

Institutes (LSHM-CT-2005-019063).

Competing interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

INTRODUCTION Top

Neurocognitive deficits and olfactory changes are frequently observed after chemotherapy and

cranial radiotherapy in adult patients [1], [2]. These changes may result from damage to the

neural stem cell (NSC) populations of the subgranular zone of the dentate gyrus (DG), the

hippocampus and the subventricular zone (SVZ) lining the forebrain lateral ventricles [3], [4].

NSCs continually generate new neurons, which are recruited to the DG and the olfactory bulb

(OB) of adult mammals [5]. New neuronal progenitors generated in the SVZ migrate along the

rostral migratory stream (RMS) towards the OB. Within the OB, they integrate into the granule

cell layer (GCL), the external plexiform layer (EPL) or the glomerular layer (GL), giving rise to

both gamma-aminobutyric acid (GABA)- and dopamine-containing interneurons [6]–[10]. This

ongoing neurogenesis is essential for maintenance of the integrity of the OB circuitry. The

blocking of this process depletes the population of OB interneurons [11].

Activity-dependent factors regulate OB neurogenesis, suggesting that adult neurogenesis is not

exclusively constitutive [12]–[15]. The new cells added to the OB and DG circuits undergo

functional integration [16], [17], and this process is thought to be important for learning and

memory [5]. Spatial memory deficits have been observed following the disruption of

neurogenesis in transgenic mice [11], [18], [19]. However, the functional relevance of adult

neurogenesis in olfaction remains unclear. Some studies have suggested that new neurons are

not required for olfaction, whereas others have implicated adult-generated neurons in a number

of olfactory functions. For example, olfaction has been shown to be unaffected in Bax-knockout

mice [20] and in mice producing a neuron-specific enolase-diphtheria toxin [11], despite the

significantly lower than normal level of neurogenesis in both transgenic models. By contrast,

both mice lacking neural cell-adhesion molecule (NCAM) and mice with the brain-derived

neurotrophic factor (BDNF) Val66Met knock-in display impaired OB neurogenesis and odor

discrimination [21], [22]. Olfactory discrimination is also impaired in aging rodents, mice

heterozygous for leukemia inhibitory factor receptor (Lifr +/−), and waved-1 mutant mice (a

hypermorph of TGF-alpha), in which OB neurogenesis level is reduced [23]. Finally, a correlation

has been found between the degree of OB neurogenesis and olfactory memory [24], [25],

providing further support for the hypothesis that adult neurogenesis plays an important role in

olfaction.

These inconsistencies may result from the use of different ablation techniques, affecting both the

DG and OB regions. Precise techniques for disrupting adult neurogenesis in specific areas of the

brain may make it possible to assign behavioral functions to each neurogenic system. Focal

irradiation of the SVZ leads to a dose-dependent loss of cell types in this region and repopulation

may take several months [26]. Here, we used focal SVZ irradiation in adult mice to disrupt the

production of new OB neurons without affecting the rest of the brain or the body. We examined

the functional effects of irradiation on odorant detection, discrimination and olfactory memory.

We found that the continuous recruitment of adult-generated OB neurons was not required for

any of the olfactory functions tested except for long-term olfactory memory, which was less

robust after irradiation.

RESULTS Top

Focal irradiation of the adult SVZ strongly reduced OB neurogenesis

We evaluated the effects of SVZ irradiation (Figures 1A and 1B) on the production of new

neurons, by quantifying doublecortin (DCX) staining in the SVZ and OB. DCX is a microtubule-

associated protein produced by neuronal progenitors and immature neurons. It can therefore be

used as a reliable marker for the quantification of adult neurogenesis [27], [28].

Figure 1. Focal irradiation decreased DCX

immunoreactivity in the SVZ.

(A, B) Focal gamma-ray irradiation of the SVZ. Adult

mice were anesthetized and placed in a stereotaxic

frame for cranial irradiation. A lead shield protected

their body during exposure of the SVZ to gamma

rays. A total dose of 15 Gray was delivered in three

equal fractions administered at two-day intervals. H,

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007017?imageURI=info:doi/10.1371/journal.pone.0007017.g001

hippocampus. (C) DCX staining of neuroblasts in a coronal section of the SVZ from a sham-treated mouse (left) and

from an irradiated mouse 7 months after SVZ irradiation (right). Note the weaker DCX staining in the SVZ of the

irradiated (IRR) mouse. LV, Lateral ventricle. (Scale bar: 100 µm.). (D) Densitometric analysis of DCX

immunoreactivity in the SVZ of sham-treated and irradiated mice 7 months after irradiation. OD, optical density.

Student's t test; *** p<0.0001 (n = 6 ).

doi:10.1371/journal.pone.0007017.g001

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Several months after SVZ irradiation, neurogenesis levels were significantly lower than normal in

both the SVZ (Figure 1C and 1D) and OB (Figure 2A and 2B). A complete statistical analysis is

provided in the Supplementary Table S1. This finding is consistent with a previous study showing

that SVZ progenitor cells are sensitive to irradiation [26]. Levels of DCX immunoreactivity were

significantly lower in the SVZ of irradiated mice (about 30% those of sham-treated mice, Figure

1D; DCX optical density for sham treatment: 0.096±0.009, for SVZ-irradiation treatment:

0.03±0.003, p<0.0001). Similarly, DCX immunoreactivity throughout the entire OB was 70%

weaker in irradiated mice than in sham-treated mice (1.177±0.134 in sham-treated mice;

0.327±0.022 in SVZ-irradiated mice, p<0.0001; see Figure 2A and 2B).

Figure 2. Irradiation decreased the number of

DCX+ cells in the OB.

(A) Representative images showing DCX+ cells in

coronal sections of the OB, 7 months after SVZ

irradiation. The GL, EPL, GCL and RMSob are

indicated. (Scale bar: 100 µm). (B and C)

Densitometric analysis of DCX immunoreactivity in

total OB (B), including the RMSob (C, left), GCL (C,

middle) and GL, (C, right) of sham-treated and

irradiated mice 7 months after irradiation. OD,


optical density. Student's t test; *** p<0.0001. ** p<0.01 (n = 12). (D) Densitometric analysis of DCX staining along

the rostrocaudal axis of the OB, in sham-treated mice and irradiated mice 7 months after irradiation (n = 12). All cell

layers along the entire rostrocaudal axis of the OB were equally affected by SVZ irradiation. (E) Immature neurons

visualized by DCX staining in the GCL and GL of sham-treated and irradiated mice, 7 months after irradiation. (Scale

bar: 20 µm.)



All the cell layers in the OB were similarly affected (Figure 2C). Irradiation decreased DCX

immunoreactivity in the RMS layer at the core of the OB (RMSob) by 75% (sham-treated:

0.722±0.065 and SVZ-irradiated: 0.185±0.015, p<0.0001). Similarly, DCX immunoreactivity in

the GCL and GL was 70% lower after irradiation (GCL: 0.224±0.031 in sham-treated,

0.072±0.006 in SVZ-irradiated, p<0.0001; GL: 0.096±0.017 in sham-treated, 0.031±0.002 in

SVZ-irradiated, p<0.01). This pattern was observed along the entire length of the rostrocaudal

axis of the OB (Figure 2D). We then used DCX immunoreactivity to assess the number of

dendrites and the dendritic morphology of the newly generated cells reaching the OB. These

features were not affected by irradiation (Figure 2E, see also Supplementary Figure S1).

We confirmed the lower level of cell proliferation in the irradiated SVZ, by quantifying

bromodeoxyuridine (BrdU) staining (Figure 3A and 3B) at two time points after the final

irradiation session (Figure 3C). Animals were injected with BrdU three days after the final

irradiation session. They were then killed 11 days after BrdU injection (14 days after irradiation).

Far fewer BrdU+ cells were found in the OB of irradiated mice(60% fewer) than in that of sham-

treated mice (4,387±969 BrdU+ cells in sham-treated mice, 1,792±859 after irradiation;

p<0.005). A similar pattern was seen in all layers (for GCL: 3,241±432 and 1,405±697, p<0.005;

for EPL: 193±14 and 115±19, p<0.01; and for GL: 490±95 and 273±120, p<0.05, for sham-

treated and SVZ-irradiated mice, respectively; Figure 3D).


Figure 3. Irradiation reduced the recruitment of new neurons.

(A, B) New cells were labeled with BrdU 3 days after the last focal irradiation and survival was determined 11 days

later. Photomicrographs show OB coronal sections labeled with BrdU, for sham-treated and irradiated mice. (Scale

bar: 30 µm). (C) New cells were labeled with BrdU 8 or 120 days after the first session of irradiation. The mean

number of BrdU+ cells in the entire OB was determined for sham-treated and irradiated mice 11 days after the final

BrdU injection. Student's t test; ** p<0.01. * p<0.05 (n = 3–6 mice). (D) BrdU+ cell number in the OB, including the

GCL, EPL and GL, for sham-treated and irradiated mice, with BrdU injected 8 days after the first session of

irradiation. Student's t test; ** p<0.01. * p<0.05 (n = 4–6).


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We then injected mice with BrdU 115 days after the final irradiation session and killed them for

analysis 11 days after BrdU injection (Figure 3C). The number of BrdU+ cells was decreased to a

similar extent in irradiated mice (60% fewer positive cells in irradiated mice; 1,684±276 vs.

724±124, p<0.05; Figure 3C). As a control, we analyzed the number of BrdU+ cells in the

hippocampus of SVZ-irradiated mice. The number of positive cells in this part of the brain was

similar in irradiated and sham-treated animals (Supplementary Figure S2), demonstrating the

confinement of exposure to gamma radiation to the targeted area.

Finally, we analyzed the survival rate of newly generated neurons in the OB, by counting BrdU+

cells 31 days after BrdU injection. Consistent with previous studies (e.g., [29]), the number of

BrdU+ cells halved between day 11 (D11) and D31 in the control OB (p<0.005). Similar decreases

were observed in the GCL, EPL and GL (Figure 3D). Surprisingly, in sharp contrast to what was

observed for the controls, the number of BrdU+ cells did not decrease significantly between D11

and D31 (p>0.05) in irradiated animals. Thus, local irradiation of the SVZ significantly impaired

the recruitment of newly generated OB neurons, and the neurons that actually reached the OB

escaped cell apoptosis.

Spontaneous odorant discrimination was not affected by SVZ

irradiation

Given the strong effects of irradiation on neuron production, we investigated possible effects on

olfaction. As irradiation has transient side effects on the functioning of the mature nervous

system due to local inflammation [30], we carried out all behavioral experiments at least two

months after irradiation, when inflammation markers had disappeared (data not shown). As a

further control, we checked that there was no change in locomotor activity or anxiety levels in

irradiated animals (data not shown). Using both non-operant and operant conditioning

paradigms, we investigated odorant detection, discrimination and olfactory memory. We first

subjected mice to a spontaneous discrimination task involving cross-habituation, to measure

their ability to distinguish between different odorants in the absence of associative learning or

previous odor reinforcement (Figure 4). Mice were initially trained through six successive

exposures to linalool (Figure 4A). Both groups showed a progressive decrease in investigating

the same odorant in repeated exposures, a process called habituation. Two-way ANOVA revealed

a significant effect of repeated exposure (p<0.0001), but no effect of treatment (irradiation) or

exposure x treatment interaction (p>0.05) was found. Mice were then subjected to a

habituation/dishabituation session in which they were exposed to three different odorants every

day over a six-day period (Figure 4B–4G). After the fourth sequential exposure to the odorant to

which they had been habituated, a similar, but different, odorant was introduced. Mice were

exposed to the odorant to which they had been habituated twice more before the introduction of

a third odorant from an odorant family different from that to which the first two odorants

belonged. We found no difference in the time spent investigating each odorant, for any of the

sessions, between sham-treated and irradiated mice (p>0.05). Moreover, the extent of

dishabituation was similar for the two groups (p>0.05): all mice detected even small differences

between similar odorants (Figure 4B). Conversely, neither irradiated nor sham-treated mice were

able to distinguish spontaneously between the limonene and terpinene enantiomers (Figures 4C

and 4D, respectively). A complete statistical analysis is provided in Supplementary Table S2.

Thus, spontaneous olfactory discrimination was not affected by SVZ irradiation.

Figure 4. Spontaneous discrimination was not

affected by irradiation.

Sham-treated and irradiated mice were tested daily,

in successive sessions of habituation/dishabituation

and memory tests. Histograms indicate the mean

time spent investigating an odorant during 90

seconds of exposure, with a two-minute period of

rest between consecutive exposures. (A) Habituation

with 6 successive exposures to linalool. Both groups

showed progressively less interest in investigating

the same odorant in repeated exposures, a process

called habituation. (B–G) Sessions of habituation (4

successive exposures to the odorants indicated)

followed by dishabituation (a single period of

exposure to an odorant similar to that used for

habituation), recall of habituation (two successive

exposures to the odorant used for habituation) and a

final dishabituation with a single exposure to a

dissimilar odorant. The time spent investigating the

test odorant is shown. The extent of dishabituation was similar for the 2 groups: all mice detected even small

differences between similar odorants (3B). Neither irradiated nor sham-treated mice could distinguish spontaneously

between the limonene and terpinene enantiomers (3C and D). No significant effects of irradiation were observed

(Two-way ANOVA; p>0.05, n = 9–10 mice). (H) 30-minute olfactory memory was not affected by irradiation. A mint

odorant was introduced into the cage for five minutes. Two minutes later, the same odorant was introduced again

for five minutes. The odorant was introduced into the cage for a final two-minute period after a 30-minute rest

period (memory test). Histograms indicate the mean time of investigation. No effect of irradiation was observed

(two-way ANOVA; p>0.05 n = 9–10 mice).



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Short-term olfactory memory was studied by exposing mice twice to mint odorant (habituation)

and then exposing them to this same odorant again after a 30-minute interval (Figure 4H). Both

groups spent less time investigating the odorant during the second and third exposure periods

(effect of exposure: p<0.0001; irradiation: p>0.05, and interaction: p>0.05), demonstrating that

short-term memory was similar in irradiated mice (tested over a period of 30 minutes) and in

controls. Thus, decreasing the number of newly generated neurons reaching the OB has no effect

on olfactory discrimination or short-term memory.

The recognition of social olfactory cues did not require adult OB

neurogenesis

All the odorants used for the behavioral tests were artificial. We therefore checked that we had

not missed a potential consequence of reducing OB neurogenesis due to our use of synthetic

odorants (i.e., ethologically non-relevant molecules). We used a social interaction test to quantify

the spontaneous investigation of an unfamiliar mouse by a resident mouse. We found that the

two groups of mice spent similar amounts of time investigating the intruder (23.2±2.0 s and

23.5±3.4 s for sham-treated and irradiated mice, respectively; p>0.05; see also Supplementary

Table S2). Thus, the disruption of adult neurogenesis did not impair the processing of social

olfactory information.

Odorant detection did not depend on adult OB neurogenesis

We hypothesized that the absence of an obvious phenotype in irradiated animals might be due

to the use of high concentrations of odorants. We therefore used an automated operant

conditioning procedure (a go/no-go test) to investigate olfactory sensitivity, by determining the

detection threshold for (+)-carvone, using the descending limits method. (+)-carvone was the

rewarded (S+) stimulus, and the solvent, mineral oil, was the unrewarded (S-) stimulus. Water-

deprived mice were first trained to distinguish between a high concentration (10−3) of (+)-

carvone and mineral oil. They were then subjected to daily blocks of trials involving exposure to

progressively lower concentrations (10−4, 10−5 and 10−6). Performance accuracy decreased with

decreasing concentration for both groups (Figure 5A; p<0.05). An analysis of odorant

concentration-performance curves for the last block of exposures revealed that both irradiated

and sham-treated mice performed the test with an accuracy of more than 80% for

concentrations of 10−3 and 10−4 (+)-carvone (see Supplementary Table S3 for a complete

statistical analysis). Furthermore, the rate of successful task completion was similar for the two

groups (effect of treatment: p>0.05; block of trials: p<0.001; interaction: p>0.05). By contrast,

the accuracy with which both irradiated and sham-treated mice performed the task fell to levels

consistent with chance alone (50% correct detection) at 10−5 and 10−6 (+)-carvone (treatment:

p>0.05; block of trials: p>0.05; interaction, p>0.05). The (+)-carvone detection threshold was

similar in irradiated and control mice (treatment: p>0.05; interaction: p>0.05). We therefore

conclude that SVZ irradiation does not impair odorant detection.

Figure 5. Irradiation has no effect on

performance in reinforced discrimination

tasks.

(A) Odor detection thresholds were not altered by

irradiation. Accuracy (% of correct responses) is

shown for the detection of successively lower

concentrations of (+)-carvone (10 blocks of 20 trials).

(+)-carvone was the rewarded (S+) stimulus and the

solvent, mineral oil (MO), was the non-rewarded (S-)

stimulus. Water-deprived mice were first trained to

distinguish between a high concentration of (+)-

carvone and MO. They were then subjected to daily

blocks of trials in which they were exposed to

progressively lower concentrations. Acquisition rate was similar for the 2 groups. A score of 50% corresponds to the

success rate expected on the basis of chance alone (dashed line, A–C). No significant differences were observed for


irradiated mice (p>0.05, n = 7). (B) The acquisition of discrimination ability in separate 2-odorant discrimination

tasks and performance in the corresponding 8-odorant task were not affected by irradiation. The accuracy of

performance in the discrimination tasks is shown as a % of correct responses for 8 blocks of 20 trials for odorant pair

A (1% anisole, S+ vs 1% cineole, S-), odorant pair B (0.1% n-amyl acetate, S+ vs 1% linalool, S-), odorant pair C (1%

butanoic acid, S+ vs 1% beta-ionone, S-), odorant pair D (1% (+)-limonene, S+ vs 1% (+)-carvone, S-) and 4 blocks

of 40 trials for 8-odorant tasks. In the two-odorant tests, the stimuli, A, B, C and D (giving eight possible

permutations) were introduced in a random order. No effect of SVZ irradiation was observed (two-way ANOVA;

p>0.05, n = 9–10). (C) The ability to distinguish between pairs of mixtures of two odors was not affected by

irradiation. Mixtures contained 1% (+)-carvone (indicated by (+)-C or S+) and 1% (−)-carvone (indicated by (−)-C or

S-). Five alternating mixtures with different ratios were used and animals were rewarded only when a go-response

was observed in the presence of mixtures in which (+)-carvone was the dominant compound. Concentrations (%) of

odors are given for the following pairs of mixtures: 8/2 vs 2/8: 0.8% (+)-C+0.2% (−)-C (S+) vs 0.2% (+)-C+0.8% (−)-

C (S-); 7/3 vs 3/7: 0.7% (+)-C+0.3% (−)-C (S+) vs 0.3% (+)-C+0.7% (−)-C (S-); 6/4 vs 4/6: 0.6% (+)-C+0.4% (−)-C

(S+) vs 0.4% (+)-C+0.6% (−)-C (S-); 5.2/4.8 vs 4.8/5.2: 0.52% (+)-C+0.48% (−)-C (S+) vs 0.48% (+)-C+0.52% (−)-C

(S-). Performance accuracy is shown as a % of correct responses for 10 blocks of 20 trials. No effect of SVZ

irradiation was observed (two-way ANOVA; p>0.05, n = 7).



Irradiated mice successfully completed two-odorant discrimination

tasks

We used the same procedure to explore further the ability of mice to distinguish between

different pairs of odorants (Figure 5B). Both groups acquired the ability to distinguish between

pairs of odorants in our separate two-odorant discrimination tasks (a complete statistical analysis

is provided in Supplementary Table S3). The acquisition rate was similar in the two groups (pair

A–D: effect of treatment: p>0.05; block of trials: p<0.001; interaction: p>0.05). Mice from both

groups reliably distinguished between all eight odors introduced in a random order within the

same session (treatment: p>0.05; block of trials: p<0.001; interaction: p>0.05). Thus, SVZ

irradiation does not affect acquisition of the ability to complete two- to eight-odorant

discrimination tasks successfully.

Irradiated mice performed odorant-mixture tasks accurately

Mice were then exposed to more complex problems, using a discrimination task based on binary

odorant mixtures. The introduction of mixtures of various proportions of (+)-carvone and (−)-

carvone increased the complexity of the task. Five alternating mixtures with different ratios were

used and animals were rewarded only when a go-response was observed in the presence of

mixtures in which (+)-carvone was the dominant compound (see Supplementary Table S3 for

statistical analysis). Performance accuracy varied significantly with the ratio of the mixture

(Figure 5C; p<0.001). Mice from both groups successfully learned to distinguish between pure

odorants (+)-carvone and (−)-carvone and between the following pairs of mixtures: 80%–20% vs.

20%–80% and 70%–30% vs. 30%–70%. However, performance accuracy was substantially lower

(close to the levels expected on the basis of chance alone), in both groups, for distinguishing

between 60%–40% and 40%–60% or 52%–48% and 48%–52% mixtures. Thus, the ability to

distinguish between mixtures of odorants was similar in the two groups of mice, even for difficult

tasks (for pairs of (+)/(−)-carvone mixtures, 8/2 vs. 2/8, 7/3 vs. 3/7, and 6/4 vs. 4/6: effect of

treatment, p>0.05; block of trials, p<0.001; treatment x block interaction, p>0.05; for 5.2/4.8-

4.8/5.2 mixtures: treatment and block, p>0.05; treatment x block interaction, p>0.05). SVZ

irradiation therefore does not impair the ability to complete difficult olfactory discrimination tasks

successfully.

Long-term olfactory memory was sensitive to SVZ irradiation

Finally, we examined the ability of irradiated mice to remember two odorants learned 30 days

before. Both groups were first trained to distinguish between two new odorants: anisole (S+) and

cineole (S-). The mice were then subjected to the same discrimination task 30 days later. In this

second session, no reward was given for correct responses. The lack of reinforcement following

the introduction of the S+ stimulus excluded the possibility of an accurate performance by the

animals simply reflecting a rapid transfer of training between tasks. We measured the

percentage of correct responses for both groups during the last block of the acquisition period

and during the first block of the memory task performed 30 days later (Figure 6A–C). For each

group, we also calculated the mean number of errors made during the session (Figure 6D). These

experiments confirmed that both sham-treated and irradiated mice were able to acquire odor-

associated memory in an operant two-odorant task. However, a significant difference in

performance was observed between sessions (p<0.01), together with a significant interaction

between treatment and session (p<0.05; a complete statistical analysis of the data is provided in

the supplementary Table S4). Performance accuracy was significantly lower in the second

session for irradiated mice (p<0.05) but not for sham-treated animals (p>0.05). Thus, memory of

learned odorants was better retained over a one-month period in sham-treated than in irradiated

mice. Irradiated animals made significantly more errors in the memory task than sham-treated

animals (Figure 6D, p<0.05). All errors made during this session were associated with responses

triggered by S-, suggesting that the mistakes made by irradiated mice were due to an impaired

memory of odors, but not of the go/no-go task procedure. This effect on olfactory memory was

replicated with four different odorant pairs (data not shown). Thus, although the ability to detect

and discriminate between odorants was not affected by irradiation, irradiated animals

remembered odorants less well one month later.

Figure 6. Impaired long-term memory in

irradiated mice.

Mice underwent 8 blocks of 20 trials every day for 4

days, to train them to distinguish between 1%

anisole (rewarded odorant) and 1% cineole (non-

rewarded odorant). Mice were tested on the same

task after a rest period of one month, in one block of

20 trials, but with no reward given for a correct

response. Representative results for experiments performed in triplicate are shown. (A–C) Mean values (%) for

correct responses in the last block of training (acquisition) and in the first block of testing (memory test) are shown


for each sham-treated (A) and irradiated mouse (B) and for all mice (C). (D) Means of errors in trials 1 to 20 of the

memory test session. Two-way ANOVA followed by unpaired or paired Student's t tests, as appropriate; * p<0.05 (n

= 9–10).



DISCUSSION Top

Neurogenesis in the healthy adult brain is principally limited to two systems: the hippocampal

dentate gyrus and the SVZ-OB. Its conservation across all mammalian species and tight

regulation [5], [31]–[33] suggest that adult neurogenesis may affect behavior. In this study, we

investigated the functional consequences for olfaction of reducing adult neurogenesis. We found

that impairment of the ongoing recruitment of adult-generated OB neurons altered long-term

olfactory memory, but had no effect on odorant detection or the discrimination, learning and

recognition of social olfactory cues.

Focal irradiation of the SVZ reduced the number of newborn neurons

in the OB

Adult neurogenesis encompasses cell production, cell fate determination, survival, integration,

and the acquisition of functional neuronal properties in the adult brain [5]. Consistent with

previous studies [26], [30], DCX staining demonstrated that SVZ irradiation reduced the

production of newborn neurons in the SVZ. DCX staining at the time of behavioral testing or

seven months after administration of the final dose of radiation revealed a long-lasting effect on

OB neurogenesis. Focal irradiation led to a permanent 70% decrease in the number of new

neurons produced in the SVZ or integrated into the OB circuit, consistent with most newly

generated OB neurons being generated by the SVZ in adults. Further studies are required to

identify the types of cell in the SVZ sensitive or resistant to irradiation. It will be particularly

interesting to determine whether GFAP-positive “B cells” in the SVZ are resistant to radiation-

induced cell death.

Consistent with previous findings, we found that about 50% of newly generated cells in control

mice died four weeks after their generation. We also showed, for the first time, that cell survival

depended on the overall number of newly produced neurons reaching the bulb: in irradiated

animals, only 30% of 11-day-old cells reached the bulb, and all were still alive 19 days later (see

Figure 3D). Further immunohistological experiments with other markers of neurons, astroglial

and oligodendroglial cells are required for the identification of a possible selection of a

subpopulation of radiation- and apoptosis-resistant SVZ/OB cells.

Despite the prolonged survival of the newly generated cells, irradiated animals made more errors

than controls in the long-term memory task, suggesting that the continuous recruitment of new

neurons, and not the total number of neurons per se, is a key element in long-term olfactory

memory. Further studies are required to determine whether the surviving cells arising from the

irradiated SVZ are functionally different from those produced in the non-irradiated forebrain.

Similarly, further studies are required to decipher the mechanisms regulating the survival of

newly generated neurons. It is possible that newly generated neurons in the OB compete for

survival factors (e.g. trophic factors) just as they compete with existing neurons for many of their

synaptic inputs. Such competition would account for the longer survival of new neurons in

irradiated animals.

The production of new neurons for olfaction

The functional relevance of adult neurogenesis remains an unresolved issue in neural stem cell

biology. One general strategy used to address this problem involves studying the effects on

behavior of inhibiting proliferation in a neurogenic area. In this study, we investigated three

types of olfactory function, to assess the effects of reducing adult neurogenesis on odorant

detection, discrimination and memory. Both spontaneous and reinforced discrimination tests

were used to investigate potential differences in olfactory functions [34]. Irradiated mice exposed

to synthetic odorants discriminated between these odorants as efficiently as sham-treated mice.

This finding is consistent with previous studies showing that neonatal irradiation or the genetic

blockade of adult neurogenesis does not impair olfactory discrimination [11], [35]. However, this

result contrasts with previous data obtained in NCAM-knockout mice [21] and BDNF Val66Met

knock-in mice [22], both of which displayed disrupted neurogenesis. This discrepancy may be

due to the impairment of neurogenesis during embryogenesis rather than the disruption of

neurogenesis during adulthood.

Irradiation had no effect on social investigation on the basis of olfactory cues (reviewed in [36]).

By contrast, Iwata et al. [37] observed social interaction deficits in animals subjected to

irradiation of the entire forebrain, leading to various undesirable side effects. For example,

irradiated rats displayed abnormal locomotor activity, introducing a potential bias into behavioral

tests. No such abnormal behavior was observed in our model, probably due to the more

restricted ablation of adult neurogenesis.

Most newly produced neurons remaining in the OB after learning are still present several weeks

later and can develop thousands of spines [9], [14], [15], [29]. New neurons may be specifically

involved in formation of the synaptic network serving as the structural basis for long-term

synaptic changes (the cell-autonomous hypothesis). This hypothesis is supported by previous

results demonstrating that newly generated neurons are unique in that 1) they have lower

thresholds for synaptic plasticity [38], [39], 2) they induce “synaptic disquietude” [40], 3) they

increase complexity at the ‘gate to long-term memory’ [41], and 4) they trigger unique

responses during odorant familiarization [42]. Thus, bulbar neurogenesis may increase plasticity

in several ways, including the addition of new cells, the structural remodeling of neural circuits,

and synaptogenesis, and changes in synaptic strength. All these forms of plasticity are consistent

with the neurons generated during adulthood being required for long-term olfactory memory.

Alternatively, OB neurogenesis may play an important role in the functioning of pre-existing

networks (the host circuit hypothesis). The activity of established circuits depends on sensory

inputs (i.e., the sensory space) and centrifugal fibers (i.e., the internal state). It is possible that

new neurons are the main targets of experience-induced changes in the activity of sensory

inputs and/or centrifugal fibers, acting as key elements in the retrieval or recall of memory

traces. Further experiments are required to test these two hypotheses specifically.

Our findings suggest that there is a correlation between adult neurogenesis and long-term

olfactory memory. Similar correlations also emerged from theoretical studies demonstrating the

involvement of adult neurogenesis in memory storage, rather than in perception or learning [43],

[44]. Aimone et al. also suggested that young neurons facilitate the formation of temporal

association in memory [45], and play key role in the encoding of memory [46]. Computer-based

studies have indicated a role for constitutive adult neurogenesis in mnesic function, providing a

theoretical background for future experimental approaches.

Our data support a causal link between the number of new neurons in the OB and long-term

olfactory memory, but we cannot exclude the possibility that another area of the brain is affected

by SVZ irradiation and participates in the observed changes in long-term olfactory memory. We

also cannot rule out the possibility of changes in the neuronal or synaptic activity of the

preexisting OB neurons in irradiated animals. Further studies, involving the selective and

reversible inhibition of OB neurogenesis, are required to determine whether there is a genuine

causal relationship and investigated the feasibility of attenuating and recovering memory

function.

Neurogenesis may allow an increase in the complexity of the network for memory consolidation

or long-term adaptation processes during adulthood. Improvements in information processing

resulting from the incorporation of newly generated neurons may facilitate olfactory learning and

memory formation, consistent with the interdependence between memory performance and the

degree of neurogenesis. Previous studies showing a transient role for the OB in memory storage

support this notion [47]. The natural replacement of bulbar neurons provides a rationale for the

transfer of memory traces out of the bulb. The loss of OB neurons may be programmed to occur

after the transfer of traces from these neurons to other parts of the brain. Alternatively, a

rejuvenating population of neurons capable of rapidly forming synaptic connections may be

highly suitable for the function of the OB in the transient processing of information sent

elsewhere for storage. Our findings suggest that new neurons are involved in long-term olfactory

memory, consistent with the assumption that new neurons provide unique functions for olfaction

[48].

MATERIALS AND METHODS Top

Animals

We used eight-week-old male C57BL/6J (Janvier, Le Genest-Saint-Isle, France) mice. Animals were

housed in groups of four or five and maintained in standard conditions (12 h/12 h light/dark

cycle, ad libitum access to dry food and water; for olfactometer experiments, animals were

subjected to partial water deprivation, as described below) in Pasteur Institute animal care

facilities officially registered for experimental studies on rodents (Ministry approval number for

animal care facilities: A 75-15-08; approval number 75-585 for animal experimentation). All

experimental procedures complied with the European Communities Council Directive of 24

November 1986 (86/609/EEC) and European Union guidelines, and were reviewed and approved

by our institutional animal welfare committee.

Irradiation

Mice were irradiated with a medical Alcyon irradiator (gamma-rays 60Co). They were anesthetized

with ketamine (75 mg/kg, Merial, Lyons, France) and medetomidine (1 mg/kg, Pfizer, Paris,

France) by the intraperitoneal (i.p.) route. They were placed in a stereotaxic frame (Stoelting,

Wood Dale, Illinois, USA) and exposed to cranial irradiation or not irradiated (sham-treated). Two

lead shields protected the body of the mouse during exposure of the SVZ to gamma rays. The

first shield consisted of a 10 cm-thick lead brick with a 12 mm diameter circular hole positioned

above the mouse's head. The second lead shield was 5 cm thick, with a rectangular opening of

3×11 mm, corresponding to the area of the SVZ (bregma AP: 1.5 and L: 5.5). The OB, RMS and

olfactory epithelium were unaffected by the procedure. Radiation (five Gray) was delivered at a

rate of 1 Gray/min on days 1, 3 and 5. After exposure, mice were woken up by i.p. injection of

atipamezole (1 mg/kg, Pfizer, Paris, France).

BrdU injections

Mice were injected i.p. with a DNA synthesis marker, BrdU (75 mg/kg, Sigma-Aldrich, St. Louis,

MO). They received four injections, at two-hour intervals, on a single day.

Immunohistochemistry

Mice were deeply anesthetized with sodium pentobarbital (100 mg/kg, Sanofi, Bagneux, France)

and perfused transcardially with a solution containing 0.9% NaCl and heparin (5×103 U/ml,

Sanofi-Synthelabo, Le Plessis-Robinson, France) at 37°C, followed by 4% paraformaldehyde (PFA)

in cold phosphate buffer (PB), pH 7.3. Brains were dissected out and post-fixed by incubation at

4°C in 4% PFA in PB, overnight for BrdU and for one week for DCX staining. Slices were

transferred to phosphate-buffered saline (PBS) and kept at 4°C until use. Immunohistochemistry

was carried out on 40 µm-thick free-floating serial coronal sections of the brain cut with a

vibrating microtome (VT1000S, Leica, Rueil-Malmaison, France) and collected in 0.2% sodium

azide (Sigma) in PBS. Brain sections were washed in PBS and treated with 0.2% Triton X-100, 4%

bovine serum albumin (both purchased from Sigma) in PBS for 2 h, to non-specific protein

binding and to permeabilize membranes. For BrdU staining, sections were treated with 2 N HCl

for 30 minutes at 37°C. BrdU and DCX were detected by incubation with a rat monoclonal anti-

BrdU antibody (C18, 1: 200; Immunologicals Direct, UK) or a goat anti-DCX antibody (1:200;

Santa Cruz Biotechnology, Santa Cruz, CA, USA). Labeled cells were detected with a peroxidase-

conjugated secondary antibody (ABC system, Vector Laboratories, Inc., Burlingame, CA, USA),

using biotinylated donkey anti-rat or horse anti-goat IgG (1:200, Vector Laboratories) and 3,3′-

diaminobenzidine (0.05%) as a chromogen (Sigma).

Image analysis for BrdU+ cell counting

We obtained reconstructed images with a 20× objective for one in every six coronal sections of

the OB (six sections in total) for each animal (Compix Imaging; Hamamatsu Photonics, Massy,

France). BrdU+ cells were automatically counted with a dedicated stereological computer

program [49]. The internal and external borders of the GL and GCL were drawn manually and

cells detected in the entire layer or in the GL, EPL and GCL were counted. Values are expressed

as the mean total BrdU+ cell count in six sections of the OB per animal. For hippocampal analysis,

values are expressed as the mean total number of BrdU+ cells counted manually in eight sections

of the dentate gyrus per animal.

Measurement of optical density

DCX expression was quantified by measuring optical density with a dedicated stereological

computer program [49], for one in every six coronal sections of the OB and in equivalent

selected sections containing the SVZ. After manual selection of the brain area to be analyzed,

the density of staining was calculated by dividing the pixel count by the overall area (pixels per

mm2).

Spontaneous discrimination of synthetic odorants

Olfactory discrimination.

For the olfactory discrimination task, we used a slightly modified version of the habituation–

dishabituation test described elsewhere [21]. The test cages were boxes of 36×23.5×13 cm

(length×width×height), with two compartments separated by an aluminum partition (holes 0.5

cm diameter). The lower compartment was 2.5 cm high. Animals were exposed to odors by

placing a filter paper dish (70 mm diameter, #1440 070; Whatman, Florham Park, NJ, USA),

impregnated with 0.4% odorant in 10 µl of odorless mineral oil (odorants and mineral oil obtained

from Sigma) in the lower compartment. Four days before the experiment, the animals were

familiarized with the test cage and the procedure by exposing them to mineral oil. Mice

underwent one session per day. The mouse was placed in the test cage for 10 minutes and

exposed to mineral oil for 2 minutes before each session. Mice were trained with six exposures to

0.4% linalool. They were then exposed to odorants as follows:

Four successive exposures to the first odorant (habituation odor);

One exposure to a second similar odor;

Two exposures to the habituation odor;

One exposure to a dissimilar odorant (dishabituation).

Each exposure lasted 90 seconds. An interval of 2 minutes was left between trials. We recorded

the time that the animals spent investigating the odorant for each experiment. Animals were

considered to have recognized an olfactory stimulus when they spent significantly less time

investigating an odorant introduced into the cage for a second time.

Short-term olfactory memory.

Mice were exposed to mint odorant twice (5 minutes each), with a two-minute interval between

the two exposures. They were then exposed to this odorant again after a rest period of 30

minutes. The time spent investigating the odorant was recorded for each animal.

Social interaction.

Social interaction was tested in the same test cages used for spontaneous discrimination. Each

mouse was tested for 5 minutes with a C57BL/6 mouse of the same age, sex (male) and weight,

reared in the same conditions (in the same animal facilities, in similar cages, each containing 4

to 5 mice). Social interaction was measured as the time the test subject (sham-treated or

irradiated) spent interacting with the other mouse (interacting social behavior included following

the other animal, anogenital sniffing and allogrooming).

Olfactory performance in automated

olfactometers.

Mice were maintained on a 1 ml/day water deprivation diet for 10 days and then trained in a

go/no-go discrimination task in Knosys (Bethesda, MD) computer-controlled olfactometers, as

previously described [50]. Mice were trained to respond to the presence of an odorant dissolved

in mineral oil (positive stimulus, S+) by licking the water delivery tube situated within the

odorant sampling port, and to refrain from responding to the presence of another odorant

(negative stimulus, S-). These two types of trials were carried out in a modified random order,

such that an equal number of each type occurred in each block of 20 trials and one type of trial

did not occur more than three times consecutively. A response in an S+ trial and an absence of

response in an S– trial were scored as correct. Accuracy was scored for each block of 20 trials.

Mice underwent a session of eight to 10 blocks of trials per day. All odorants were diluted in

mineral oil and their concentrations are given as the dilution of the odorant in the saturator

bottles.

Odorant detection threshold.

Mice were trained, in the air dilution olfactometer, to detect successively lower concentrations of

(+)-carvone diluted in mineral oil. Each concentration was given for 10 blocks of 20 trials each

day. In each session, (+)-carvone vapor served as the S+ stimulus and mineral oil served as the

S– stimulus. The concentrations of (+)-carvone used in these tests were 0.001, 0.0001, 0.00001

and 0.000001%.

Olfactory discrimination tasks.

Mice were trained in a series of two-odorant discrimination tasks using the eight-channel

olfactometer. Each mouse was subjected to eight blocks of 20 trials for each of the following

tasks:

Task 1: S+ was 1% anisole and S- was 1% cineole.

Task 2: S+ was 0.1% n-amyl acetate and S- was 1% linalool.

Task 3: S+ was 1% butanoic Acid and S- was 1% beta-ionone.

Task 4: S+ was 1% (+)-limonene and S– was 1% (+)-carvone. Mice not fulfilling the

performance criterion of 90% correct responses in two successive 20-trial blocks

underwent further training in daily 200-trial sessions until this level of performance was

reached.

Task 5: Eight-odorant discrimination. Upon completing tasks 1–4, each mouse was given

additional training, in which they were exposed to the eight odorants used in tasks 1–4 in

two blocks of 40 trials. Stimuli were introduced in a modified random order such that,

within each block of 40 trials, mice were exposed to the S+ and S– stimuli five times each.

Odorant mixture discrimination

tasks.

Mice were trained to distinguish 1% (+)-carvone from (−)-carvone in 10 blocks of 20 trials (Task

1). Then they were given 10 blocks of 20 trials for each of the following two-odorant mixture

tasks:

Task 2: S+ was 0.8% (+)-carvone +0.2% (−)-carvone and S- was 0.2% (+)-carvone +0.8% (−)-

carvone.

Task 3: S+ was 0.7% (+)-carvone +0.3% (−)-carvone and S- was 0.3% (+)-carvone +0.7% (−)-

carvone.

Task 4: S+ was 0.6% (+)-carvone +0.4% (−)-carvone and S- was 0.4% (+)-carvone +0.6%(−)-

carvone.

Task 5: S+ was 0.52% (+)-carvone +0.48% (−)-carvone and S- was 0.48% (+)-carvone +0.52%

(−)-carvone.

Long-term memory test.

We followed the memory test procedure described by Bodyak and Slotnick [50], with minor

modifications. Mice were given four daily training sessions of eight blocks of 20 trials for a two-

odorant task (S+ was 1% anisole and S- was 1% cineole). Mice were then left for 32 days in their

home cages, with partial water deprivation for the last 10 days. They were given no water on day

31. The following day, each mouse was subjected to a 20-trial memory test for the two-odorant

task. No reinforcement was given for correct responses in this session. Mice therefore received

no feedback concerning whether their responses were correct or incorrect.

Statistical analysis.

All data are expressed as means±SEM. Statistical analyses were carried out with Prism software

(Graphpad Software, San Diego, USA). For immunohistochemistry data, we used Student's t-test.

Behavioral data were analyzed with parametric methods: unpaired Student's t-tests were used to

compare the two groups, with unpaired observations to assess social interaction. Spontaneous

discrimination and olfactometer data were analyzed by standard two-way analysis of variance

(ANOVA) followed by unpaired or paired Student's t-tests, as appropriate.

SUPPORTING INFORMATION Top

Figure S1.

Irradiation did not alter the morphology of DCX+ cells reaching the OB. Dendrites were counted

for DCX+ cells in the GCL, EPL and GL of sham and irradiated mice, 7 months after irradiation.

P>0.05 with Student's t-test (n = 25 from 5 random cells analyzed per OB layer and from 5 mice

per group).

(2.75 MB EPS)

Figure S2.

Focal SVZ irradiation inhibited the recruitment of new neurons in the OB but not in the

hippocampus. New cells were labeled with BrdU 3 days after the final focal irradiation and

survival was evaluated 11 days later. The mean number of BrdU+ cells in the GCL of the OB and

in the dentate gyrus of the hippocampus was determined for sham and irradiated mice. **

indicates p<0.01, Student's t test, n = 6 mice per group.

(4.54 MB EPS)

Table S1.

Complete statistical analysis on neurogenesis data.

(0.04 MB RTF)

Table S2.

Complete statistical analysis on spontaneous discrimination.

(0.08 MB RTF)

Table S3.

Complete statistical analysis for operant conditioning.

(0.11 MB RTF)

Table S4.

Statistical analysis on 2-odor memory test.

(0.03 MB RTF)

ACKNOWLEDGMENTS Top

We thank Jean-Baptiste Lahaye and Karine Sii-Felice for technical assistance with irradiation,

Marie-Madeleine Gabellec for histological advice, and Sébastien Wagner for designing the

computer-driven olfactometers.

AUTHOR CONTRIBUTIONS Top

Conceived and designed the experiments: FL MAM GG FDB PML. Performed the experiments: FL

MAM SL FDB. Analyzed the data: FL GG PML. Contributed reagents/materials/analysis tools: FdC

JCOM DNA. Wrote the paper: FL GG PML. Carried out irradiation, all behavioral experiments and

histological methods for quantifying adult neurogenesis: FL. Designed and carried out irradiation

experiments: MAM FDB. Designed the image analysis software: FdC JCOM. Helped with

immunohistochemistry experiments: SL.

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ARTICLE ONLINE

Early Experience in the Treatment of Intra-Cranial Aneurysms by Endovascular Flow Diversion: A Multicentre Prospective Study

James V. Byrne1*, Radu Beltechi1, Julia A. Yarnold1, Jacqueline Birks2,

Mudassar Kamran1

1 Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom, 2 Centre for Statistics in

Medicine, University of Oxford, Oxford, United Kingdom

Abstract Top

Introduction

Flow diversion is a new approach to the endovascular treatment of intracranial aneurysms which

uses a high density mesh stent to induce sac thrombosis. These devices have been designed for

the treatment of complex shaped and large size aneurysms. So far published safety and efficacy

data on this approach is sparse.

Material and Methods

Over 8 months, standardized clinical and angiographic data were collected on 70 patients

treated with a flow diverter device (SILK flow diverter (SFD)) in 18 centres worldwide. Treatment

and early follow up details were audited centrally. SFDs were deployed alone in 57 (81%) or with

endosaccular coils in 10 (14%) aneurysms, which included: 44 (63%) saccular, 26 (37%) fusiform

shapes and 18 (26%) small, 37 (53%) large, 15 (21%) giant sizes. Treatment outcome data up to

30 days were reported for all patients, with clinical (50 patients) and imaging (49 patients) follow

up (median 119 days) data available.

Results

Difficulties in SFD deployment were reported in 15 (21%) and parent artery thrombosis in 8

(11%) procedures. Procedural complications caused stroke in 1 and serious extracranial bleeding

in 3 patients; 2 of whom developed fatal pneumonias. Delayed worsening of symptoms occurred

in 5 patients (3 transient, 1 permanent neurological deficit, and 1 death) and fatal aneurysm

bleeding in 1 patient. Overall permanent morbidity rates were 2 (4%) and mortality 4 (8%).

Statistical analysis revealed no significant association between complications and variables

related to treated aneurysm morphology or rupture status.

Conclusion

This series is the largest reporting outcome of the new treatment approach and provides data for

future study design. Procedural difficulties in SFD deployment were frequent and anti-thrombosis

prophylaxis appears to reduce the resulting clinical sequelae, but at the cost of morbidity due to

extracranial bleeding. Delayed morbidity appears to be a consequence of the new approach and

warrants care in selecting patients for treatment and future larger studies.

Citation: Byrne JV, Beltechi R, Yarnold JA, Birks J, Kamran M (2010) Early Experience in the Treatment of

Intra-Cranial Aneurysms by Endovascular Flow Diversion: A Multicentre Prospective Study. PLoS ONE 5(9):

e12492. doi:10.1371/journal.pone.0012492

Editor: Maria A. Deli, Hungarian Academy of Sciences, Hungary

Received: May 21, 2010; Accepted: July 14, 2010; Published: September 2, 2010

Copyright: © 2010 Byrne et al. This is an open-access article distributed under the terms of the Creative

Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium,

provided the original author and source are credited.

Funding: This study was supported by a grant made to the Nuffield Department of Surgery, University of

Oxford by Balt Extrusion. The funders were consulted in the study design but had no executive role in the

final protocol. They had no role in data collection and analysis, decision to publish, or preparation of the

manuscript.

Competing interests: This study was supported by the manufacturer of the SILK flow diverter (Balt

Extrusion) through a grant made to the Nuffield Department of Surgery, University of Oxford. They informed

the Oxford Neurovascular and Neuroradiology Research Unit (ONNRU) of centres using the Silk Flow Diverter

and ONNRU invited the centres to participate, and independently collected and audited data. The sponsor

was shown the final manuscript and invited to make comments at the draft stage which the authors were

free to accept or reject in drafting the manuscript. The authors declare the following: JV Byrne has acted as

an unpaid scientific advisor for Siemens AG, a trainer for Boston Scientific Corporation and is a scientific

advisor board member for Codman Corporation. M Kamran is funded by the Rhodes Trust. J Birks is funded by

the Oxford Biomedical Research Centre.

* E-mail: [email protected]

INTRODUCTION Top

Coil embolisation has proved an effective treatment for most intracranial aneurysms but larger

aneurysms, fusiform shaped aneurysms, and those with wide necks are technically challenging.

Such aneurysms are also more liable to recurrence which occurs in 20–30% [1] and results in 10–

12% of patients requiring retreatment [2]–[3]. Adjuvant stenting was proposed in the mid-1990's,

initially for fusiform aneurysms [4] and more recently to support the neck region of intracranial

saccular aneurysms and to prevent recurrence [5]. The principle that a stent placed in the parent

artery can reduce blood flow in the sac of an aneurysm to the point of stagnation and thrombosis

has been exploited for sometime in other anatomies and has now been extended to the

intracranial vasculature in a new range of implants designed to be sufficiently flexible for

intracranial navigation.

These new devices, termed flow diverters, recently became available for clinical use. The SILK

stent (Balt Extrusion, Montmorency, France) was approved in 2007. This self-expanding flexible

stent is constructed of woven nitinol strands with low porosity, to retain blood flow in the parent

artery and exclude the aneurysm sac. In order to assess the safety and efficacy of the SILK flow

diverter (SFD), a multicentre registry was established. The intention of this study is to assess the

device's performance in routine clinical practice by collecting data from as many users as

possible.

MATERIALS AND METHODS Top

Methods

a). Patient recruitment.

All centres using the SFD during 8 months (March to November, 2008) were invited to send

anonymised and coded data to the Oxford Neurovascular and Neuroradiology Research Unit

(ONNRU). Thirty centres world-wide used the device during this period. Eighteen participated and

returned reports of consecutive treatments using a standard case report form (CRF). The study

protocol defining data to be collected was established prior to recruitment and prior to most

invitations.

Patients were selected for treatment locally at the treating centre on the basis that the target

aneurysm was unsuitable for conventional endovascular or neurosurgical treatments (see Figure

1). No details of the patients' age and gender were collected to maintain absolute anonymity.

The following exclusion criteria were set by the study Steering Committee: any contraindication

to antiplatelet drugs, pregnancy, breast feeding, and aneurysms considered treatable with coils

alone. The study protocol was defined as clinical audit using the UK National Research Ethics

Service guidance (Defining Research 2007) and did not require research ethics committee

approval. Each centre was responsible for obtaining appropriate permissions for data sharing.

Figure 1. Representative angiograms of

aneurysms treated with the SFD.


Fusiform vertebral artery aneurysm (a), giant saccular aneurysm at the vertebro-basilar junction (b), large

cavernous carotid artery saccular aneurysm (c) and a recurrent cavernous carotid artery aneurysm (d).



b). Data collection.

Prior to treatment, modified Rankin scores (mRS) and Glasgow Coma Scores (GCS) were

recorded. Therapists were asked to record any untoward procedural events (UPE) during

procedures, any acute clinical complication and a GCS after treatment. The CRF described UPEs

as; catheterization failure, poor SFD positioning, SFD migration, poor SFD opening on

deployment, haemorrhage during or after the procedure, complications related to other devices,

and partial or complete thrombosis of the parent artery.

A standard description of the aneurysm to be treated was made. These details included: rupture

status, location, saccular or fusiform shape, and sac dimensions (neck width, maximum

diameter, and dome to neck length). Details of prophylactic anti-thrombosis therapies were

reported. These included: the dose and type of drugs used, and the duration of prescriptions.

c). Follow-up protocols.

A standard report of each patient's clinical status was requested at the time of follow-up imaging.

In order to assess the effectiveness of the SFD at inducing thrombosis, it was requested that

follow-up catheter angiography was performed 1 month after stopping antiplatelet therapy. The

timing of interval imaging was therefore left to the centres. The follow-up period range was 9–

528 days (median 119 days). Assessments of the degree of aneurysm thrombosis after SFD

placement were rated as complete occlusion (OG1), neck remnant (OG2) and residual sac filling

(OG3), at end-of-treatment and follow-up [2].

d). Statistical analysis.

Potential associations between the procedural and delayed complications and the variables

related to aneurysm morphology, presentation, and use of adjuvant coils were explored using

Fisher's exact test (aneurysm shape, use of coils, location, and previously ruptured vs unruptured

aneurysms) and the Pearson's chi-square test (sac size).

Materials

a). Patients.

Seventy patients were entered in the study. Patients' prior degrees of disability and symptoms

due to the aneurysms were: mRS = 0 in 30, mRS = 1 in 19, mRS = 2 in 12, mRS = 3 in 2, mRS =

4 in 6 and mRS = 5 in 1 patients. Thus 30(43%) treatments were performed for patients without

symptoms, 31(44%) with symptoms but no or only mild disability and 9(13%) with moderate or

significant disablity i.e. mRS 3–5.

b). Aneurysms and SFD Sizing.

Sixty target aneurysms were unruptured and 4 of the 10 ruptured aneurysms were treated within

30 days of haemorrhage. They comprised 44(63%) saccular and 26(37%) fusiform shapes. There

were 15(21%) giant (>25 mm), 37 (53%) large (10–25 mm) and 18 (26%) small (<10 mm)

aneurysms. Details of target aneurysms are presented in Table 1. Multiple aneurysms were

identified in 5 patients (4 with 2 and 1 with 3 aneurysms). One patient with 2 aneurysms had

both treated with the SFD in separate procedures (without complication) and another patient was

treated for a co-incidental aneurysm with coils alone at the same procedure. These treatments

have not been included in the analysis of primary treatment outcomes. The remaining co-

incidental aneurysms were not treated during the study period.

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012492?imageURI=info:doi/10.1371/journal.pone.0012492.t001

Table 1. Aneurysm locations presented as maximum sac size, neck width, and morphology.

doi:10.1371/journal.pone.0012492.t001


c). Centres.

The number of treatments performed in contributing centres varied from 1–9. Treatments per

centre were: 1 in 2, 2 in 6, 3 in 2, 4 in 1, 5 in 3, 7 in 2, 8 in 1 and 9 in 1. Thus half the centres

performed only 1–3 procedures and for most these were their first using the SFD.

d). SFD Deployment Technique.

Sixty treatments were performed with SFD alone and 10(14%) with adjuvant endosaccular coils.

Additional coils were used for parent vessel occlusion during 1 treatment. During the study

period many centres were supported by a proctor introduced by the manufacturer and

treatments were performed under general anaesthesia according to the current instructions-for-

use of the SFD. Briefly, the technique involves positioning a delivery microcatheter (Vasco 21,

Balt, Montmorency, France) with its tip distal to the aneurysm and then pushing the SFD, which

is applied to a delivery microwire, to the tip of the delivery microcatheter. The system is then

aligned with the aneurysm under x-ray fluoroscopy and the SFD deployed by unsheathing it from

the constraint of the microcatheter. This involves a combination of pushing the delivery wire and

retrieving the microcatheter to allow the SFD to expand and to compensate for any resulting

fore-shortening. The SFD can be retrieved into the microcatheter and removed or repositioned

when less than 80% of its length has been extruded. No retrieval is possible thereafter.

e). Antiplatelet regimens.

All centres prescribed heparin and antiplatelets during treatments. Antiplatelet prescriptions in

the majority of patients were a combination of aspirin and clopidigrel (46 pre-treatment and 49

post-treatment) or aspirin and dipyridamole (19 pre-treatment and 11 post-treatment). Single

therapy with aspirin (2 pre-treatment and 3 post-treatment) or clopidigrel (3 pre-treatment and 7

post-treatment) was used in a minority. Reports of post-treatment prescriptions were incomplete.

Reported intervals to stopping clopidigrel or dipyridamole after treatments were: after 2 months

in 5 patients, after 3 months in 3 patients, after 4 months in 17 patients, after 5 months in 5

patients, and after 6 months in 9 patients.

RESULTS Top

a). Procedures

Sixty seven (96%) primary treatments were completed. In 3(4%) treatments, an SFD could not be

deployed for technical reasons and the procedures were abandoned. These were: failure to

catheterise the aneurysm bearing artery, inability to open the SFD correctly and failure to

advance a long SFD through the delivery catheter. In the last case, the patient was successfully

treated with two shorter SFDs after the study period ended. Another 3(4%) patients required

repeat procedures because of suboptimal SDF deployment. In 2, the initial SFD shortened and

another SFD was placed at a second procedure without complication. In the third, positioning 2

SFDs in a dolichectatic artery failed due to foreshortening and a second procedure was

performed a few days later with an additional LEO stent (Balt Extrusion). Arterial patency was

confirmed after 48 hours but the patient died from pneumonia 1 month later.

b). Untoward Procedural Events (UPEs)

One or more UPEs were reported in 20 patients. They were: 12(17%) poor SFD opening on

deployment, 7(10%) partial or complete parent artery thrombosis (PAT), 6(8%) poor SFD

positioning, 4(6%) SFD migration, 3(4%) post procedure extracranial haemorrhages and 1(1.5%)

complication related to another device. No intracranial haemorrhage was reported.

Thus, the commonest UPE was poor SFD opening on deployment. In 4 procedures, poor SFD

positioning and in 3 procedures SFD migrations were concurrently reported. Thus overall in

15(21%) of procedures SFD deployment difficulties (DD) were reported and amalgamated for

statistical analysis. The consequences of these events were that 3 patients required repeat

procedures, as described above. In 1 other patient, the operator decided to occlude the parent

artery with coils because of incomplete SFD opening. This was without clinical worsening.

The 7 reports of PAT occurred together with reports of SFD DD in 6 procedures (poor opening in

3, positioning in 1 and both in 2). At the remaining procedure, branch artery thrombosis was

treated successfully with abciximab. Subsequent extracranial bleeding complicated 3

procedures. In 1 patient this occurred after endovascular access via the cervical carotid artery. A

resulting neck haematoma was so serious that the patient required a week of intensive care

treatment and the relevant internal carotid artery was occluded on follow-up angiography. A

significant acute groin haematoma was reported in 1 further patient and 1 patient developed

serious gastric bleeding 48 hours post-procedure and died of pneumonia 2 weeks later.

c). Procedural Morbidity

The GCS of patients immediately prior to treatment were: GCS 15 n = 61 (plus 2 abandoned),

GCS 10–14 n = 5 (plus 1 abandoned) and GCS<10 n = 1. In the 48 hours after treatment, 1

patient worsened (GCS 13 to 5) and 2 patients improved (GCS 14 to 15). Worsening followed a

thromboembolic complication, which caused a new permanent hemiparesis. This was attributed

to thromboembolism caused by adjuvant endosaccular coils. Post-procedure MRI showed a new

infarct in the temporal lobe and delayed angiography showed the SFD to be patent. There were

no procedural deaths reported in the acute period. However, 2 patients whose treatments were

complicated by UPEs, died of pneumonia within 1 month of the procedure. Thus the immediate

procedural related morbidity was 1 new neurological deficit and 2 deaths.

d). Delayed Morbidity

Follow-up reports on clinical outcomes were returned on 50(71%) patients. Delayed worsening of

symptoms and/or new abnormal neurological signs were reported in 6(12%) of these 50 patients.

Transient events in 3 patients occurred within 30 days of treatment and were attributed to

increased mass effect following aneurysm thrombosis. This caused exacerbation of headaches

and in 2 patients (with aneurysms of the cavernous carotid artery) worsening opthalmoplegia.

Permanent deficits developed in 3 patients and were attributed to delayed thrombosis of the SFD

in the first patient (who developed hemiparesis 10 days post-procedure), increased mass effect

in the second (who developed a new facial palsy a few days after treatment of a giant basilar

artery aneurysm and died 2 weeks later due to increased brain stem compression) and delayed

aneurysm bleeding in the third patient. In this last case, follow-up angiography at 5 months

showed residual aneurysm filling. Two months later the patient experienced increasing headache

and acute hemiparesis. CT showed aneurysm enlargement and acute haemorrhage (Figure 2).

The patient died 4 weeks later despite surgical by-pass. He had required long-term

anticoagulation with warfarin because of a prosthetic heart valve and remained on aspirin after

clopidigrel was stopped 2 months following SFD deployment.

Figure 2. Partially thrombosed aneurysm after

treatment with the flow diverter.

CT angiograms showing a residual lumen within a

large partially thrombosed fusiform aneurysm of the

middle cerebral artery. Follow-up CTA (a) was performed 4 months and (b) 6 months after SFD (arrows) placement.

The second follow-up study shows enlargement of the residual aneurysm lumen (arrow heads) and was performed

after a new haemorrhage (not shown).



Thus the overall morbidity reported amongst 50 patients with follow-up was 2 permanent

neurological deficits and 4 deaths, i.e. morbidity 2(4%) and mortality 4(8%). All clinical

complications are summarized in Table 2.



Table 2. Clinical complications observed in patients treated with SFD.



e). Angiographic outcomes

The end-of-treatment (EOT) assessments of the degree of aneurysm occlusion for 68 treated

aneurysms (67 patients) were: OG1 in 7(10%), OG2 in 4(6%) and OG3 in 57(84%) aneurysms.

Reports of follow-up angiograms were returned for 49(72%) aneurysms. The degrees of

aneurysm occlusion on follow-up were: OG1 in 24(49%), OG2 in 13(26%) and OG3 in 12(25%).

Thus all but 2 aneurysms were unchanged or improved on follow up (1 OG1 and 1 OG2 dropped

to OG3 on follow up) (Table 3).

Table 3. Angiographic outcomes for the

aneurysms treated with SFD.



Follow up imaging showed parent artery occlusion (PAO) in 7(14%) and a degree of arterial

narrowing in 3(6%). Three PAOs were associated with UPEs (neck haematoma, DD, and

deliberate PAO with coils), 1 was attributed to non-compliancy with antiplatelet treatment and 3

occurred for no apparent reason. The degree of arterial narrowing reported in 3 cases was not

graded but reported as severe in 1 patient. In 2 cases of arterial stenosis on follow up imaging,

UPEs had occurred during treatments (poor SDF opening and thrombosis requiring salvage

abciximab).

The frequency of complete occlusion (i.e. OG1) and a possible effect of the timing of follow-up

imaging was investigated by dividing the cohort at 20 week intervals post-treatment and


comparing the within-group percentage occlusion grades. This analysis showed that the

proportion of cured aneurysms increased with time intervals (Table 3 and Figure 3).

Figure 3. Timings and results of angiographic

follow up.

Plot of angiographic outcomes against follow up times in weeks.



f). Statistical Analysis

No statistically significant relationship was found between the variables related to aneurysm

morphology, rupture status, use of adjuvant coils and the occurrence of procedural and delayed

complications (Table 4).

Table 4. Effect of patient and procedure

related variables on SFD deployment difficulty,

flow disturbance, and delayed neurological

complications.



DISCUSSION Top

The concept of a stent capable of inducing thrombosis of intracranial aneurysms has stimulated

the development of flow diverters over the last 15 years. Several researchers have demonstrated

their potential to disrupt endosaccular blood flow in experimental aneurysms, and systematic



haemodynamic studies have informed their design [6]. These established 70% porosity (defined

as the proportion of open area to total area of the stent) as the optimum [7]. Lower porosity

covered stents have generally proved too inflexible for intracranial use and results of stent-alone

treatments using conventional high porosity stents have been inconsistent [8]. Some operators

have proposed the use of double or overlapping conventional stents to increase the effective

stent porosity [9] and new covered devices are being tested [10].

Against this background, flow diverters designed with low porosity and high flexibility were first

used in humans in late 2006. To date, the few reports of their efficacy have been encouraging.

Lylyk et al. [11] reported a series of 53 patients treated in Buenos Aires using the Pipeline

(Chestnut Medical Technologies, Inc., Menlo Park, CA). The aneurysm types were predominantly

saccular with only 12% fusiform and nearly half were small in size. Clinical experience with the

same flow diverter was collected on 31 patients treated in the “Pipeline Embolization Device in

the Intracranial Treatment of Aneurysms (PITA) Trial” conducted between January and November

2007 [12]. One of the 4 contributing centres recently reported on 8 additional aneurysms [13].

Few procedure-related neurological complications were reported; a new stroke rate of 6% in the

PITA trial and none in the Buenos Aires report. The latter reported delayed exacerbation of

aneurysm related cranial nerve palsies in 3 patients.

Complication rates reported in this study were similar with only one acute new neurological

deficit despite technical difficulties associated with SFD deployment in 21% of procedures. Like

the Buenos Aires experience, a minority of our patients developed delayed neurological

symptoms. The cause of transient worsening of symptoms after induced aneurysm thrombosis is

generally attributed to aneurysm expansion. However, the low porosity of flow diverters

theoretically increases their likelihood of occluding covered side branch arteries which might also

cause worsening of symptoms [13]. In this registry, partial or complete parent artery thrombosis

affected 7 procedures and was attributed to the SFD deployment difficulties rather than

inadequate antithrombotic prophylaxes since these were given during all the procedures.

However, in 1 case it was caused by the device covering a large branch artery. A cause of 1

delayed complication was parent artery occlusion found on follow-up imaging. The relative

contribution of aneurysm expansion or branch artery occlusion in patients developing new

neurological deficits is therefore difficult to assess. Our experience suggests a real vulnerability

of parent artery blood flow after SFD deployment, perhaps due to a combination of its relatively

low radial force and high metal density, which high dose antiplatelet therapy mitigates.

Treatment using SFDs after acute SAH raises several concerns. Firstly, after SAH there is a

natural reluctance to prescribe antiplatelets prior to securing the aneurysm against rebleeding.

In the minority of patients treated after acute SAH in this study, it was the usual practice to

withhold prophylaxis with antiplatelet treatment until the SFD was deployed; accepting a small

additional risk of thrombotic complications. The second issue is the longer delay to complete

thrombosis of the aneurysm after SFD treatment than after clipping or coiling. During this period,

the patient is at risk of more prolonged bleeding, should the aneurysm re-rupture, because of

antiplatelet medication. One solution is to additionally pack the aneurysm sack with coils. In this

cohort, adjuvant coils were only used in 1 of the 3 saccular aneurysms treated after acute SAH.

Thus, when it proves difficult or impossible to place coils, treating physician have to assume that

the alteration of local haemodynamics induced by the SFD reduces the risk of rebleeding.

Currently, there is insufficient experience to prove this assumption.

The relationship between complications and anti-thrombotic prophylaxis was clear in the patients

who suffered haemorrhagic complications but its contribution to delayed events is complex. In

this cohort, patient non-compliance caused delayed spontaneous artery occlusion and flow

disturbances observed during treatments would probably have resulted in more frequent

thrombotic events without the use of heparin or antiplatelets. Its contribution to the subsequent

evolution of endosaccular thrombosis is unclear and needs further systematic study. It is

tempting to attribute concurrent long-term anticoagulation and aspirin therapy as the cause of

delayed aneurysm enlargement and haemorrhage, seen in one patient. On the same basis,

antiplatelet therapy might contribute to symptomatic worsening attributed to sac swelling

observed in the early post-procedure period. Another unanswered question raised by our results,

is whether it is a factor in the asymptomatic failure of sac occlusion, seen on follow up imaging,

in a minority of aneurysms.

Limitations to this study are incomplete follow-up imaging, no longitudinal data and the relatively

short follow up period. The 50% rate for complete occlusion up to 3 months is similar to the

Buenos Aires experience [11]. Their longitudinal follow-up suggested that induced thrombosis

was progressive, with the 3-month 56% rate of complete occlusion rising to 95% at 12 months

[11]. Progressive improvement in occlusion rates were reported by Zentano et al. [14] and no

recurrence was reported for Pipeline treated aneurysms [13]. Our findings are similar but

because our data are based on a single follow up time point, between-patients variations cannot

be differentiated from variations over time. The subsequent behaviour of those aneurysms

followed up early is unknown. The aneurysms reported here comprised a heterogeneous group,

with a higher proportion of fusiform type and posterior circulation aneurysms than the series of

Lylyk et al. or Szikora et al. [11], [13]. A concern is two aneurysms whose occlusion grades

dropped relative to the EOT grade and a large study of sufficient numbers will be needed to show

the long-term stability of thrombosis of different aneurysm types. This concern has resulted in a

recent change in the manufacturer's instructions for use and the SFD should now only be used to

support embolisation with coils.

The conclusions that can be drawn from the results of this study need to be considered with

reference to its objectives and methodology. The intention was to collect data on the use of the

SFD during a period when it was being first used in a large number of hospitals, by experienced

practitioners who had either not previously used the device or had only done so in very few

patients.

The centres contributed data on a voluntary basis and deserve our thanks. A registry can

highlight early technical and clinical problems which would take much longer to identify without

amalgamated data. Complete procedure reports allowed early recognition of deployment

problems leading to a redesign of the SFD and an alteration in its instructions for use. The

associations tested, though not statistically significant, should help to inform the design of future

studies and devices. Several technical improvements have since been made to improve the

SFD's visibility on x-ray fluoroscopy and deployment control. A new guide pusher has now been

developed in addition to a new radio-opaque marker, which is more visible than the original

version. However, conclusions about long-term efficacy must be tentative, because a possible

bias to under reporting of poor outcomes cannot be excluded, without complete follow-up data.

Further studies are needed to define the place of flow diverters in the endovascular treatment of

aneurysms and it is too early to predict their ultimate role.

ACKNOWLEDGMENTS Top

We thank all the contributing centres to the SILK registry. Representatives of these centres were:

E. Akgul, S. Bakke, A. Bonafe, P. Brouwer, R. Chapot, S. Cekirge, P. Fourie, G. Gal, T. Goddard, R.

Juszkat, K. Kupcs, M. Leonardi, L. Lemme-Plaghos, O. Levrier, S. Margus, L. Remonda, W. Weber.

AUTHOR CONTRIBUTIONS Top

Conceived and designed the experiments: JVB. Analyzed the data: JB MK. Contributed

reagents/materials/analysis tools: RB JY. Wrote the paper: JVB MK. Principal investigator: JVB.

Collected the data: RB JY MK.

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Cranial function in a late Miocene Dinocrocuta gigantea (Mammalia: Carnivora) revealed by comparative finite element analysisAbstract

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Abstract

INTRODUCTION

MATERIAL AND METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

REFERENCES

Appendix

Carnivoran ecomorphologies evolved repeatedly during the Cenozoic. Whereas extreme forms (e.g. sabretoothed predators) probably represent similarities in ecology, other morphologies are more subtle with respect to the extent of their shared niche space. Finite element models of the skulls of Dinocrocuta gigantea, Canis lupus, and Crocuta crocuta were constructed to test the interpretation of D. gigantea as a bone cracker, an interpretation made on the basis of its large, conical premolars, and robust cranial morphology. Dinocrocuta gigantea is also of interest because it represents a lineage that has been placed in its own family, sister to Hyaenidae. Thus, functional similarity in craniodental performance could represent rapid convergence. The findings obtained indicate that the crania of D. gigantea and C. crocuta perform better in stress dissipation and distribution than that of C. lupus, regardless of P3 or P4 biting. In particular, the domed frontal region of the bone crackers received lower and more evenly distributed stress than C. lupus. Thus, the craniodental forms of the two bone-crackers are linked by functional advantage over that of C. lupus. Further examination of lineages such as borophagine canids could elucidate the extent of functional convergence of the bone-cracking ecomorph across diverse groups. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 51–67.

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INTRODUCTION

Top of page

Abstract

INTRODUCTION


RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

REFERENCES

Appendix

Our understanding of functional morphology in fossil carnivorans has been greatly augmented by studies of morphospace and ecomorphology in both extant and extinct predator guilds (Van Valkenburgh, 1988, 1989, 1999, 2001; Werdelin, 1996). General categories have been established based on correlations between craniodental form and diet in living carnivorans, and a similarity in craniodental form between living and fossil taxa. One example of an iteratively evolved mammalian carnivore ecomorphology is the bone-cracking predator, which has been characterized on the basis of craniodental morphology (Werdelin, 1989) and enamel microstructure specialization (Rensberger, 1995; Stefen & Rensberger, 2002; Ferretti, 2007). Bone cracking carnivorans are best represented today by members of the family Hyaenidae. Although the generalizations are well established, no study has compared hyaenid craniodental forms in their response to mechanical stress imposed on the skull during bone cracking behavior. This study presents a comparative analysis of a purported fossil bone cracking carnivoran, Dinocrocuta giganteaSchlosser, 1903, the extant bone cracking spotted hyena Crocuta crocutaErxleben, 1777, using the extant grey wolf Canis lupusLinnaeus, 1758 as a non-bone-cracking hypercarnivore for comparison. It is hypothesized that the craniodental morphology of Dinocrocuta is more suited for bone-cracking in terms of its stress-dissipating architecture because of the highly domed frontal region, than the spotted hyena. Both would perform better under loading conditions simulating bone-cracking than the grey wolf, which is a meat specialist with capability for bone-crushing with molars (for a definition of crushing versus cracking, see Werdelin, 1989) but with a very shallow forehead.

To test the functional hypothesis regarding the capability of the cranium for bone cracking, the engineering technique finite element modelling is utilized (Laitman, 2005; Richmond et al., 2005; Ross, 2005). The cranial models are compared solely on craniodental form, with element volumes standardized. Because the goal of the present study is to examine the distribution and concentration of reaction forces in the cranium, stress (force per unit area) is used instead of

strain (change in length relative to original length), a measure of deformation. The measure used to evaluate craniodental function in stress dissipation is the Von Mises stress, which is a scalar function incorporating principal stress in the three orthogonal planes of a three-dimensional (3D) object. Von Mises stress is also used as a criterion to evaluate how close an object is to failure (i.e. it is directly comparable to yield strength of the object; Irons & Ahmad, 1980). The expectation is that both the median Von Mises stress of the entire cranium, as well as the maximum Von Mises stress in the frontal-parietal region, hypothesized to serve an important function in stress dissipation (Buckland-Wright, 1978; Werdelin, 1989), would be lowest in Dinocrocuta, higher in Crocuta, and highest in Canis.

As a more recently applied methodology in the field of evolutionary biology, finite element modelling has demonstrated high potential as a tool to understand kinematics of forms (Rayfield et al., 2001; Rayfield, 2004; Dumont, Piccirillo & Grosse, 2005; Laitman, 2005). The fundamental principle of this technique is to sufficiently recreate a representation analysable by computers from an extremely complex natural structure such as the skull. To build a finite element model, the basic steps include: (1) the morphology of object of interest (shape reconstruction); (2) the characteristics of the material composition of the object that governs the behavior of that object under mechanical loads (material properties); and (3) a scenario in which the object is being loaded with mechanical force, and how much force is involved (boundary conditions). Modelling of complex objects simplifies the problem at hand by making assumptions. Bone, a living and dynamic tissue, would require highly complex models to be portrayed accurately. By starting only with shape, or craniodental morphology, the present study eliminates many details of bone biomechanics (e.g. different material properties for cancellous versus cortical bone; different properties in different load directions or anisotropy; bone sutures, contact between bone and tooth, etc.). However, the simplifying assumptions employed in the study means that fossils are made comparable to extant specimens by standardizing the methods of modelling. Furthermore, in any comparative analyses involving fossil material, the quality of the data is often constrained by fossil quality because many anatomical features might be incomplete or missing altogether. Assumptions made under these considerations are discussed in more detail in the model building protocol below.

The institutional abbreviations used in the present study are: IVPP, Institute of Vertebrate Paleontology and Paleoanthropology, Beijing, China; LACM: Natural History Museum of Los Angeles County (mammalogy department), Los Angeles, CA, USA; UCLA, University of California, Los Angeles, CA, USA.


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Abstract

Jump to…

INTRODUCTION


RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

REFERENCES

Appendix

Data acquisition and processing

An undeformed skull of D. gigantea (IVPP V15649) was used. Although not all fine internal structures of the bone are preserved, the specimen represents the best preserved skull of the species. The skull retains cranium and associated mandibles; the specimen comes from the late Miocene beds of Fugu, Shaanxi Province, in northern China. The associated cranium and mandibles retain a complete dentition, which is fully erupted but unworn, representing a sub-adult or young adult equivalent compared to C. crocuta. From the developmental stage of the cranial bones in comparison with other specimens of the same species, it was determined that this individual also does not have a fully grown cranial region. Thus, a skull of the extant spotted hyena C. crocuta (LACM 30655) in a similar stage of ontogenetic development was chosen for analysis and comparison. In addition, an adult grey wolf C. lupus (LACM 23010) skull was chosen as a hypercarnivore comparison.

All three skulls were scanned by computed tomography (CT) using a Siemens Definition 64 scanner (Siemens Medical Solutions) at UCLA Medical Center. Specimens were scanned with 0.6 mm slice thickness and a 0.6 mm interslice distance. This produced 616 images for D. gigantea, 464 for C. crocuta, and 499 for C. lupus. The data were then imported into the image processing software programs Amira (Visage Imaging, Inc.) and VGStudio Max (Volume Graphics GmbH), in which a combination of automated thresholding operations and manual delineation were used to identify the craniodental morphology from the image scans. For the D. gigantea skull, the internal cavities were filled with inorganic matrix during burial, and the matrix has approximately the same density as the fossilized bone. Thus, manual editing of bone boundaries, visible as small gaps and fissures between fossil bone and rock matrix, was performed for all image slices. To ensure a faithful reconstruction of the fossilized bone, the slices were edited thoroughly in all three planes (axial, coronal, and sagittal). Once the regions of interest were defined, 3D representations of the crania were reconstructed.

The reconstructions were then imported into the rapid prototyping software program Geomagic Studio 9.0 (Geomagic, Inc.), which allowed operations that improved the quality and consistency of the reconstructions. The cranial reconstructions were refined, holes filled, and then decimated

to 300 000 triangles, which formed the basic elements of the 3D surface reconstruction. The fossil specimen required extensive modification such as removing sharp artefacts in internal bone boundaries created during manual delineation. The mandible reconstructions were modified and cleaned separately from the cranium, but a correct orientation of articulation preserved to allow for modelling of muscle forces in the final models.

Finite element model building

The refined reconstructions were then imported into the finite element analysis software STRAND7 (G+D Computing Pty Ltd, Sydney, Australia). In this software, the reconstruction was again checked for errors such as sharp angles between triangles and very large aspect ratios. In addition, the basic triangles of the surface reconstruction were zipped and extraneous nodes removed to create a single continuous surface. This structure was then transformed into a solid mesh of four-noded tetrahedral elements. The solid mesh reconstructions of Dinocrocuta and Canis were then standardized to that of the C. crocuta mesh, which had an elemental volume of 326 625 mm3. This was carried out in the STRAND7 software program by dividing the elemental volume (calculated by the software using the model summary function) of the C. crocuta model by the volume of the model to be standardized to get a ratio; next, the model of interest was re-scaled with the cubic-root value of this ratio in each of the x, y, and z axes to obtain the standardized volume. This step standardized the amount of craniodental material represented by the finite elements, and allowed differences in analytic results to be attributed to shape differences represented by the cranial models. This approach is appropriate for the hypothesis being tested because, once the amount of craniodental material present in all three cranial models is standardized, the analyses could address how remaining morphological differences such as frontal shape and thickness affect the stress-dissipating function of the structures during unilateral premolar biting.

After a reconstruction of the original morphology was achieved, the solid mesh needed to be assigned (1) material properties and (2) boundary conditions (constraints and loads). Although voxel-based techniques are now able to import density differences (as Hounsfield units) in CT data directly into the final model for assigning multiple material properties (McHenry et al., 2007), this was simply not practical for more typical fossil specimens. For most fossil skulls, including the D. gigantea investigated in the present study, many minute details of the cranium have been obliterated by diagenesis, and cavities are often filled by different minerals. These modifications are amplified in the scanning process and localized diagenesis can create density differences in the CT data even along small distances of a single bone. Thus, a direct import of the density data from CT scans does not create a correct representation for such specimens. To enable direct comparison with extant skulls, the craniodental reconstruction was assumed to represent a homogeneous, isotropic, elastoplastic material. For static analyses, the only required material properties under this assumption are Young's modulus (E) and Poisson's ratio (ν).

The range of Young's modulus values provided in Erickson, Catanese & Keaveny (2002) for birds and mammals is in the range 15–30 GPa, and finite element models used in this study were run

with this range of values at 5-GPa intervals. All returned similar results, but only data using E = 20 Gpa (i.e. the mean for birds and mammals; Erickson et al. (2002): table 1) are presented here. Given the variation in Poisson's ratio depending on state of fatigue of the test specimen (Pidaparti & Vogt, 2002) and the large range of values (from 0.1 to over 0.5) published for both cranial and post-cranial bones of mammals (Reilly & Burstein, 1975; Peterson & Dechow, 2003; Peterson, Wang & Dechow, 2006; Wang, Strait & Dechow, 2006; Shahar et al., 2007), a range of Poisson's ratio values were tested. When the same models were run using ν = 0.1 to 0.5 in 0.1 intervals, the median Von Mises stress values decreased between 1% and 4% for every 0.1 increase in ν (data not shown). A sensitivity study is currently in progress to further quantify the effect of changing ν values on model outcomes. All data presented here used a mid-range Poisson's ratio of 0.3. Because the data are interpreted comparatively, the conclusions made here are not likely to be altered by slight changes in the exact Poisson's ratio used. All three models were assigned identical material properties. Using an estimated density of 2 mg mm−3 for dog cortical bone (Cowin, 1989), together with standardized bone volumes, the final models had a skull mass close to 653.25 g (see Appendix, Table A1). Model skull mass exceeds the actual dry skull mass by 137% in C. crocuta and by 193% in C. lupus. This difference imparts additional strength in the skull models by treating all skull bone (and teeth) as cortical bone; therefore, the absolute stress values obtained in the analyses are probably lower than would be present in actual skulls.

The boundary conditions included constraints at three locations on the cranial models: (1) left temporomandibular joint; (2) right temporomandibular joint; and (3) tooth of interest. The temporomandibular joints were fixed from any movement by ten arbitrary fixed nodes on each joint, approximately representing the length of contact between the glenoid and the mandibular articular processes. In addition, the tip of the tooth of interest (e.g. P3 for bone-cracking) was fully constrained from movement. The main cusp of P3, paracone of P4, and paracone and metacone of M1 were constrained in the respective models. These boundary conditions are meant to represent the simulation of an instantaneous linear static load applied to a food item (i.e. bone) at the moment of peak force applied through the tooth of interest by actions of the temporalis and masseter muscles.

Finally, the loads applied to the models were the contracted muscles of the temporalis and masseter on both sides of the cranium. The pterygoid muscles were not modelled because empirical data are lacking for extant carnivorans, and thus introducing estimated forces would only increase uncertainty in these models, in particular for Dinocrocuta. The muscle forces were simulated by creating thin plates over the area of bone where the respective muscles originate. The insertion sites of the temporalis and masseter were identified by bone rugosities where muscles attach on the mandibles and from muscle dissection of an extant specimen of Hyaena hyaena (LACM freezer catalogue number 42206) conducted at the LACM. The software program BONELOAD (Grosse et al., 2007) was used to create tangentially oriented forces on the cranium, simulating the wrapping of the muscles around the cranium (Fig. 1). The insertion directions of those two masticatory muscles on the mandibles were pointed toward the center of the ascending ramus for temporalis, and at the midpoint of the lateral ridge ventral of the mandibular fossa that

extends posteriorly to the angular process for masseter, respectively. This method simulates the approximate pulling direction of the contracting musculature, but does not account for differences in muscle fiber angles that might exist between groups within each muscle.

Figure 1. Finite element model of Dinocrocuta gigantea with muscle insertion areas of the temporalis and masseter created (dark grey areas) using the BONELOAD program (Grosse et al., 2007). Anterior dorsolateral view. The length of the cranium is approximately 322 mm. Other models were constructed similarly by demarcating regions of temporalis and masseter attachment. Mandibles were included for identification of resultant muscle force directions, and then removed before analyses were run.

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As all three models were scaled to approximately the same element volume, that of C. crocuta, and the specimen of Crocuta used represents a sub-adult with fully erupted permanent dentition but with still developing cranial bones, a bite force of 318.15 N at the P4 was used in all analyses. This bite force was derived from the regression equation of empirical bite force data taken by Binder & Van Valkenburgh (2000) calculated for a 12-month-old captive spotted hyena:

Each model was loaded with an arbitrary 1000 N total muscle force, with the proportions of muscle activation set as described above. Because bite force measurements were taken with a fork force transducer by Binder & Van Valkenburgh (2000), which converts vertical displacement of the forks to change in detected current, the resulting values represent force perpendicular to the plane of occlusion. Thus, the resulting bite force produced at the cusp tip of P4 paracone, perpendicular to the tooth occlusal plane, was analysed. The new resultant total muscle force was calculated as below (Dumont et al., 2005), and then distributed according to the muscle activation scheme:

where (Ft)new is the resultant muscle force required to produce Fexp; Fexp is the experimentally measured force, in this case 318.15 N from Binder & Van Valkenburgh (2000); and FRN is the resulting bite force from the initial arbitrarily chosen total muscle force, Ft.

The relative proportions of muscle activation between the temporalis and masseter, and between the working and balancing side muscles in unilateral biting, can affect the model results. In all the models constructed, a 60% difference between the balancing side and working side muscle activation was used. This value is based on Dessem's (1989) empirical data for domestic dog,

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which showed that, during unilateral bone-crushing with M1, the balancing side muscles acted at 60% of the maximum recorded electromyographic activity. Although quantitative data have been collected on muscle recruitment in cats (Gorniak & Gans, 1980), the Dessem study included M1 bone-crushing behavior, under which loading condition better approximates bone-cracking than one feeding on soft tissue only (as in the cat study). Thus, the total required muscle force to produce a bite force of 318.15 N was distributed with the balancing side muscle force being 60% of working side muscle force. Next, the division of forces between the temporalis and masseter muscles on each side was made proportional to the estimated cross-sectional areas of the respective muscles, using the photography protocol outlined in the dry skull method (Thomason, 1991; Wroe, McHenry & Thomason, 2005). Photos of dorsal and ventral cranium were taken perpendicular to the plane of muscle cross-section as in Thomason (1991), and the area of the plane measured using IMAGEJ (Rasband, 1997–2007). A summary of the muscle force values used in the analysis is provided in the Appendix (Table A1).

Data analysis

The biting scenarios examined were: (1) unilateral P3 biting, simulating a bone-cracking bite; (2) unilateral P4 biting, simulating a shearing bite for all three models; and (3) unilateral M1 biting, simulating a crushing molar bite in the wolf C. lupus. All scenarios were analysed for both left and right unilateral biting to identify any asymmetric biases. In addition, the muscle forces derived from the P4 bite force calculation were used to model both P3 and M1 biting. Three types of data were collected from the models for each biting scenario: (1) scaled median Von Mises stress of tetrahedral elements in the entire cranium, and their respective deviations; (2) scaled median and maximum Von Mises stress of tetrahedral elements in the frontal-parietal region (‘frontal dome’) where morphological changes have been hypothesized to represent functional adaptation; and (3) change in raw Von Mises stress of tetrahedral elements along the sagittal plane of the frontal dome, representing changes in stress as it is dissipated from the originating tooth to the rest of the cranium.

For the same amount of stress and volume, the models having larger number of tetrahedral elements will have lower raw stress per element. Therefore, the contribution of each element to the overall stress should be made proportional before comparisons are made. Scaled median stresses were calculated after multiplying the tetrahedral element stress results by their respective volumes and then dividing by the median tetrahedral volume (i.e. to eliminate the effect of high stress simply from isolated small elements in the solid mesh, thus leaving high stress correlated with model shape). The scaled values were approximately linear to tetrahedral volume after transformation; thus, differences between models can be attributed to morphological difference in the models.

Because stress distribution in the models was expected to be highly skewed, with most of the elements under little stress, and a few elements sustaining high stress, the descriptive statistics employed include the median, standard error (SE) of the median, median absolute deviation (MAD) from the median, and interquartile range (IQR). All of the above statistics are robust

measures, which are insensitive to outliers caused by model singularities and sharp features; at the same time, these descriptive measures are sufficient in using the entire dataset. Because of variation in model quality across different specimens as well as fossil versus extant taxa, another robust measure of central tendency, the trimmed mean (summing average of dataset by trimming a set percentage from the ends of the data), was not used. The reason is difficulty in objectively delineating singularities versus ‘real’ data consistently in all three models. All statistical summaries were calculated in the software program JMP IN (SAS Institute).

For mid-sagittal plane point sampling, data were collected in each of seven mid-sagittal landmarks. Moving posteriorly, stresses from single nodes were sampled from the mid-sagittal point at the position of: (1) the mid-sagittal anterior edge of nasal bones; (2) the central point of the infraorbital foramen; (3) the most anterior point of the orbits; (4) the tip of the post-orbital processes; (5) the point of maximal post-orbital restriction of the frontal-parietal region; (6) the anterior-most point of the sagittal crest; and (7) the posterior-most point of the sagittal crest. The single nodes recorded stress at these specific landmarks; each was sampled five times. In addition, mean stresses for ten nodes within a circular area around each landmark, covering approximately 6 mm in diameter, were sampled.

RESULTS

Top of page

Abstract

INTRODUCTION


RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

REFERENCES

Appendix

Within-model comparisons

For Canis lupus, no consistent stress pattern could be discerned from values measured on the entire cranium for the different biting scenarios (Table 1). Furthermore, left and right biting with the same tooth position vary only slightly. P3 biting appears to result in higher scaled median stress compared to P4 biting, but not M1. The dispersion of stress values is comparable across all

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scenarios as described by the MAD and IQR. The results from the frontal dome, however, show that the scaled median stresses for all biting scenarios are at least doubled from the recorded values for the entire cranium. In addition, the dispersion of stress values also increases in the frontal region. The maximum stress is comparable for all biting scenarios. IQR increases slightly for scaled stress in the frontal region. In the Dinocrocuta model, all stress measures are comparable across biting scenarios for the entire cranium (Table 2). Only small differences were found between left and right sides. The scaled median stress increases slightly for the frontal dome. IQR decreases from the entire cranium to the frontal region in all cases. Both the scaled and absolute maximum stresses are comparable across all scenarios. The median scaled stress of the entire cranium has an overlapping range between P3 and P4 analyses in the Crocuta model (Table 3). By comparison, the frontal dome has a slightly increased median stress. The maximum stress in the inter-orbital region is comparable for P3 and P4 biting. The IQR of scaled median stress increases slightly for the frontal region for all scenarios except left P4 biting. Only slight differences were found between left and right sides.

Table 1. Descriptive statistics of Von Mises stress in the Canis lupus finite element model

lP3 rP3 lP4 rP4 lM1 rM1

Data shown are for both the entire cranium as well as the frontal region. Scenarios tested include third premolar (P3), fourth premolar (P4), and first molar (M1) biting. Both right and left side unilateral loading cases were analysed. IQR, interquartile range; MAD, median absolute deviation from the median; MPa, megapascal; SE, standard error of the median. For a definition of the terms, see text.

Entire model

Median scaled ± SE (MPa)0.5440 ± 0.0019

0.5061 ± 0.0018

0.4872 ± 0.00170.4257 ± 0.0015

0.5203 ± 0.0019

0.4447 ± 0.0017

Scaled MAD (MPa) 0.45 0.42 0.41 0.36 0.44 0.38

Scaled IQR (MPa) 1.28 1.20 1.17 1.01 1.26 1.12

Frontal dome


1.0956 ± 0.0194

1.1824 ± 0.02280.8423 ± 00155

1.2435 ± 0.0245

1.1508 ± 0.0222



Scaled MAD (MPa) 0.78 0.68 0.77 0.53 0.82 0.74

Scaled IQR (MPa) 1.79 1.58 1.86 1.26 2.00 1.81

Maximum scaled (MPa) 18.33 19.41 20.45 16.07 21.49 23.02

Table 2. Descriptive statistics of Von Mises stress in the Dinocrocuta gigantea finite element model

lP3 rP3 lP4 rP4

Data shown are for both the entire cranium as well as the frontal region. Scenarios tested include third premolar (P3) and fourth premolar (P4) biting. Both right and left side unilateral loading cases were analysed. Abbreviations are as shown in Table 1. For a definition of the terms, see text.

Entire model


0.4813 ± 0.00150.4519 ± 0.0015

0.5108 ± 0.0016

Scaled MAD (MPa) 0.41 0.41 0.39 0.44

Scaled IQR (MPa) 1.24 1.20 1.17 1.28

Frontal dome


0.6576 ± 0.01400.6141 ± 0.0133

0.5904 ± 0.0136

Scaled MAD (MPa) 0.50 0.46 0.43 0.44

Scaled IQR (MPa) 1.18 1.07 1.02 1.04



Maximum scaled (MPa) 6.27 5.63 6.74 6.23

Table 3. Descriptive statistics of Von Mises stress in the Crocuta crocuta finite element model

lP3 rP3 lP4 rP4

Data shown are for both the entire cranium as well as the frontal region. Scenarios tested include third premolar (P3) and fourth premolar (P4) biting. Both right and left side unilateral loading cases were analysed. Abbreviations are as shown in Table 1. For a definition of the terms, see text.

Entire model

Median scaled ± SE (MPa) 0.4074 ± 0.0019 0.3333 ± 0.0017 0.3951 ± 0.0018 0.3704 ± 0.0017

Scaled MAD (MPa) 0.35 0.29 0.33 0.31

Scaled IQR (MPa) 1.21 1.06 1.11 1.09

Frontal dome

Median scaled ± SE (MPa) 0.6950 ± 0.0132 0.5862 ± 0.0118 0.6083 ± 0.0113 0.5769 ± 0.0116

Scaled MAD (MPa) 0.48 0.42 0.41 0.40

Scaled IQR (MPa) 1.25 1.12 1.07 1.10

Maximum scaled (MPa) 57.77 40.44 52.76 48.68

Between-model comparisons

For the entire cranium, median raw stress is highest in Canis and comparable in the other two models for P3 and P4 biting (Figs 2, 3). Scaled median stress of P4 biting is similar in Dinocrocuta and Canis, but higher during P3 biting for Canis (Tables 4, 5). Crocuta had the lowest scaled median stress values for all biting scenarios. The dispersion of stress values (MAD, IQR) of raw stress is

highest in Canis and lowest in Crocuta; however, the scaled stress dispersion is comparable between Dinocrocuta and Canis, and somewhat lower in Crocuta. For the frontal dome, both raw and scale median stress are lowest overall in Dinocrocuta; in the Canis model, the raw and scaled median stress increased by 50% or more over those of the Dinocrocuta and Crocuta models. The same trend is observed for both raw and scaled measures of dispersion (MAD, IQR). The raw maximum stress of Crocuta frontal dome is approximately three times of that in Dinocrocuta, and that of Canis is approximately five times as much as in Dinocrocuta. The scaled maximum stress is highest in Crocuta, and lowest in Dinocrocuta. The highest overall scaled maximum stress in Crocuta is during left P3 biting, and it is close to ten times the scaled stress in Dinocrocuta.

Figure 2. Dorsal views of Von Mises (VM) stress distribution during left P3-biting scenario in the cranium of (A) Crocuta crocuta, (B) Dinocrocuta gigantea, and (C) Canis lupus. All legends are scaled to have a range of 0–8 MPa for optimized visualization. The deeper blue areas represent small or no stress and the red areas represent highly stressed regions. White patches represent areas where stress exceeds 8 MPa. The crania are scaled to approximately the same length in the figure. Right P3 biting and right and left P4 biting scenarios produced similar stress distributions that are not statistically different.


Figure 3. Ventral views of Von Mises (VM) stress distribution during left P3-biting scenario in the cranium of (A) Crocuta crocuta, (B) Dinocrocuta gigantea, and (C) Canis lupus. Legends are as shown in Fig. 2.


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Table 4. Comparative statistics of Von Mises stress in the finite element models of the Crocuta crocuta, Dinocrocuta gigantea, and Canis lupus skulls

Crocuta crocuta Dinocrocuta gigantea Canis lupus

The comparative data are for left P3 unilateral biting. Abbreviations are as shown in Table 1. For a definition of the descriptive statistics, see text.

Entire model

Median scaled ± SE (MPa) 0.4074 ± 0.0019 0.4715 ± 0.0016 0.5440 ± 0.0019

Scaled MAD (MPa) 0.35 0.41 0.45

Scaled IQR (MPa) 1.21 1.24 1.28

Frontal dome


Scaled MAD (MPa) 0.48 0.5 0.78

Scaled IQR (MPa) 1.25 1.18 1.79

Maximum scaled (MPa) 57.77 6.27 18.33



The comparative data are for left P4 unilateral biting. Abbreviations are as shown in Table 1. For a definition of the descriptive statistics, see text.

Entire model


Scaled MAD (MPa) 0.33 0.39 0.41



Scaled IQR (MPa) 1.11 1.17 1.17

Frontal dome


Scaled MAD (MPa) 0.41 0.43 0.77

Scaled IQR (MPa) 1.07 1.02 1.86

Maximum scaled (MPa) 52.76 6.74 20.45

Point sampling along mid-sagittal plane of dorsal cranium

Seven points were chosen to document the change in absolute stress along the mid-sagittal plane, including the frontal region which is highly domed in Dinocrocuta (Fig. 1). All five samples returned similar stress trends; one representative trend from each biting scenario is presented in Figure 4A, B, C. In the Crocuta model, P3 biting creates peak stress in the region between anterior borders of the orbits, whereas P4 biting peaks between the post-orbital processes (Fig. 4A). In Dinocrocuta, the pattern is the same as in Crocuta; the only difference is that the first peak at the anterior orbit boundary is not as pronounced relative to the inter-orbital region of the post-orbital processes as in Crocuta (Fig. 4B). In Canis, two peaks are present in P3 biting: one between the infraorbital foramina, the other at the anterior tip of the sagittal crest (Fig. 4C). For P4 biting, there are also two peaks, but the first one has shifted from infraorbital foramina to inter-orbital region between post-orbital processes. M1 biting shows a similar pattern as the P4 data for Canis. When the mean stresses along the identical landmarks are sampled across a circular area of ten nodes, the same patterns are observed in Crocuta and Dinocrocuta (Fig. 4D, E). However, in Canis, the stress peaks previously observed around the infraorbital foramina and the anterior tip of the sagittal crest became less obvious in the mean sample, but P3 biting still exhibits more abrupt stress increases in those regions (Fig. 4F).

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Figure 4. Von Mises stress gradients from the anterior to posterior cranium along the mid-sagittal plane in analogous anatomical sampling points. Stresses from single node samples of (A) Crocuta crocuta, (B) Dinocrocuta gigantea, and (C) Canis lupus and mean stresses from node group samples of (D) Crocuta crocuta, (E) Dinocrocuta gigantea, and (F) Canis lupus are shown. The data points (from left to right) represent stress recorded along the mid-sagittal plane in lateral alignment with (1) anterior border of nasal bones, (2) infraorbital foramina, (3) anterior boundary of the orbits, (4) the inter-orbital region between the post-orbital processes, (5) post-orbital restriction of the frontal-parietal region, (6) anterior-most point of the sagittal crest, and (7) posterior-most point of the sagittal crest. The points are plotted as percentages of skull condylobasal length (CBL) in the anterior–posterior direction. Left side, filled symbol; right side, open symbol; P3 biting, solid line; P4 biting, dashed line; M1 biting, dotted line.


DISCUSSION

Top of page

Abstract

INTRODUCTION


RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

REFERENCES

Appendix

All within-model results demonstrate that, given the same muscle force input, P3 and P4 biting generate similar stress reactions in all three models, in addition to M1 biting in the Canis model. Although the more posteriorly placed teeth have more mechanical advantage by leverage, the differences in stress distribution that might represent adaptations to specific biting regimes (e.g. P3 bone cracking) are not obvious from the analyses. Thus, the crania of the three carnivorans investigated in the present study cannot be said to have functional advantages for any particular biting scenario tested. It could be that the teeth are simply too close in proximity for the analyses to detect differences in performance (e.g. larger differences in stress magnitude and distribution are to be expected for P4 versus canine biting in all cases by the principle of lever mechanics). Another likely explanation could be that the cranium, a product of complex selective pressure for

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different functions (e.g. protection of brain, tuning of sensory organs, bite force, gape, variation in dental function across tooth row), would not appear optimized for specifically P3 bone-cracking, even if it represents a mechanically demanding task.

Canis lupus

In the Canis model, the median scaled stress of the interorbital region was more than double that of the entire cranium. In part, this could be interpreted as the poor ability of the inter-orbital region to dissipate stress in Canis. What was unexpected, again, is that this doubling of median stress is observed for all three biting cases. One might expect the inter-orbital region to respond differently when biting with the slender P3 versus biting with the carnassial or the bone-crushing M1. By visually checking for artificial sharp features and highly distorted triangular elements using error-checking functions in the STRAND7 Finite element software, it was concluded that no major errors ensued during the creation of the finite element model; thus, it is unlikely that some critical error in modelling caused this pattern. Canine bite force estimations based on the dry skull method suggest that Canis lupus has a higher bite strength than Crocuta crocuta (Wroe et al., 2005). In that study, however, it was also suggested that bite strength does not necessarily imply bone-cracking, which might be more closely associated with structural adaptations in teeth and cranial bones. The findings from the present study suggest structural adaptation in the cranium of the bone cracking Crocuta compared to Canis for the scenarios tested. The high stress observed in the dorsal cranium of Canis is in accordance with the suggestion of Wroe et al. (2005) who demonstrated that larger theoretical bite forces in Canis are not necessarily realized because of cranial structure constraints to bone-cracking, in addition to any structural improvement in teeth.

An important point to be noted is that, given the differences in cranial morphology and masticatory muscle attachment sites, the skull of Canis required the highest muscle input to produce the same bite force compared to the other two models (see Appendix, Table A1). The high input force is probably what created the elevated stresses observed throughout the Canis model. Furthermore, upon examining the regions of highest stress in all three models, it was found that the ends of the zygomatic arches generally exhibit relatively high stress values (Figs 2, 3). The presence of these high stresses are also likely to be partially attributed to muscle force action because the downward pull of the masseter muscles would tend to bend the arches in the ventro-medial direction. Furthermore, the models constructed in the present study do not contain any sutures, which have been shown to represent sites of high strain and may be important in stress distribution across cranial bones (Herring & Teng, 2000).

CrocutaandDinocrocuta

Discussions of correlation between craniodental form and performance in hyaenids have often highlighted the caudally extended frontal sinus present in members of the group. The extent of the development of the posterior frontal sinus in hyaenids is unique among carnivorans (Joeckel, 1998), and functional relevance to stress dissipation during bone cracking has been speculated (Werdelin, 1989). More recently, it was shown, through examination of theoretical morphology,

that Joeckel's (1998) hypothesis appears to hold, at least for C. crocuta (J. Tanner, pers. comm.). In the Crocuta model, the thin bones delineating the frontal sinus are preserved and included; however, the Dinocrocuta model has a single, continuous cavity inside the cranium. A caudally extended frontal sinus is definitely present in Dinocrocuta, but too poorly preserved to be included in the model (Fig. 5). Furthermore, the hypothesis of Joeckel (1998) places functional significance on the formation of a shell-like forehead by the presence of caudally elongate frontal sinus, and not the presence of the sinus per se. Even though the bony plate between the frontal sinus and brain cavity is incompletely preserved in Dinocrocuta, it is not expected to be load-bearing because of its contact with the brain roof in life. Thus, if the enlargement of the sinus is causal in creating the frontal dome of the bone-cracking carnivorans, it could explain the similarity in stress distribution between Dinocrocuta and Crocuta. The presence of frontal sinus structure in the Crocuta model, however, created regions of concentrated stress in the bony struts surrounding the sinus. The curvature is acute in some of the bony struts; thus, stress does not conduct smoothly through the area. A contributing factor to this result could be the lack of soft tissue in the models. The frontal sinus of the domestic dog has been shown to contain a covering of respiratory epithelium (Reznik, 1990; Craven et al., 2007) in the same region where the Crocuta model has concentrated stress (Fig. 5). A dissection of the cranium of H. hyaena (LACM freezer catalogue number 42 206) confirmed the presence of this epithelium throughout the frontal sinus of that hyaenid. Although the material properties of the respiratory epithelium are not known, its close association with the inner surface of the frontal sinus might nevertheless impart a certain degree of structural continuum across which stress could be distributed. Further testing with incorporation of soft tissue material into the model is needed to clarify whether the dissipation of stress occurs in those small regions.

Figure 5. Computer tomography images of (A) Crocuta crocuta, (B) Dinocrocuta gigantea, and (C) Canis lupus taken as lateral views of the mid-sagittal section of the cranium. The frontal sinus (fs) is indicated in all three crania, and caudal expansion of the frontal sinus in C. crocuta and D. gigantea is noted by arrows. The internal cavities of the D. gigantea specimen are filled with matrix, which is light grey in colour in the frontal sinus area. All crania are scaled in the figure to the same approximate length.


Mid-sagittal point-sampling stress

Clearer patterns arise when the mid-sagittal point-sampling data are graphed across biting scenarios for each model (Fig. 4A, B, C). Both Crocuta and Dinocrocuta show a much smoother

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increase in stress just caudal of the nasal opening and extending to the sagittal crest. The down-sloping stress values caudal of the biting point in Crocuta supports the hypothesis that the curvature of the cranium matches the path of stress distribution to function in dissipation (Fig. 4A). A similar pattern is observed in Dinocrocuta; however, it is less clear, with peaks distributed between the anterior orbital border and the post-orbital constriction (Fig. 4B). In general, both the Dinocrocuta and Crocuta models demonstrate gradual change in stress levels across the dorsal cranium, approximately matching the shape of the frontal dome in their stress distribution patterns. By stark contrast, point sampling of the Canis model shows multiple stress peaks across different scenarios along the mid-sagittal plane. The anterior stress peaks demonstrate that the Canis cranium is not as suited for P3 biting as Crocuta or Dinocrocuta (Fig. 4, Table 4). A peak in stress could be better buffered by the cranium if it can be transmitted caudally to the active temporalis muscles that are undergoing tension during biting (Buckland-Wright, 1978). The interpretation appears to stand for both the Dinocrocuta and Crocuta models, and may explain the very low stresses in the region of temporalis muscle action (Fig. 2).

Mid-sagittal sampling of P4 biting in Canis returned much lower stresses using the right P4 than the left (Fig. 4C, F); re-examination of the results indicate that the heightened stress in left P4 biting is not concentrated in small regions but, instead, is a general elevation of stress across the entire cranium. The same models were used for the P3 and M1 scenarios as well, which did not return such asymmetry. It is unclear why this difference exists; however, other than the magnitude, the general trend of the stress peaks remains valid for all analyses, and does not affect the interpretation made here. Single node stress gradients in the Canis model are congruent with theoretical expectation (Fig. 4C). The stress gradient along the mid-sagittal plane of the Canis model matches the basic pattern of bending stress calculation from a beam model of a dry Canis skull (Thomason, 1991): there is a small peak in the region above the infraorbital foramina on the mid-sagittal plane. The second, higher peak occurs near the region between the post-orbital processes. No data were shown for the posterior cranium by Thomason (1991). Although the absolute magnitude of stress is higher in Thomason's calculations, it is of a different biting scheme compared to the present study (i.e. canine biting instead of P3-M1 biting). This lends additional support to the models, and also that the analyses demonstrate the relatively less well ‘designed’ cranium of the Canis compared to the other two models for the biting scenarios tested (Tables 4, 5). With the added advantage of zig-zag Hunter–Schreger enamel banding (Rensberger & Stefen, 2006), which is not present in C. lupus (Stefen, 1999), the robust craniodental morphology of Dinocrocuta is thus likely to be both capable and functional for bone cracking.

An interesting pattern is revealed when single node stress is compared with mean stress over a 6-mm diameter area in the point-sampling analysis. Whereas the stress patterns for Crocuta and Dinocrocuta remain unchanged between the two sampling methods (Fig. 4A, B, D, E), the same cannot be said for Canis. In Canis, a steep slope is still present leading posteriorly to the anterior border of the sagittal crest, but the mean stresses of nodes around a larger area at each landmark appear much smoother than the single node stresses (Fig. 4C, F). This could be explained by the presence of sharper stress gradients in those regions in the Canis model compared to the other

two models, thereby allowing single node sampling to pick up local extremes. In both Crocuta and Dinocrocuta, the increase in stress from the lateral sides toward the mid-sagittal plan is visibly more gradual (Fig. 2A, B), whereas, in the Canis model, small patches of higher stress appear more abruptly (Fig. 2C). Stresses collected from a group of nodes around a landmark would thus tend to average out small areas of high stress. These findings further suggest that the Canis skull model experiences not only elevated stress along the entire mid-sagittal plane relative to the Crocuta and Dinocrocuta models (Tables 1–3), but also shows steep and unevenly distributed stress gradients in the nasal and interorbital region. These results provide potential avenues for validation and testing with in vivo strain gauge experiments (Herring et al., 2001), which may help to explain additional nuances in the differences between Crocuta and Canis observed here.

Broader implications and future directions

The Dinocrocuta cranium represents an individual with unworn permanent dentition and incompletely developed sagittal crest and frontal dome. As the finite element model of Dinocrocuta was made from a relatively young individual, the patterns observed here might be affected by ontogeny. From observations made on specimens of more mature individuals, the shape of the forehead becomes much more pronounced and ‘vaulted’, and may have been better aligned with vertically oriented stress, thereby channeling them dorsoposteriorly. Crocuta, on the other hand, shows relatively less modification of the frontal shape through ontogeny. The smooth curvature in Crocuta thus might persist throughout growth, altering the stress distribution curve to a lesser degree than in Dinocrocuta (Fig. 4A, B).

Much previous theoretical work has been conducted on the mechanics of the mammalian mandibles (Greaves, 1982, 1983, 1985, 2000), which firmly established the ‘one third rule’. The rule provides that, given all considerations to maximize bite force, function, and mechanical stability, the resultant muscle force vector from the action of masticatory muscles fall 30% of the way along the jaw length away from the jaw joint. To maintain stability and prevent frequent torsional loading in the temporomandibular joint, no biting should occur within 30% of the length from the jaw joint. In addition, the proper occlusion required for the shearing carnassials (upper P4 and lower m1) places an evolutionary constraint on the location of those teeth and in turn the arrangement of other cheek teeth relative to them (Savage, 1977). These are factors that might explain the number, position, as well as the use of cheek teeth in Dinocrocuta. From attrition patterns of tooth cusps observed in a sample of eight D. gigantea skulls from Gansu Province, China, it can be concluded that all cheek teeth are used and worn, just as in extant spotted hyenas. Thu, the cranium must respond to the overall biting function over evolutionary time, and not just to the tooth doing the maximum amount of work (i.e. P3 bone cracking). This again would explain why there is little difference between biting scenarios within the premolar toothrow.

The finite element approach has great potential in reconstructing past ecomorphology by testing anatomically inferred form–function relationships (Rayfield, 2007). More specifically, the comparison of living and fossil taxa using this technique holds promises for testing current functional hypotheses and refining ecomorphological definitions. For example, subtleties in cranial

mechanics of carnivorans (e.g. borophagine canids, amphicyonids) and creodonts (e.g. hyaenodontids) that are inferred bone-crackers could be elucidated in this manner by comparison to their closest living relatives (e.g. caniform carnivorans) and with living bone-crackers (i.e. Crocuta crocuta). On the other hand, the undifferentiated cranial response to premolar bites tested in the present study highlights difficulties in identifying bone-cracking adaptations when the cranium might be more generally adapted as a result of multiple evolutionary constraints. Thus, the application of finite element analysis could be further refined to test new evolutionary questions that stem from each additional analysis. Among the anticipated developments: (1) more rigorous statistical testing techniques for analysing finite element stress and strain data; (2) a more fundamental understanding of ecomorphology by finite element analyses of wide-ranging theoretical morphologies; and (3) the application of comparative finite element analysis to an entire clade of closely related species to examine function in a phylogenetic context, will all continue to improve the utility of the finite element method in our understanding of functional morphology evolution.

Conclusion

Analyses using three finite element models showed that the crania of C. crocuta and D. gigantea experienced lower stress for the same P3 and P4 biting scenarios using identical bite force than C. lupus. Differences in biting scenarios were small within each model and the same holds true for stress dissipation in the frontal region. Of the bone cracking carnivorans, Dinocrocuta experienced lower overall stress in the inter-orbital region as well as lower maximum stress. Point sampling of stresses along the mid-sagittal plane of the models demonstrate the ability of Dinocrocuta and Crocuta crania in smoothly conducting stress into the inter-orbital region, probably allowing the stress to be dissipated through the shell-like dorsal cranium and/or tension in the temporalis muscles. This is in stark contrast to the multiple peaks of stress in the Canis model, which does not spread stress evenly. Through the examination of functional morphology using finite element analysis, the present study demonstrates that the morphology of the frontal region plays an important role in conducting stress, regardless of premolar usage. The actual capability of a bone-cracking individual may be balanced by a continuous shift in dental and cranial mechanical advantage and requirements during its ontogeny. The examination of purported bone-cracking taxa in other mammalian lineages would shed light on the extent of functional similarity that underlies morphological convergence.

ACKNOWLEDGEMENTS

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Abstract

INTRODUCTION

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RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

REFERENCES

Appendix

I thank Xiaoming Wang and Gary Takeuchi of LACM for discussion, guidance and encouragement; Betsy Dumont, Sean Werle, and Ian Grosse for training and hospitality during the Finite Element Analysis in Biology workshop in June 2007 at the University of Massachusetts, Amherst; Betsy Dumont and Ian Grosse for access to their BONELOAD program for modelling jaw musculature in the skull models; Michael McNitt-Gray at UCLA Medical Center for CT scanning the specimens; Graham Slater at UCLA for discussion and access to software; the editor and referees for their dedicated reading of the manuscript and stimulating ideas that greatly improved the content of this paper; Jim Dines at LACM for extant specimen loans; Zhanxiang Qiu at the Institute of Vertebrate Paleontology and Paleoanthropology in Beijing, China for an extended loan of the Dinocrocuta gigantea skull; Jill McNitt-Gray, Reyes Enciso, Henryk Flashner, and Faizal Kamaruddin at the University of Southern California (USC) for comments and help with software programs; and the Dinosaur Institute at LACM for research space. This research was funded by a USC Zumberge grant, American Society of Mammalogists grant-in-aid of research, a National Science Foundation Graduate Research Fellowship and National Science Foundation of China (40730210).

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Appendix

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Abstract

INTRODUCTION


RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

REFERENCES

Appendix

Summary of the three finite element models analysed in the present study

The muscle cross-sectional areas were measured from posterodorsal and ventral view photos as in Thomason (1991). Balancing muscles were given 60% of the magnitude of working muscle forces. The frontal dome region was identified by a section of similar volume between the post-orbital processes of the three models.

Table A1. Finite element model parameters of the three skulls studied.

Dinocrocuta Crocuta Canis

*

Estimated from condylobasal length using regression equations for > 100 kg (Dinocrocuta) and 10–100 kg (Crocuta and Canis) categories in Van Valkenburgh (1990).

†

Nowak (1999).

‡

Estimated using dog femur cortical bone density of 2 mg mm−3 (Cowin, 1989).

Body mass (kg)* 199.53 36.31 46.24

(Species body mass range; kg)† – 40∼86 18∼80



Model condylobasal length (mm) 198.15 209.91 275.66

(Actual condylobasal length; mm) 322.36 213.76 244.90

Model maximum width (mm) 147.40 137.45 163.58

(Actual maximum width; mm) 251.72 139.56 142.66

4-noded tetrahedral elements 1 532 146 973 734 1 120 780

Total element volume (mm3) 324 610.7 326 625.0 324 513.3

Model skull mass (g)‡ 649.22 653.25 649.03

(Actual dry skull mass; g) – 477.85 335.50

Temporalis cross-section (mm2) 6.28 × 103 2.42 × 103 3.81 × 103

Masseter cross-section (mm2) 5.67 × 103 1.90 × 103 2.93 × 103

Temporalis contribution (%) 52.54 55.92 56.56

Masseter contribution (%) 47.46 44.08 43.44

Modelled bite force (N) 318.15 318.15 318.15

Required muscle force (N) 1 095.55 1 376.36 1 537.92

Total muscle plate area (mm2) 1.13 × 104 1.36 × 104 5.51 × 104

Balancing temporalis (N) 215.46 288.62 326.19

Balancing masseter (N) 194.62 227.51 250.53

Working temporalis (N) 359.10 481.04 543.65



Working masseter (N) 324.37 379.19 417.54

Frontal dome elements 14 432 22 043 16 391

Frontal dome volume (mm3) 4.40 × 103 4.43 × 103 4.59 × 103

Documents

Cellular and Behavioral Effects of Cranial Irradiation of the Subventricular Zone in Adult Mice