Mechanics of the exceptional anuran ear

The anuran ear is frequently used for studying fundamental properties of vertebrate auditory systems. This is due to its unique anatomical features, most prominently the lack of a basilar membrane and the presence of two dedicated acoustic end organs, the basilar papilla and the amphibian papilla. Our current anatomical and functional knowledge implies that three distinct regions can be identified within these two organs. The basilar papilla functions as a single auditory filter. The low-frequency portion of the amphibian papilla is an electrically tuned, tonotopically organized auditory end organ. The high-frequency portion of the amphibian papilla is mechanically tuned and tonotopically organized, and it emits spontaneous otoacoustic emissions. This high-frequency portion of the amphibian papilla shows a remarkable, functional resemblance to the mammalian cochlea.
Introduction
The anatomy and physiology of the amphibian ear show both remarkable resemblances and striking differences when compared to the mammalian auditory system. The differences between the human and the amphibian auditory system are too significant to warrant direct generalizations of results from the animal model to the human situation. However, studying hearing across species helps to understand the relation between the structure and function of the auditory organs (Fay and Popper 1999). Thus, we hope and expect that the knowledge gained about the amphibian auditory system fits into our understanding of auditory systems in general.
Over the course of history, a number of diverse amphibian species developed. Currently only three orders remain: anurans, urodeles, and caecilians. Their evolutionary relationship, as well as the evolutionary path of the individual orders, is still under debate. However, they are generally grouped into a single subclass, Lissamphibia of the class Amphibia (Wever 1985).
The ancestral lineage of amphibians separated from the mammalian lineage, approximately 350 million years ago, in the paleozoic era. Many of the important developments in the auditory systems emerged after the ancestral paths separated (Manley and Clack 2003). This implies that shared features, like the tympanic middle ear, developed independently in different vertebrate lineages.
The anurans -frogs and toads- form the most diverse order of amphibians. The living species are classified into two suborders, Archaeobatrachia and Neobatrachia (Wever 1985). Both within and between these suborders, there is a large variation in the anatomy and physiology of auditory systems. The most thoroughly studied species belong to the family Ranidae, as is reflected in the work referenced in this paper.
The hearing organs of anurans are often falsely assumed to be more primitive than those of mammals, crocodiles, and birds. The relatively simple structure and functioning of the amphibian ear offer an excellent possibility to study hearing mechanisms (e.g., Ronken 1990; Meenderink 2005). On the other hand, the sensitivity of the frog inner ear, which appears to be able to detect (sub)angstrøm oscillations (Lewis et al. 1985), shows that the frog ear functions as a sophisticated sensor.
While the ears of most vertebrate species contain one dedicated acoustic end organ, the frog ear has two, the amphibian papilla and the basilar papilla.
1 Like in other vertebrates, these organs contain hair cells for the transduction of mechanical waves into electrical (neural) signals. In mammals, birds and lizards, the hair cells are set on a basilar membrane. The frog inner ear lacks such a flexible substrate for its sensory cells. The hair bundles of the frog’s auditory organs are covered by a tectorial membrane, as they are in all terrestrial vertebrates except for some lizards species (Manley 2006).
In mammals, the mechanical tuning of the basilar membrane is the primary basis for frequency selectivity. In the absence of the basilar membrane, the frog’s auditory organs must rely solely on the tectorial membrane and on the hair cells themselves for frequency selectivity.
Recently, Simmons et al. (2007) and Lewis and Narins (1999) published reviews of the frog’s ear anatomy and physiology. In the current paper, we focus on the mechanics of the inner ear, specifically on the mechanics of the tectorial membrane. Only one publication exists on direct mechanical/acoustical measurements of structures in the frog inner ear (Purgue and Narins 2000a). Therefore, many of our inferences will result from indirect manifestation of inner ear mechanics, as observed in anatomical, electro-physiological and otoacoustic-emission studies. Nevertheless, these studies provide a consistent view of the mechanics of the anuran inner ear.
Anatomy
Middle ear
The ears of most terrestrial vertebrates can be divided into three principal parts: the outer ear, the middle ear and the inner ear. In mammals, the outer ear consists of a pinna and an ear canal, which terminates at the tympanic membrane. In most frog species the outer ear is absent,
2 and the tympanic membrane is found in a bony ring, the tympanic annulus, in the side of the skull.
The tympanic membrane defines the distal boundary of the middle ear cavity. This air-filled cavity is spanned by the ossicular chain, which serves to transfer vibrations of the tympanic membrane to the oval window of the inner ear. In the frog, the ossicular chain consists of two structures, the extra-columella and the columella (Jørgensen and Kanneworff 1998; Mason and Narins 2002a). The cartilaginous extra-columella is loosely connected to the center of the tympanic membrane. Medially, it flexibly connects to the partially ossified columella. The columella widens to form a footplate at its medial end, where it attaches to the oval window of the inner ear. Acoustic stimuli primarily enter the inner ear through the oval window.
The middle-ear’s primary function is to compensate for the impedance mismatch between the air and the fluid-filled inner ear. There are two contributions to this compensation (Jaslow et al. 1988; Werner 2003). The first contribution results from the small area of the oval window relative to the area of the tympanic membrane. This causes a concentration of the external force exerted on the tympanic membrane. The second contribution involves a lever action of the columella footplate. The footplate attaches to the otic capsule along its ventral edge. This bond is suggested to be the location of the hinging point of the middle ear lever in the frog (Jørgensen and Kanneworff 1998; Mason and Narins 2002b). The lever action serves as a force amplification mechanism and contributes to the impedance matching between the outside air and the fluids in the inner ear. Both effects result in pressure amplification between the tympanic membrane and the columella footplate, thus overcoming the impedance mismatch between air and the inner-ear fluids.
An additional bony disk, the operculum, is flexibly attached to the oval window in amphibians. The presence of an operculum in anurans is unique among vertebrates. The operculum’s position in the oval window can be modulated through the m. opercularis, which also connects it to the shoulder girdle.
The function of the operculum is not entirely clear. Possibly, it serves to transfer substrate vibrations to the inner ear (Lewis and Narins 1999; Mason and Narins 2002b). The putative path for these vibrations includes the front limbs, the shoulder girdle and the m. opercularis (Hetherington 1988; Wever 1985).
Alternatively, the operculum-columella system is proposed to protect the inner ear’s sensory organs from excessive stimuli. This protection hypothesis takes two different forms. Wever (1985) suggests that the operculum and the columella footplate can be locked together through muscle action. In this manner, the flexibility of the connection to the oval window decreases and the input impedance increases, which in turn decreases the input signal amplitude of the pressure wave in the inner ear. It has also been suggested that the action of the m. opercularis could uncouple the operculum and the footplate (Mason and Narins 2002b). This would allow the operculum to move out of phase with the footplate. The out-of-phase motion could absorb part of the inner ear fluid displacement caused by the motion of the footplate. Effectively this creates an energetic by-pass and decreases the amplitude in the inner ear.
A tympanic middle ear, as described above, is considered to be the typical situation (Jaslow et al. 1988), which can be found in the family Ranidae. However, a wide range of variations in middle ear structures is found across species. In some species, a bony disk occupies the tympanic annulus rather than a membrane, for example, Xenopus leavis (Wever 1985), and there are a number of "earless" frogs. The tympanic membrane and the tympanic annulus are absent in these species. A functioning inner ear and a partial middle ear usually exist, although the middle ear cavity may be filled with connective tissue (e.g., Telmatobius exsul, Jaslow et al. 1988), or not exist at all (e.g., species in the Bombina family, Hetherington and Lindquist 1999; Wever 1985). Remarkably, some of these "earless" frogs have a mating call and exhibit neurophysiological responses (Bombina bombina, Walkowiak 1988; Atelopus, Lindquist et al. 1998) at typically auditory frequencies, which implies they have another path for the transfer of airborne sound to the inner ear (Jaslow et al. 1988), for example, through the lungs (Narins et al. 1988; Lindquist et al. 1998; Hetherington and Lindquist 1999).
Inner ear
The inner ear in the frog has two membranous windows: the oval window and the round window. As mentioned above, acoustic energy primarily enters the inner ear through the oval window. The round window is the main release point of this energy (Purgue and Narins 2000a). A similar lay-out can be found in other terrestrial vertebrates. However, the round window of the frog does not open into the middle ear as it does in mammals. Rather, it can be found in the top of the mouth cavity, under a lining of muscle tissue.
Within the inner ear, there are two intertwined membranous compartments: the perilymphatic and the endolymphatic labyrinths (see Fig. 1). The perilymphatic labyrinth connects to both the oval window and the round window. Starting at the oval window and going medially, it passes through a narrow foramen, and widens into the otic cavity, forming the periotic cistern. Continuing medially it narrows again into the periotic canal. This canal connects the periotic cistern with the perilymphatic space at the round window (Purgue and Narins 2000b). Fig. 1Schematic drawing of a transverse section through the frog ear (adapted from Wever 1985). The division into the middle, and inner ear is indicated above the image; a selection of features is indicated in the image. The colored arrows indicate the paths of vibrational energy: green arrows represent the columellar path, red arrows the putative opercular path, and blue arrows indicate the path through the inner ear after combination of the columellar and opercular paths. The grey areas represents endolympatic fluid, dark yellow perilymphatic fluid. The green areas indicate the tectorial membranes in the papillae. (Color figure is available in the online version)
Between the lateral perilymphatic cistern and the round window, part of the endolymphatic space can be found. The endolymphatic space also includes the semi-circular canals located dorsally from the otic system. It contains the sensory organs of hearing and balance. In the frog inner ear, there are eight sensory epithelia (Lewis and Narins 1999; Lewis et al. 1985), located as follows:
three cristae in the semi-circular canals, which are sensitive to rotational acceleration of the head, and one each in:
the utricule, which detects linear acceleration,the lagena, which detects both linear acceleration and non-acoustic vibrations (Caston et al. 1977),the sacculus, which is sensitive substrate vibrations up to approximately 100 Hz, and also detects high level low-frequency airborne sound, (Narins 1990; Yu et al. 1991)the amphibian papilla, which detects low-frequency acoustic stimuli (Feng et al. 1975), andthe basilar papilla, which is sensitive to high-frequency airborne stimuli (Feng et al. 1975). Hair cells are the sensory cells in all of these organs. Like all hair cells, these cells have a stereovillar bundle on their apical surface. Deflection of the bundle as a result of an acoustical vibration or a mechanical acceleration, initiates an ionic transduction current into the cell. This initial current causes a cascade of ionic currents, eventually resulting in the release of neurotransmitter at the basal surface of the cell. The released neurotransmitter triggers neural activity in the nerve fiber dendrites that innervate to the basal portion of the hair cell (Pickles 1988; Yost 2000; Keen and Hudspeth 2006).
As in most vertebrates, a tectorial membrane covers the sensory cells of the auditory end organ. This membrane is a polyelectrolyte gel, which lies on the stereovilli (Freeman et al. 2003). The function of the tectorial membrane is not well understood, and may vary between classes. However, since the stereovilli in most vertebrate ears connect to this membrane, it obviously plays an important role in the conduction of acoustic vibrations to the hair cells.
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Basilar papilla
The basilar papilla is found in a recess that opens into the saccular space at one end, and is limited by a thin contact membrane at the other. The contact membrane separates the endolymphatic fluid in the papillar recess from the perilymphatic fluid at the round window (Lewis and Narins 1999; Wever 1985). The recess perimeter is roughly oval in shape; in the bullfrog, Rana catesbeiana, its major axis is approximately 200 μm long, while the minor axis measures approximately 150 μm (Van Bergeijk 1957). In the leopard frog, Rana pipiens pipiens, it is of similar size (personal observation, RLMS & JMS).
The oval perimeter of the lumen is formed from limbic tissue; a substance unique to the inner ear, and similar to cartilage (Wever 1985). The sensory epithelium is approximately 100 μm long. It occupies a curved area that is symmetrical in the major axis of the elliptical lumen. It contains approximately 60 hair cells (measured in Rana catesbeiana), from which the stereovilli protrude into the lumen and connect to the tectorial membrane (Frishkopf and Flock 1974). Typically the orientation of the hair cells, as defined by the direction to which the v-shape of the stereovilli bundle points (Lewis et al. 1985), is away from the sacculus in Ranidae.
The tectorial membrane spans the lumen of the papillar recess. It occludes about half the lumen, and consequently takes an approximately semi-circular shape when viewed from the saccular side (Frishkopf and Flock 1974; Wever 1985). The membrane has pores at the surface closest to the epithelium, into which the tips of the hair bundles project (Lewis and Narins 1999).4
Amphibian papilla
The amphibian papilla can be found in a recess, that extends medially from the saccular space and, in frogs with derived ears, bends caudally to end at a contact membrane. Like the basilar papilla’s contact membrane, the membrane separates the endolymphatic fluid in the papilla recess from the perilymphatic fluid at the round window.
The sensory epithelium is set on the dorsal surface of this recess (Lewis and Narins 1999). The epithelium itself has a complex shape; it consists of a triangular patch at the rostral end, and an s-shaped caudal extension towards the contact membrane (see Fig. 2). The exact shape and length of the caudal extension varies across species, with the most elaborate extensions occurring in species of the family Ranidae (Lewis 1984), while some species lack the s-shaped extension altogether (Lewis 1981). Fig. 2Schematic drawing of the amphibian papilla of the bullfrog, Rana catesbeiana, (adapted from Lewis et al. 1982), rotated to match orientation of Fig. 1). TM Tectorial membrane, AP amphibian papilla. a General overview of the AP; the dashed outline indicates the location of the sensory epithelium, b hair cell orientation in the sensory epithelium; dashed line indicates the position of the tectorial curtain. The numbers along the perimeter indicate the characteristic frequency of the auditory nerve fibers connecting to that site (in Hz)
In the epithelium, the hair cell orientation follows a complicated pattern (see Fig. 2b). In the rostral patch the cells are orientated towards the sacculus. On the rostral half of the s-shaped extension, they are oriented along the s-shape. However, on the caudal half, the orientation rotates 90° to become perpendicular to the s-shape (Lewis 1981).
An elaborate tectorial membrane is found on the hair bundles. A bulky structure covers the rostral patch, while the membrane gets thinner along the caudal extension (Lewis et al. 1982). A tectorial curtain spans the papilla recess approximately halfway between the sacculus and the contact membrane (Shofner and Feng 1983; Wever 1985). The curtain, also called the sensing membrane (Yano et al. 1990), spans the entire cross-section of the lumen. A small slit in the tectorial curtain may function as a shunt for static fluid pressure differences (Lewis et al. 1982).
Response of the auditory end organs
As mentioned in the section “Anatomy”, the oval window serves as the primary entry point of acoustic energy into the inner ear; the round window presumably serves as the primary release point. After the energy passes through the oval window, it enters the periotic cistern. Between this relatively large perilymphatic space and the round window there are two possible routes: through the endolymphatic space, or through the periotic canal, bypassing the endolymphatic space and the sensory organs altogether (Purgue and Narins 2000a). The bypass presumably serves to protect the sensory organs against low-frequency over stimulation (Purgue and Narins 2000b).
The vibrational energy that ultimately stimulates the auditory end organs predominantly may enter the endolymphatic space through a patch of thin membrane in its cranial wall near the sacculus. This entry-point was identified by Purgue and Narins (2000b), by mechanically probing the perimeter of the endolyphatic space. After entering the endolymphatic space, the energy may pass either through the basilar papilla’s or through the amphibian papilla’s lumen to the round window.
Measurements of the motion of the respective contact membranes show that there is a frequency-dependent separation of the vibrational energy between paths through the amphibian and the basilar papilla (Purgue and Narins 2000a; see Fig. 3c). The accompagnying dynamic model of the energy flow through the bullfrog’s inner ear (Purgue and Narins 2000b) indicates that this separation may occur based on the acoustic impedances of the paths. Fig. 3Overview of measurements of the frog inner ear; comparison between Rana (left) and Hyla (right). The dashed lines indicate the separation between the amphibian papilla and the basilar papilla. a,  b Distributions of characteristic frequencies of auditory nerve fibers in Rana pipiens pipiens, and Hyla cinerea. c Example of the response of the contact membrane in R. catesbeiana; black line represents the amphibian papilla, open markers the basilar papilla. d, e Distributions of spontaneous otoacoustic emissions in ranid species (combined data from R. pipiens pipiens and R. esculenta), and hylid species (combined data from H. cinerea, H. chrysoscelis, and H. versicolor). f Example of stimulus frequency otoacoustic emissions in R. pipiens pipiens at indicated stimulus levels. g, h Examples of DP-grams measured in Rana pipiens pipiens, and Hyla cinerea. a, b, d, e, g and h are taken from Van Dijk and Meenderink (2006). There they were reproduced from Ronken (1990), Capranica and Moffat (1983), Van Dijk et al. (1989, 1996), Meenderink and Van Dijk (2004), and Van Dijk and Manley (2001), respectively. c is taken from Purgue and Narins (2000a), and f is an adapted presentation of data from Meenderink and Narins (2006) (graph created with data provided by Dr. Meenderink)
The perilymphatic path through the periotic canal may serve as a shunt for acoustical energy to the round window. As its impedance exponentially increases with frequency, low-frequency vibrations will most effectively utilize this path. The endolymphatic path, on the other hand, presumably has a relatively constant impedance throughout the frog’s auditory range. The respective lumina of the amphibian and basilar papilla have a frequency-dependent impedance of their own. According to the model mentioned above, these impedances are dominated by the characteristics of the contact membranes (Purgue and Narins) (2000b). The respective peak displacements of the contact membranes correspond to the detected frequencies in the associated organs (Purgue and Narins 2000a).
Basilar papilla
The basilar papilla’s tectorial membrane is presumably driven by a vibrating pressure gradient between the the sacculus and the basilar papilla’s contact membrane. No reports have been published on direct measurements of the mechanical response of the tectorial membrane, or on the basilar papilla’s hair bundle mechanics. However, the hair cell orientation in the basilar papilla implies that the tectorial membrane’s primary mode of motion is to and from the sacculus.
Auditory nerve fiber recordings from the frog basilar papilla show a frequency selective response (see Fig. 4 for examples of tuning curves). The range of characteristic frequencies in nerve fibers from the basilar papilla is species dependent. In the leopard frog, they are approximately between 1,200 and 2,000 Hz (Ronken 1990); in the bullfrog they are slightly lower, between 1,000 and 1,500 Hz (Shofner and Feng 1981; Ronken 1991). In the Hyla-family, the characteristic frequencies appear to be significantly higher; in Hyla cinerea, the green treefrog, they range from 2.8 to 3.9 kHz (Ehret and Capranica 1980; Capranica and Moffat 1983), and in Hyla regilla roughly from 2 to 3 kHz (Stiebler and Narins 1990; Ronken 1991). Where studied in other species, the characteristic frequencies of the basilar papilla’s nerve fibers fall roughly within the bounds defined by the bullfrog at the low end and the green treefrog at the high end (Scaphiopus couchi: approximately 1−1.5 kHz, Capranica and Moffat 1975; Ronken 1991; Eleutherodactylus coqui: approximately 2−4 kHz, Narins and Capranica 1980, 1976; Ronken 1991; Physalaemus pustulosus group: around 2.2 kHz, Wilczynski et al. 2001). Fig. 4Tuning curves measured in the auditory nerve in R. catesbeiana (unpublished measurements by JMS & PvD, 1992; various specimens). The numbers in the graph indicate  values
In each individual frog, the tuning curves of the auditory nerve fibers appear to have a nearly identical shape and characteristic frequency (Ronken 1990; Van Dijk and Meenderink 2006). This suggests that the entire basilar papilla is tuned to the same frequency. Because of this collective tuning, characterized by one characteristic frequency and a single tuning-curve shape throughout the organ, the basilar papilla may be referred to as a "single auditory filter". In comparison, the mammalian cochlea, and the anuran amphibian papilla (see below), consist of a combination of a large number of auditory filters (Pickles 1988).
The quality factor, Q10dB (e.g., Narins and Capranica 1976; Shofner and Feng 1981), is lower than that of other vertebrate hearing organs in the same frequency range, (Evans 1975; see Fig. 5), and ranges from approximately 1 to 2 in both the leopard frog and the bullfrog (Ronken 1991; see Fig. 5). For other anuran species, the ranges are somewhat different, with the lowest minimum values (approximately 0.5) reported for Hyla regilla, and the highest maximum values (approximately 2.8) in Scaphiopus couchi. Thus, the basilar papilla’s frequency selectivity is relatively poor. Fig. 5Comparison of the filter quality factor  versus the characteristic frequency (CF, in kHz) of nerve fibers from the cat cochlea (adapted from Evans 1975) and the leopard frog (adapted from Ronken 1991). In the leopard frog graph, the triangular symbols correspond to nerve fibers from the amphibian papilla; the circles to fibers from the basilar papilla. The black line indicates the upper limit of the amphibian papilla’s frequency domain. The grey area in the upper (cat) graph corresponds to the area of the lower (frog) graph. The loops indicate the approximate perimeter of the fiber populations in the lower graph for the amphibian papilla and the basilar papilla.
As illustrated in Fig. 3, there is no correspondence between the range of characteristic frequencies in the basilar papilla and the range of spontaneous otoacoustic emission frequencies (Van Dijk and Manley 2001; Van Dijk and Meenderink 2006; Van Dijk et al. 2003; Meenderink and Van Dijk 2004, 2005, 2006; Meenderink and Narins 2007). Since it is generally assumed that otoacoustic emissions of a particular frequency are generated at the detection site for this frequency, this suggests that the basilar papilla does not generate spontaneous emissions. However, it does emit distortion product otoacoustic emissions (Van Dijk and Manley 2001), and stimulus frequency otoacoustic emissions (Palmer and Wilson 1982; Meenderink and Narins 2006). The peak amplitudes of the distortion product otoacoustic emissions match the characteristic frequencies of the auditory nerve fibers innervating the basilar papilla (Meenderink et al. 2005).
The amplitude and phase characteristics of the distortion product otoacoustic emissions can be qualitatively modeled by assuming the basilar papilla to be a single passive non-linear auditory filter (Meenderink et al. 2005). Thus, nerve fiber recordings, otoacoustic emission measurements and a model based on these measurements show that the basilar papilla functions as a single frequency band auditory receptor. This frequency band is relatively broad, and the center frequency may depend on species and individual animals.
The hypothesis that considers the basilar papilla as a single resonator was originally put forward by (Van Bergeijk 1957). He investigated the mechanical response of the tectorial membrane in a scale model consisting of a thin rubber tectorium spanning a lumen in a stiff wall. A number of different vibration modes existed in this model. Although Van Bergeijk’s model is vastly oversimplified, the basic idea that the mechanical tuning of the tectorial membrane may be the basis of the basilar papilla’s frequency selectivity is still viable.
Amphibian papilla
As in the basilar papilla, the tectorial membrane in the amphibian papilla is presumably driven by a vibrating pressure difference between the sacculus and the round window. Due to the more elaborate tectorial membrane and the more complex pattern of hair cell orientations, the motion of the membrane may be expected to be more complex than that of the basilar papilla’s tectorial membrane. The tectorial curtain is in the sound path through the papilla, and presumably plays a role in conveying vibrations to the tectorial membrane and the hair bundles.
Electrophysiological recordings from and subsequent dye-filling of single fibers of the auditory nerve show that the amphibian papilla has a tonotopic organization (Lewis et al. 1982). The fibers innervating the triangular patch have low characteristic frequencies, down to approximately 100 Hz. The frequencies increase gradually along the caudal extension. In the bullfrog, the upper frequency is about 1000 Hz; an overview of the tonotopic organization is given in Fig. 2b.
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The frequency selectivity of the amphibian papilla’s nerve fibers is similar to that of mammalian auditory nerve fibers with the same characteristic frequency. This is in contrast to the significantly poorer frequency selectivity in the basilar papilla’s nerve fibers (Ronken 1990; Evans 1975; see also Fig. 5).
In the low-frequency, rostral part of the papilla, the hair cells are electrically tuned (Pitchford and Ashmore 1987; Smotherman and Narins 1999). This tuning stems from the electrical properties of the cell membrane’s ion channels. The hair cell tuning characteristics parallels the tonotopy of the single nerve recordings. Therefore, frequency selectivity in the rostral part of the amphibian papilla appears to be primarily determined by the electrical characteristics of the hair cells.
However, there is a fundamental discrepancy between the tuning characteristics of the hair cells and the auditory nerve fibers. While hair cells exhibit a second-order resonance (Pitchford and Ashmore 1987) auditory neurons display a higher order filter characteristic (Lewis 1984). Nevertheless, due to the parallels in the tonotopic organization, the assumption that the frequency selectivity is determined by the electrical tuning seems viable for the rostral part of the amphibian papilla. The higher-order responses in the neural signal may result from coupling between hair cells, which may be mechanical, for instance through the tectorial membrane.
Neurons innervating the rostral portion of the amphibian papilla display non-linear two-tone suppression similar to that in other vertebrates (Capranica and Moffat 1980; Benedix et al. 1994). Another manifestation of non-linear behavior can be found in the response to noise: second-order Wiener kernels of low-frequency neurons show off-diagonal components, which are an indication of non-linearity (Van Dijk et al. 1994, 1997). The spectro-temporal receptive fields constructed from these Wiener kernels exhibit suppressive side bands besides the main characteristic frequency band of the fiber (Lewis and Van Dijk 2004).
Hair cells caudal to the tectorial curtain do not display electrical resonance (Smotherman and Narins 2000). Therefore, the tuning of this high-frequency, caudal region of the papilla must result from the mechanical properties of the tectorial membrane and the hair cells.
Based on the hair cell orientation, displayed in Fig. 2b, the tectorial membrane motion in the amphibian papilla is expected to be far more complex than in the basilar papilla. Assuming that the hair bundles are orientated in such a way that they are maximally deflected by the connected tectorial membrane, the rostral patch of the membrane should be moving to and from the sacculus, if the appropriate stimuli are presented. The rostral part of the s-shaped extension is moving along its major axis, whereas the extension caudal to the tectorial curtain should be moving in a transverse direction.
The amphibian papilla appears to be the only source of spontaneous otoacoustic emissions in the frog inner ear (Van Dijk et al. 1989, 1996; Long et al. 1996; Van Dijk and Manley 2001; Fig. 3d-e). The frequency distribution of these emissions corresponds to the range of best frequencies of the neurons projecting to the portion of the amphibian papilla caudal to the tectorial curtain. It is generally assumed that an otoacoustic emission of a specific frequency is generated at the location in the inner ear where that frequency is detected. Under this assumption, the presence of spontaneous otoacoustic emissions indicates that the caudal portion of the amphibian papilla exhibits spontaneous activity. Presumably, this activity is related to active amplification of input signals in this area.
The caudal region of the amphibian papilla is also involved in the generation of distortion product otoacoustic emissions (Van Dijk and Manley 2001; Meenderink and Van Dijk 2004), and stimulus frequency otoacoustic emissions (Meenderink and Narins 2006). The distortion product otoacoustic emissions from the amphibian papilla are more vulnerable to metabolic injuries than those from the basilar papilla (Van Dijk et al. 2003). Also, both the spontaneous (Van Dijk et al. 1996) and distortion product (Meenderink and Van Dijk 2006) otoacoustic emissions display a clear dependence on body temperature. These results combine to indicate that the s-shaped extension of the amphibian papilla caudal to the tectorial curtain functions as an active hearing organ.
Discussion
Our aim in this review is to outline what is known about the mechanical response properties of the amphibian and basilar papilla. Only one published report exists of the direct mechanical measurements of structures associated with these papillae (Purgue and Narins 2000a). The measurements show that the response of the contact membrane is frequency dependent for each papilla. The movement of the contact membrane may be assumed to reflect the fluid motion within the respective papilla. The contact membrane of the amphibian papilla shows a maximum response when the ear is stimulated with relatively low acoustic frequencies, while the basilar papilla contact membrane exhibits a maximum response to higher frequencies.
The amphibian and the basilar papilla are the only hearing organs found in terrestrial vertebrates in which the hair cells are not on a flexible basilar membrane. Instead the hair cells are embedded in a relatively stiff cartilaginous support structure. Any frequency selective response, therefore, most likely originates from the mechanical or electrical properties of the hair cells, or the mechanical properties of the tectorial membrane, or a combination of these factors. Since there are no direct mechanical measurements of either the hair cells in the papillae or the tectorial membranes, we cannot come to any definite conclusions regarding their properties. However, the available morphological and functional data allow for some hypotheses.
The most conspicuous functional characteristic of the amphibian papilla is its tonotopic organization (Lewis et al. 1982). Rostral to the tectorial curtain, the hair-cell orientation is essentially parallel to the tonotopic axis. In this low-frequency region of the amphibian papilla, the tectorial membrane apparently moves in a rostro-caudal direction. In contrast, the hair-bundle orientation suggests that the tectorial-membrane motion is perpendicular to the tonotopic axis in the high-frequency, caudal region of the papilla. The tectorial membrane’s caudal end, therefore, appears to vibrate in a markedly different direction than its rostral end.
In the low-frequency region of the amphibian papilla, the hair cells display electrical tuning. The tuning properties of the hair cells parallel the tonotopic organization are measured from the afferent nerve fibers (Pitchford and Ashmore 1987). This strongly suggests that the tuning characteristics of the nerve fibers are primarily determined by the electrical hair-cell resonances. The auditory nerve-fiber recordings reflect the presence of high-order filtering (Lewis 1984), whereas hair cells essentially function as second-order resonances. It is, therefore, likely that coupling between the hair cells shapes the frequency responses in the nerve fibers. Such coupling may be mechanical, for example, by the tectorial membrane, or electrical, or a combination of mechanical and electrical.
Hair cells in the high-frequency, caudal region do not display any electrical resonance (Smotherman and Narins 1999). This implies that the frequency selectivity must be based on mechanical tuning, probably by the tectorial membrane. The caudal region of the amphibian papilla shares some notable characteristics with the mammalian cochlea (see also Lewis 1981): the papilla is elongated, and it exhibits a tonotopic gradient along the long axis;the orientation of the hair cells is perpendicular to the tonotopic axis, indicating that the hair cells are stimulated most efficiently by a deflection perpendicular to the tonotopic axis;frequency selectivity, very probably, relies on mechanical tuning;frequency selectivity is similar, with Q10dB-values ranging from 0.8 to 2.2; andboth spontaneous and distortion product otoacoustic emissions are generated. These emissions are physiologically vulnerable. The presence of spontaneous otoacoustic emissions shows that at least part of the amphibian papilla’s caudal extension functions as an active hearing organ. In this respect it is similar to the mammalian cochlea and other vertebrate hearing organs (Lewis and Narins 1999). One active mechanism in the mammalian cochlea is the prestin-mediated active somatic length changes in the outer hair cells (Brownell et al. 1985; Yost 2000; Zheng et al. 2000; Liberman et al. 2002; Dallos 2003). However, this mechanism is probably exclusively present in mammalian outer hair cells. Active hair bundle movements have been reported as an alternative active mechanism in anuran saccular hair cells (Martin and Hudspeth 1999; Martin et al. 2003; Bozovic and Hudspeth 2003); this mechanism may be present in the auditory organs as well. Although the fundamental active mechanism may differ between species, the functional result seems to be very similar across vertebrates: high auditory sensitivity and good frequency selectivity (Manley 2000).
The basilar papilla seems to function in a much simpler manner. Both neural recordings and otoacoustic emission measurements suggest that it functions as a single auditory filter. Since the hair cells in the basilar papilla are unlikely to be electrically tuned, its frequency selectivity most likely results from mechanical tuning, probably via the tectorial membrane.
The basilar papilla is remarkable in that no spontaneous otoacoustic emissions have been recorded in its frequency range. The absence of such emissions can either be caused by the fact that they are not generated within the papilla, or by the fact that the transmission of such emissions to the tympanic membrane is inhibited. However, distortion product otoacoustic emissions can be recorded in this range (e.g., Van Dijk and Manley 2001). This implies that the outward transmission is not inhibited, and therefore that spontaneous emissions are most likely not generated within the basilar papilla.
Furthermore, the amplitude of the basilar papilla’s distortion product otoacoustic emissions depends less on temperature than that of the amphibian papilla’s (Meenderink and Van Dijk 2006). Also, emissions from the basilar papilla are less sensitive to the disruption of oxygen supply (Van Dijk et al. 2003). Apparently, emissions from the basilar papilla are relatively independent of the metabolic rate, and therefore, it has been suggested that the basilar papilla is not an active hearing organ (Vassilakis et al. 2004; Van Dijk and Meenderink 2006).
In conclusion, the frog inner ear takes an exceptional place among the hearing organs of terrestrial vertebrates. It includes two auditory end organs, which both lack the basilar membrane present in every other terrestrial vertebrate species. Instead the hair cells are embedded in a relatively stiff structure. They are stimulated by the motion of the tectorial membrane. Although the basilar and amphibian papilla are similar in this respect, they appear to function by different mechanisms. In fact, even within the amphibian papilla two distinctly different functional regions can be identified. The low-frequency portion, rostral to the tectorial curtain, contains hair cells that exhibit electrical tuning. The hair cells are most sensitive to deflection along the tonotopic axis, thus this is presumably the tectorial membrane’s direction of vibration. By contrast, the region caudal to the tectorial curtain shows more similarities to, for example, the mammalian cochlea: the hair cell orientation is perpendicular to the tonotopic axis, and the presence of spontaneous otoacoustic emissions suggests that it functions as an active hearing organ. Finally, the basilar papilla is yet different: it appears to function as a single passive auditory filter. Thus the frog inner ear includes two auditory end organs with three functional regions.

anuran
auditory system
amphibian
frog
inner ear mechanics


Modulation in voluntary neural drive in relation to muscle soreness

The aim of this study was to investigate whether (1) spinal modulation would change after non-exhausting eccentric exercise of the plantar flexor muscles that produced muscle soreness and (2) central modulation of the motor command would be linked to the development of muscle soreness. Ten healthy subjects volunteered to perform a single bout of backward downhill walking exercise (duration 30 min, velocity 1 ms−1, negative grade −25%, load 12% of body weight). Neuromuscular test sessions [H-reflex, M-wave, maximal voluntary torque (MVT)] were performed before, immediately after, as well as 1–3 days after the exercise bout. Immediately after exercise there was a −15% decrease in MVT of the plantar flexors partly attributable to an alteration in contractile properties (−23% in electrically evoked mechanical twitch). However, MVT failed to recover before the third day whereas the contractile properties had significantly recovered within the first day. This delayed recovery of MVT was likely related to a decrement in voluntary muscle drive. The decrease in voluntary activation occurred in the absence of any variation in spinal modulation estimated from the H-reflex. Our findings suggest the development of a supraspinal modulation perhaps linked to the presence of muscle soreness.
Introduction
In an exercise inducing muscle damage, inadequate neural drive can be an attempt of the neuromuscular system to protect the muscle-tendon unit from additional damage (Strojnik and Komi 2000; Nicol et al. 2006). This inadequate neural drive could be the result from a combination of three factors: the conscious and unconscious will of the subject to reduce the exercise intensity; an inability of the motor cortex to generate sufficient output to maximally activate the muscle; and/or a decreased transmission of the supraspinal input to the muscle by the spinal motor axons.
A decrement in H-reflex amplitude has been observed immediately after an exhausting voluntary contraction of a single muscle group (i.e., Duchateau et al. 2002; Garland and McComas 1990; Kuchinad et al. 2004). It has been proposed that this decline in the transmission of the action potentials from the Ia afferent to the α-motoneuron may be a consequence of a presynaptic inhibition mediated by group III and IV afferents (Bigland-Ritchie et al. 1986; Duchateau et al. 2002; Garland and McComas 1990; Garland 1991; Woods et al. 1987) induced by muscle damage (Avela et al. 2006). Furthermore, increased group III and IV muscle afferent inputs could induce H-reflex depression when muscle soreness progresses as muscle pain is believed to reflect activity in group III and IV muscle afferents (O’Connor and Cook 1999). But only a few studies have observed a decrease in H-reflex amplitude after exercise of multiple muscle groups as occurs for example in running (Avela et al. 1999; Bulbulian and Bowles 1992; Racinais et al. 2007b) raising the question of whether spinal modulation occurs after whole body exercise. Recently it has been shown that walking backward induces muscle soreness in the muscles of the lower limb (Nottle and Nosaka 2005). Accordingly, this exercise model allows the study of the effects of muscle soreness on alteration in neural drive. Thus the goal of this study was to determine whether the impaired exercise performance of muscles with delayed onset muscle soreness (DOMS) is due to an alteration in neural drive related to spinal modulation.
Methods
Subjects
Ten healthy subjects (eight males and two females; age 27 ± 1 years; weight 68 ± 2 kg; height 174 ± 2 cm; data in mean ± SEM) gave informed, written consent to participate in this study. The procedures complied with the Helsinki declaration for human experimentation and were approved by the local Ethics Committee. None of the subjects suffered from muscle soreness or ankle injuries. Subjects were asked to avoid caffeine intake within the 8-h preceding the test and to avoid all vigorous activity during the 24-h preceding the test. Subjects were also asked to refrain from analgesic intake all along the protocol, which could have disturbed DOMS perception.
Experimental procedures
Subjects visited our laboratory on four consecutive days (Fig. 1). The first day consisted of a neuromuscular test session (described subsequently) followed by a backward walking exercise (description below) followed by further neuromuscular testing. On the second, third and fourth days, subjects returned to the laboratory at the same hour of day that they had finished the walking exercise and performed the neuromuscular testing.
Fig. 1Experimental design. Thin arrow indicates stimulation at Hmax intensity, simple thick arrow indicates stimulation at Mmax intensity, double thick arrow indicates doublet at Mmax intensity
Neuromuscular tests
The neuromuscular tests are described in Fig. 1. All the neuromuscular tests began with the determination of the stimulation intensity required to elicit a maximal H-reflex (Hmax). Afterwards, three Hmax (interspaced by 20 s) and three maximal M-wave amplitudes (Mmax) interspaced by 8 s were elicited from the relaxed muscle. The amplitude of the three twitches evoked at Hmax and Mmax intensities were averaged for subsequent analysis in both the soleus and gastrocnemius medialis. Thereafter, subjects were instructed to perform three maximal voluntary torque (MVT) contractions of the plantar flexor muscles, each for 5 s. Subjects were verbally encouraged to perform maximally. A superimposed stimulus (Hmax intensity) was evoked during each MVT plateau to obtain the H-reflex during contraction (Hsup). Then, another superimposed stimulus (Mmax intensity) was evoked in order to obtain a superimposed M-wave (Msup) during voluntary contraction. Finally, a doublet (two electrically evoked twitches, 10 ms apart, Mmax intensity) was evoked during each plateau (superimposed twitch) and another doublet was evoked 4 s after each MVT (potentiated twitch). The ratio of the amplitude of the superimposed twitch torque over the amplitude of a twitch evoked at rest 4 s after the MVT was used to assess the level of voluntary activation (VA) (Allen et al. 1995). According to the twitch interpolation method (Allen et al. 1995), the percentage of VA was calculated as follow: VA (%) = (1 − Superimposed Twitch/Potentiated Twitch) × 100.
Muscle soreness assessment
A subjective evaluation of the extent of DOMS in the plantar flexor muscles was performed before each neuromuscular test by completing two subjective scales for evaluation of DOMS. The first was a visual scale of 9 cm without any graduation (horizontal line ranging from no pain at the left to extreme pain at the right). The second was a Lickert scale with seven items (from 0: no pain to 6: severe pain limiting movement, Vickers 2001).
Backward downhill walking exercise
Subjects exercised by walking on a motorized treadmill (S2500, HEF Techmachine, France) for 30-min at a constant velocity of 1 ms−1 with a negative grade of −25%. To increase the eccentric loading on the plantar flexor muscles, the walk was performed backward (Nottle and Nosaka 2005) whilst wearing a vest loaded with an additional weight equivalent to 12% of body weight.
Measurement and calculations
Torque measurement
The MVT of the plantar flexor muscles was recorded by a dynamometric pedal (Captels, St Mathieu de Treviers, France). Subject position was standardized with hip, knee and ankle angulations of 90°, and foot securely strapped on the pedal.
Evoked potentials
The tibial nerve was stimulated with a cathode electrode with a diameter of 9 mm placed in the popliteal cavity (Contrôle Graphique Medical, Brie-Comte-Robert, France). Subjects were in a standardized position with motionless head (Zehr 2002) and a standardized environment (i.e., same time-of-day, silent room, constant lighting). Furthermore, a constant pressure was applied to the electrode with the use of a strap. This was controlled by an air pressure-recorder (Kikuhime, TT MediTrade, Soro, Denmark) located under the strap. The anode (10 cm × 5 cm, Medicompex, Ecublens, Switzerland) was positioned distal to the patella. Electrical stimulations (400 V, rectangular pulse of 0.2 ms) were delivered by a high-voltage stimulator (Digitimer DS7AH, Digitimer, Hertfordshire, England). The amperage was adjusted for each subject during the familiarization session. During this first session, the amperage was increased progressively (10 mA increment) until a plateau in twitch mechanical response [peak twitch (Pt)] and Mmax were observed. With increasing stimulation intensity, the H-reflex response initially increased progressively before decreasing and then disappearing. Thereafter, the intensity needed to obtain Hmax was adjusted by 1 mA. The stimulation intensity needed to obtain Hmax was determined before each test session but with a simplified procedure based on the knowledge of the intensity used during the first test session. This adjustment seemed necessary for the H-reflex in view of the important variation occurring in Hmax for a small variation in stimulation conditions (e.g., intensity, localization of the cathode).
Recordings
Evoked Pt torque was recorded in relaxed muscle by the same ergometer than MVT. Both MVT and Pt were measured with the knee at 90° to reflect the changes occurring in the soleus. Reflex waves for both the soleus, which provides the highest responses due to the activation of the slow twitch fibre by H-reflex (Buchthal and Schmalbruch 1970), and the gastrocnemius medialis, which is particularly susceptible to be affected by the walking exercise were recorded with 9 mm diameter bipolar Ag/AgCl electrodes (Contrôle Graphique Medical, Brie-Comte-Robert, France) with an inter-electrode distance of 25 mm. The reference electrode was placed on the wrist. Low impedance between the two electrodes (<5 kΩ) was obtained by abrading and washing the skin with emery paper and cleaning with alcohol. Signals were amplified and filtered (band pass 30–500 Hz, gain = 1,000), and recorded at high frequency (2,000 Hz). The compound muscle action potentials were recorded using MP30 hardware (Biopac Systems Inc., Santa Barbara, CA, USA) and dedicated software (BSL Pro Version 3.6.7, Biopac Systems Inc., Santa Barbara, CA, USA). The same equipment was also used to drive the stimulator.
Calculation
The Pt may be considered as an index of the contractile properties and Mmax amplitude represents an index of sarcolemmal excitability. Because no evidence exists to show that data recorded at rest reflects the responses of the neuromuscular system during contraction, Hsup amplitude was recorded during MVT to complement Hmax amplitude at rest. To ensure that any changes in the evoked Hmax and Hsup amplitudes were not due to changes at the muscle fiber membrane or neuromuscular junction (Cupido et al. 1996), we normalized these recordings to the M-wave amplitude recorded under the same testing conditions, that is the Hmax/Mmax and Hsup/Msup ratios. These ratios may be considered as a global index of the spinal modulation produced by presynaptic inhibition, motoneuron excitability, collision in antidromically activated axons and Renshaw cell inhibition, acting individually or in concert.
Statistical analysis
Each variable was tested for normality using the Skewness and Kurtosis tests with acceptable Z values not exceeding ±1.5. With the assumption of normality confirmed, parametric tests could be performed. The effect of the walking exercise was analyzed for each variable by a one-way analysis of variance for repeated measures (five test sessions). The contrast method was applied as post hoc to further investigate the effect of both the exercise and the recovery. Because VA level failed to display a normal distribution, a Friedman test was used instead of the ANOVA. Statistical analyses were performed with Systat software (Systat, Evanston, IL, USA). Data are reported as mean ± SEM and the level of statistical significance was set at P < 0.05.
Results
Maximal voluntary torque, voluntary activation and contractile properties
The MVT significantly changed across the 4 days following the walking test (F4,36 = 3.8, P < 0.02, Fig. 2a). Post hoc analysis showed a significant decrease in MVT after the walking exercise and which persisted during the next two days (F1,9 = 12.33, P < 0.01). A significant recovery in MVT was observed on the third day (48-h versus 72-h after: F1,9 = 9.64, P < 0.02).
Fig. 2Evolution of voluntary torque (a), voluntary activation (b), electrically evoked peak twitch (c) and subjective delayed onset muscle soreness (d, black rectangle: analogic visual scale, white rectangle: Lickert scale) across the experimental sessions. Data in mean ± SEM, Asteriskindicates value or group of values significantly different from the other values of the graph (P < 0.05)
In line with the evolution observed in MVT, post hoc analysis showed a significant decrease in VA level after the walking exercise (pre versus post-exercise: P < 0.02, Fig. 2b) which failed to recover by 48-h (post-exercise versus 24-h and 48-h after exercise, NS). However there was a significant recovery by 72-h (post-exercise versus after 72-h, P < 0.005).
The electrically evoked Pt also displayed a significant variation following walking exercise (F4,36 = 14.07, P < 0.001, Fig. 2c). Post hoc analysis revealed a significant decrease in Pt after the exercise (F1,9 = 58.34, P < 0.001) followed by a significant recovery thereafter (F1,9 = 24.76, P < 0.001).
Subjective DOMS
The subjects feeling of DOMS increased significantly in the days following the walking exercise (F4,27 > 28, P < 0.001 for both scales used, Fig. 2d). Post hoc analysis displayed significantly higher subjective DOMS for the 3 days following exercise compared to the termination of exercise (F1,9 > 35, P < 0.001, for both scales). Muscle soreness reached a maximum 48-h after exercise and began to recover by 72-h (48-h versus 72-h after exercise: F1,9 > 12, P < 0.01 for both scales).
Evoked potentials
An example of evoked potentials recorded in a representative subject is displayed in Fig. 3 and the mean values are displayed in Table 1. The walking exercise failed to induce significant changes in the evoked potentials both at rest (Mmax, F4,36 = 1.60 and 0.60 for soleus and gastrocnemius medialis respectively, NS) and during the voluntary contraction (Msup, F4,36 = 0.46 and 2.20 for soleus and gastrocnemius medialis respectively, NS). Furthermore, the reflex waves calculated both at rest (Hmax/Mmax ratio) and during contraction (Hsup/Msup ratio) also did not change significantly (all F4,36 < 0.98, NS, Table 1).
Fig. 3Example of evoked potentials recorded in a representative subject. Each drawing represents the average of three recordings obtained on a relaxed muscleTable 1Evolution of maximal compound action potential electrically evoked at rest (Mmax) and during MVT (Msup), normalized H-reflex at rest (Hmax/Mmax) and during MVT (Hsup/Msup)ExerciseRecoveryStatistical analysisBeforeAfter24-h48-h72-hSoleusMmax (mV)7.356.807.837.747.18NS0.530.490.80.580.75Msup (mV)8.878.139.058.918.75NS1.140.761.130.971.29Hmax/Mmax ratio0.360.370.380.370.36NS0.100.090.100.100.09Hsup/Msup ratio0.390.450.390.340.34NS0.100.100.080.080.06Gastrocnemius medialisMmax (mV)6.616.106.426.836.50NS1.070.760.641.00.92Msup (mV)9.407.048.247.958.14NS1.291.060.810.892.26Hmax/Mmax ratio0.180.190.220.190.19NS0.050.050.050.050.05Data in mean ± SEM
Discussion
The downhill walking exercise induced a significant decrease in the MVT of the plantar flexor muscles. Immediately after the walking exercise, the torque decrement of −15% (Fig. 2a) appeared to be caused partly by an alteration in muscle contractile properties (i.e., −23% for Pt, Fig. 2c). This alteration is typically referred to as “peripheral fatigue” [for a review, see Millet and Lepers 2004]. Furthermore, this peripheral fatigue was also associated with a decrease in VA (i.e., −5.3%, Fig. 2b) suggesting the concomitant existence of a “central modulation” [for a review, see Gandevia 2001]. The first finding of this study is that the maximum voluntary torque failed to recover before the third day (i.e., −12% and −10% after 24-h and 48-h of recovery, respectively, Fig. 2a) whereas the measure of the (peripheral) contractile properties had recovered significantly within the first 24 h after exercise (P < 0.01, Fig. 2c). This delayed recovery in MVT appeared to be mainly associated with a decrease in voluntary muscle activation (Fig. 2b). The time course of change in VA presents similarities with the time course of torque changes.
A significant decrease in the VA reaching the muscle has previously been observed after prolonged (Millet et al. 2002, 2003) and short-duration (Racinais et al. 2007a) fatiguing exercise, but our data showed that a backward downhill walking exercise did produce a decrease in muscle activation persisting for a few days after the exercise. It has been suggested that this central component could explain as well the reduction in force production by the respiratory muscles after heavy exercise (Verin et al. 2004) and represents a protective mechanism in order that peripheral muscle fatigue does not exceed a critical threshold (Amann et al. 2006). This central protection of the muscle from further peripheral fatigue and damage will be performed at the expense of a truly maximal performance in which all the motor units are activated (Gandevia et al. 1996).
It has recently been suggested that a “central governor” in the brain regulates the extent of skeletal muscle recruitment during exercise (Noakes et al. 2005). According to this theory, the sensation of fatigue is the conscious interpretation of these homoeostatic control mechanisms during prolonged exercise (Noakes et al. 2005). Our results could partly support this theory but with the proviso that the modulation of central activation in this experiment appears to be related not only to fatigue immediately after exercise but also to the development of DOMS the days following the exercise. However, regulation of muscle recruitment by the brain (i.e., supraspinal regulation) is not the only system that could explain the observed decrease in skeletal muscle activation since reflex pathways need also to be considered (i.e., spinal modulation). Previous results showed impairment in VA after eccentric exercise when VA was estimated by nerve stimulation but not by cortical stimulation (Prasartwuth et al. 2005). That suggests that the VA deficit lies within these two site of stimulations, i.e., in the motor cortex or at spinal level (Prasartwuth et al. 2005), rather than a governor upstream to the motor cortex.
In the present study, we used H-reflex amplitude as a tool to evaluate the spinal modulation (Aagaard et al. 2002; Schieppati 1987) to provide a differentiation within these possible regulatory mechanisms. The DOMS produced by our experimental protocol would have induced an increased discharge of group III and IV muscle afferents (Avela et al. 1999) and thus a pre-synaptic inhibition of the transmission from the Ia afferent stimulation to the α-motoneurons (Avela et al. 2006; Bigland-Ritchie et al. 1986; Duchateau et al. 2002; Garland and McComas 1990; Garland 1991; Woods et al. 1987). The extent to which this inhibition occurs will depend on whether the input producing the inhibition is ongoing or has ceased. But, in theory, increased group III and IV muscle afferent input induced by the DOMS should induce H-reflex depression as the soreness progresses. Indeed, muscle pain is believed to reflect activity in group III and IV muscle afferents (O’Connor and Cook 1999). However, our results failed to show significant variations in the evoked reflex-wave amplitudes throughout the experiment (i.e., Hmax/Mmax and Hsup/Msup ratios) suggesting that the motoneuron pool excitability was well preserved.
A number of previous studies have demonstrated a significant H-reflex decrease in the exercised muscle group after an exhausting voluntary contraction of that muscle group (i.e., Duchateau et al. 2002; Garland and McComas 1990; Kuchinad et al. 2004). But only a few studies have observed this decrease after more generalized muscular exercise such as running, (Avela et al. 1999; Bulbulian and Bowles 1992; Racinais et al. 2007b). Our results support these findings by showing that after a 30-min downhill walking exercise, electrically evoked reflex-wave activity was not decreased. Thus we conclude that non-exhausting walking exercise, sufficient to induce significant DOMS seems not to induce alteration of the spinal modulation.
Since the persistence of a decrease in VA during several days in this study could not be explained by spinal modulation, it seems likely that a supraspinal component must have played a part. Accordingly the changes in VA that occurred at the same time that the subjective symptoms of DOMS suggest a supraspinal regulation of muscle recruitment. Indeed it has been known for some time that exercise performance is regulated at least in part by supraspinal factors. For example, Bigland-Ritchie (1981) showed that central fatigue during repeated isometric contraction is minimized by exhorting the subject to produce a “super” effort at the end of each voluntary contraction. Our results add to that interpretation by suggesting the possibility that supraspinal modulation can also occurs after locomotory activity such as walking, even without exhaustion.
As we have already argued, the observed increase in DOMS could represent the conscious interpretation of an increased discharge of group III and IV muscle afferents (O’Connor and Cook 1999). Even though we failed to observe a significant alteration of the spinal modulation at the time of increased muscle soreness, this would not prove that the discharge of these afferents had not increased since it is still unclear whether group III and IV muscle afferents induce a post-exercise decrease in motoneurons excitability in healthy humans. Indeed, previous studies have shown that maintained firing of ischaemically sensitive group III and IV muscle afferents does not influence the altered muscle responses to cortical or corticospinal stimulation observed after fatiguing exercise (Andersen et al. 2003; Gandevia et al. 1996; Taylor et al. 2000). All these findings might suggest that, after exercise, increased output from group III and IV muscle afferents may not directly inhibit the motoneurons but may act upstream of the motor cortex to impair voluntary descending motor drive (Taylor et al. 2006).
Accordingly, a novel contribution of this study is that VA significantly recovered on the third day, when the DOMS displayed also a significant decrement (F1,9 > 12, P < 0.01, for the both scales). Although this temporal relationship does not prove causality, this finding could suggest a relationship between the persistence of the decrease in VA and the subjective symptoms of DOMS in these subjects. This observation is consistent with the data of Le Pera et al. (2001) showing that muscle pain could induce a long-lasting depression in motor activation. Their data suggest that this inhibition in motor system excitability could be linked to a decreased excitability of the motor cortex as well as spinal modulation (Le Pera et al. 2001). However, Prasartwuth et al. (2005) observed a different time course in muscle soreness after eccentric exercise and changes in VA leading these authors to suggest that muscle pain did not directly cause the change in voluntary drive.
These different data emphasised the complexity of the relation within muscle soreness and voluntary muscle drive. It has been recently observed that motoneuron excitability in elbow flexors, but not extensors, was able to recover when ischemia is maintained after fatiguing contractions (Martin et al. 2006), a finding that suggests differential influences of group III and IV muscle afferents on different motoneuron pools (Martin et al. 2006). In this study concerning the plantar flexors, we observed some statistical similarities within DOMS and VA (i.e., lowest value of VA at 48-h when DOMS was the highest and significant recovery for the both at 72-h). However, from a functional point of view, VA returned at control level at 72-h whereas DOMS at 72-h was not less than DOMS at 24-h, and VA decreased after exercise when DOMS was weak. That suggests that subjective DOMS of the plantar flexors can not be considered as an objective indicator of VA capability.
Conclusion
It was recently reported that a 90 min bout of flat running exercise produced a modification in spinal modulation (Racinais et al. 2007b). The present study showed that a 30-min downhill walking exercise failed to induce the same modification. However there was a significant decrease in VA during maximal voluntary contractions performed during the days after eccentric exercise that produced DOMS. This suggested the occurrence of a supraspinal modulation of muscle activation during this period when muscle contractile properties had fully recovered following the eccentric exercise. Furthermore, the persistence of the decrement for several days suggests that this modulation was not caused by an acute exercise-related physiological or biochemical alteration in the motor cortex, so-called central fatigue but more likely represents a modulation which may be partly linked to the muscle soreness.

eccentric
exercise
neuromuscular
doms
central fatigue


A Comparison Between Radioimmunotherapy and Hyperthermic Intraperitoneal Chemotherapy for the Treatment of Peritoneal Carcinomatosis of Colonic Origin in Rats

Background Cytoreductive surgery (CS) followed by heated intraperitoneal chemotherapy (HIPEC) is considered the standard of care for the treatment of patients with peritoneal carcinomatosis (PC) of colorectal cancer (CRC). These surgical procedures result in a median survival of 2 years at the cost of considerable morbidity and mortality. In preclinical studies, radioimmunotherapy (RIT) improved survival after CS in a model of induced PC of colonic origin. In the present studies we aimed to compare the efficacy and toxicity of CS followed by adjuvant RIT in experimental PC to the standard of care, HIPEC.
Peritoneal carcinomatosis (PC) of colorectal cancer (CRC) frequently is an end stage of colorectal cancer, occurring in 5–50% of the patients, either synchronous or metachronous.1 If untreated, patients suffering from PC have a median survival of only 6 months.2 Survival is significantly improved by radical surgical debulking procedures (cytoreduction) in combination with intraperitoneal chemotherapy, either in combination with normothermia or hyperthermia (HIPEC).3–7 The median survival after cytoreductive surgery and HIPEC is 13–34 months,4,8 and the 5-year survival rate is 19–27% at the cost of considerable morbidity and mortality rates of up to 23% and 4%, respectively.6,9
In the latest reported clinical trial on adjuvant RIT in the setting of colon cancer, Liersch et al. reported results of a Phase II trial with 131I-labeled anti-CEA antibody labetuzumab administered to patients after complete resection of colorectal liver metastases. This study, where RIT was applied in an adjuvant setting to complete resection, resulted in a promising 5-year survival rate of 51.5%.10 Radioimmunotherapy using radiolabeled monoclonal antibodies directed against tumor-associated antigens may therefore be an attractive anticancer therapy in patients with small volume disease.
We therefore have studied the application of RIT as adjuvant therapy following cytoreductive surgery (CS) in the setting of PC. In previous studies regarding PC of CRC in a rat model we showed that RIT could be an effective adjuvant treatment after CS. The efficacy of adjuvant RIT in combination with CS was investigated and compared with no treatment, CS only, and RIT only. The results of this study showed a significantly improved survival of animals treated with CS followed by RIT (median 88 days) compared with those treated with CS only (median 51 days) and RIT only (median 61.1 days).11 Based on the encouraging results, showing the observed increase in survival that was achieved with low-dose RIT and concomitant low toxicity, we now aimed to compare the efficacy of this treatment to that of today’s standard of care, HIPEC,12,13 in a preclinical setting.
MATERIALS AND METHODS
Animal Model of Peritoneal Carcinomatosis
WAG/Rij rats (10–12 weeks old, body weight 240–290 g, Harlan Horst, The Netherlands) were housed under nonsterile standard conditions (temperature, 20–24°C; relative humidity, 50–60%; 12-h light/dark cycle) in filter-topped cages (two rats per cage), with free access to food (Ssniff, Bio Services Uden, The Netherlands) and water. Rats were accustomed to laboratory conditions for at least 1 week before experimental use. Peritoneal carcinomatosis was induced by intraperitoneal inoculation of 2.0 × 106 CC-531 colon cancer cells, as described previously.14 All experiments were approved by the local Animal Welfare Committee of the Radboud University Nijmegen and were carried out in accordance with the Dutch Animal Welfare Act of 1997.
Operative Procedure
Prior to the laparotomy, all rats were given 10 mL of saline to prevent hypovolemia. Surgical procedures were performed under general anaesthesia using isoflurane 3%, O2 and N2O 1:1. Thirty minutes prior to and once daily until the third day postoperatively, rats were given buprenorphine (5 μg, 0.1 mL/rat/day) for analgesia. During the operation, rats were placed on a warmed mattress to limit body heat loss. All rats underwent a midline laparotomy. After opening the abdomen the extent of intraperitoneal tumor growth was scored semiquantitatively, 0 (no macroscopic tumor), 1 (little; located at 1–2 sites with a diameter of 1–2 mm), 2 (moderate; located at 1–2 sites and a diameter 2–5 mm), or 3 (abundant; located at multiple sites and/or diameter >5 mm) in all four quadrants of the abdomen. The sum of the tumor scores of all sites represented the peritoneal cancer index (PCI).11
Subsequently, CS, including a routine omentectomy, was performed in all rats. Irresectable tumor deposits were cauterized using an electrocoagulation device. After completion of the surgical cytoreduction, the abdominal wall was closed in two layers using continuous Vicryl 3/0 sutures for the muscular component and iron wound clips for the skin in animal treated with CS only or CS + RIT.
Monoclonal Antibody, Radiolabeling, and RIT
The murine MG1 monocolonal antibody (MAb), an anti-CC531 IgG2a monoclonal antibody that recognizes a 80 kDa cell surface antigen and localizes preferentially in tumors when injected in rats bearing CC-531 tumors,15 was purchased from Antibodies for Research Applications BV (Gouda, The Netherlands). Labeling of the antibody with 177Lu was carried out as previously described.11 In brief, the MAb was conjugated with 2-(4-isothiocyanatobenzyl)-diethylenetriaminepenta-acetic acid (ITC-DTPA) (Macrocyclics, Dallas, TX) and subsequently labeled with 177Lutetium (IDB Holland, Baarle Nassau The Netherlands) and purified by gel filtration on a PD10 column (Amersham, Pharmacia Biotech, Maarsen, The Netherlands). The purified 177Lu-MG1 preparation was diluted in PBS with 0.5% BSA for injection, the specific activity of the administered 177Lu-MG1 preparation was 0.4 MBq/μg. The labeling procedure using 177Lu was performed under strict metal-free conditions.
RIT (185 μg MG1/ rat, radiolabeled with 74 MBq 177Lu in 3.0 mL) was intraperitoneally injected immediately after surgery, as this was determined to be the most optimal time for adjuvant administration.16
Mitomycin-C
Mitomycin-C (MMC) was obtained from Nycomed Christiaens BV (Breda, The Netherlands) as a powder in glass vial (40 mg/vial). Immediately before use, MMC was dissolved in 0.9% sodium chloride to the appropriate concentrations.
HIPEC Procedure
Following CS, while the abdomen was still exposed, two multiperforated catheters (Argyle, Sherwood Medical, Ireland) were inserted laterally through the abdominal wall and subsequently fixed in the abdominal cavity. The inflow drain was placed in the right paracolic gutter, the outflow drain in the left subdiaphragmatic space. The intraperitoneal temperature was monitored with an intra-abdominal thermometer (PTFE Insulated thermocouple, VWR International, Amsterdam, The Netherlands), at the site with generally the highest tumor load (omentum). In addition, a thermometer was placed inside the rectum. After placement of the catheters, the abdominal wall was closed using a continuous suture (Ethilon 3.0, Johnson & Johnson, Ethicon) (Fig. 1). During the HIPEC procedure, rats were removed from the warmed mattress to prevent general hyperthermia. The perfusion system was filled with 250 mL saline, containing 4 mg MMC (Mitomycin-C Kyowa, Christiaens). The perfusate was heated in a tube coil using a thermostatically regulated water bath set to a temperature of 48°C and infused into the peritoneal cavity by a roller pump (Ismatec IPS-8, Ismatec SA, Glattbrugg, Switzerland) for the duration of 60 minutes at 10 mL/min. Abdominal inflow temperature was set at 44°C. In order to achieve a uniform heat distribution, gentle massage of the abdomen was applied throughout the duration of the HIPEC procedure. After completion of the perfusion, the abdominal cavity was flushed with warmed (37°C) saline for a period of 10 minutes. The abdomen was opened again to remove the catheters. Subsequently, the abdomen was closed as described previously.
FIG. 1.HIPEC Perfusion System; MMC Mitomycin C Kyowa 16 mg/L perfusate. Adapted from Ref. 19. Reproduced with permission.
Intraperitoneal Distribution of MMC and Dose Determination
Prior to the therapy experiment with HIPEC, we investigated the intraperitoneal distribution of the perfusion fluid using a methylene blue stained perfusate. The perfusate was administered in the same fashion as in the therapeutic experiment. After completion, the abdominal cavity was inspected for the presence of blue dye in all quadrants on both parietal as well as visceral peritoneum of the intra-abdominal organs and the diaphragm. Subsequently, a study to determine the dose of MMC that resulted in acceptable toxicity was performed in nine animals (three animals per group). Animals underwent a laparotomy including an omentectomy and complete bowel inspection followed by heated perfusion of the abdominal cavity with MMC at 4 mg/L or 16 mg/L. Control rats underwent laparotomy and an omentectomy only. Body weight and physical condition were monitored during 6 days following the procedure to assess treatment-related toxicity.
Treatment Efficacy
Seven days after intraperitoneal tumor induction with 2.0 × 106 CC-531 tumor cells, 45 rats, 15 per treatment group, were randomly assigned to undergo either CS only, CS + RIT or CS + HIPEC. The operative procedures and application of the adjuvant therapies were performed as described previously. Toxicity of the treatment was determined clinically and by weighing the rats. Body weight was expressed as relative body weight compared with the body weight on the day of surgery. Survival was scored, and at autopsy the extent of tumor growth was determined.
Follow-Up
The primary endpoint was 16-week survival. As part of monitoring the physical condition during the immediate postoperative period, the general condition was monitored and the body weight was measured daily during the first 2 weeks. When the humane endpoint (HEP) was reached (signs of massive hemorrhagic ascites, physical inactivity or signs of intra-abdominal tumor growth with invalidating consequences), rats were killed by O2/CO2-administration and immediately dissected. The HEP was determined by an experienced biotechnician who was blinded to the therapeutic regimen. At the time of the HEP rats were generally lethargic, showing signs of advanced PC as the presence of ascites. At dissection, the intraperitoneal tumor growth was scored as described previously. At 16 weeks postoperatively, the study was terminated and the remaining rats were euthanized and dissected. In case of absence of macroscopic tumor, all relevant organs, including the greater momentum, the mesentery, and the diaphragm were removed for histopathological staining to determine tumor presence microscopically. Sections were stained using hematoxylin & eosin (H&E) and/or immunohistochemical staining using the murine MG1 antibody in combination with a horse-anti-mouse IgG antibody, HRP conjugated (Vector Laboratories Inc., Burlingame, CA, USA).
Statistical Analysis
Statistical analysis was performed using SPSS (Chicago, IL) software and Graphpad Prism (Graphpad Software Inc., San Diego CA) for analysis. Comparison of dichotomous values was done using chi-square or Fisher’s Exact test. Nonparametric testing was performed using two-way ANOVA testing. Survival curves were analyzed using Kaplan–Meier curves and compared by means of the log-rank test. Posttesting using Bonferroni was applied to correct for multiple groups. All tests were two-sided; the level of statistical significance was set at a P value of <.05.
RESULTS
Intraperitoneal Distribution of HIPEC and Dose Determination
The intraperitoneal distribution of the perfusate administered according to the previously described procedure, showed a distribution pattern amongst all quadrants, including the diaphragm bilaterally and at the mesenterial root (Fig. 2).
FIG. 2.Intraperitoneal distribution of methylene blue stained perfusate.
The applied dose of 16 mg MMC/L resulted in a maximum mean weight loss of 13.7 ± 2.9% at 4 days postoperatively. In addition, the first 3 days following the heated perfusion, animals were lethargic and suffered from diarrhea from day 2 until day 4 postoperatively. In contrast, the maximum weight loss in the 4 mg/L was 8.3 ± 2.9% at day 3 and 7.5 ± 2.3% at day 3 in the control group (Fig. 3). None of the animals died during the immediate postoperative period. Based on these observations, HIPEC, when administered at a dose of 16 mg/L for the duration of 60 minutes at the given temperature, was considered to be the maximal tolerable dose to be used for the HIPEC procedure.
FIG. 3.The relative body weight of Wag/Rij rats after exploratory laparotomy (control) and heated intraperitoneal chemotherapy (HIPEC) given immediately postoperatively in different doses. Data represent means ± standard error of the mean (SEM).
Operative Procedure
Preoperative body weight did not differ between groups, P = .52 (Table 1) At laparotomy, tumor nodules were present in the omentum, liver hilum, the mesentery, and gonadal fatpads (1–3 mm diameter). Median PCI score at time of surgery was 5 (range 4–8) and was similar in all experimental groups. After surgical cytoreduction, residual disease remained in situ in 7 rats after cauterization and was equally distributed among the groups (P = .84). The surgical procedures without adjuvant therapy took 20–30 minutes per animal.
TABLE 1.Treatment group characteristicsPCIMedian (range)CSCS + HIPECCS + RITPreoperative body weight (g)266 (251–287)264 (245–285)262 (244–276)Tumor score per site  Greater omentum2 (2–3)2 (1–2)2 (1–2)  Liver hilum1 (0–1)1 (0–1)1 (0–1)  Perisplenic0 (0–1)0 (0)0 (0)  Mesentery1 (0–2)1 (0–2)1 (0–2)  Gonadal fatpads0 (0–2)0 (0–2)1 (0–2)  Diaphragm0 (0–1)0 (1)0 (0–1)  Parietal peritoneum1 (0–1)1 (0–1)1 (0–1)  Total5 (4–8)5 (4–6)5 (4–8)Resection macroscopically complete  Yes121313  No322Treatment group characteristics (peritoneal cancer index; PCI) found during laparotomy before the administration of the adjuvant therapy.CS, cytoreductive surgery; HIPEC, heated intraperitoneal chemotherapy; RIT, radioimmunotherapy. PCI is expressed as median and range.
There was no intraoperative mortality. However, one rat in the CS + HIPEC and one rat in the CS + RIT group were euthanized after 2 and 9 days, respectively. The animal in the CS + HIPEC group showed massive weight loss as a result of bowel necrosis and subsequent perforation, the cause of death of the animal in the CS + RIT group remained unclear. The median intra-abdominal temperature during the HIPEC procedure, measured at the site where the greater omentum was removed, was 41.0°C (range 40.4–41.6°C). In contrast, the median rectal temperature was 34.6°C (range 34.1–34.8°C) (Fig. 4).
FIG. 4.The recorded intra-abdominal and rectal temperature during the HIPEC procedure. Data represent means ± standard error of the mean (SEM).
CS and CS + RIT were well tolerated, whereas animals in the CS + HIPEC groups showed signs of physical discomfort; animals of the latter group were generally lethargic and showed pilo erection two days following the procedure. In addition, these animals all suffered from diarrhea up to 4 days after the HIPEC procedure. The relative body weight after of the various treatment groups is depicted in Fig. 5. Maximum body weight loss after CS or CS + RIT was similar (7.3 ± 2.6% vs 9.3 ± 1.8% 4 days postoperatively, P > .05). Rats that received adjuvant HIPEC had a maximum body weight loss of 12.3 ± 1.7%, which was significantly higher than that after CS alone (P < .001) or CS + RIT (P < .001). Rats generally gained weight from the fifth postoperative day onward. In the HIPEC group, however, postoperative mean body weight remained significantly lower than that of the animals in the CS group, until 5 weeks postoperatively.
FIG. 5.The relative body weight of Wag/Rij rats with small peritoneal CC-531 tumors in the first 14 days after cytoreductive surgery (CS) only, CS + radioimmunotherapy given immediately postoperatively (RIT) or heated intraperitoneal chemotherapy (HIPEC) given immediately postoperatively. Data represent means ± standard error of the mean (SEM).
Treatment Efficacy
During the experiment, 29 animals were euthanized because of massive amounts of ascites that resulted from intraperitoneal tumor growth. The mean amount of ascites at the humane endpoint was 31 ± 22.6 mL, 26.5 ± 23.8 mL, and 26.4 ± 22.6 mL in the CS, RIT, and HIPEC groups, respectively (P = .82). At the time of death, mean PCI in the CS, CS + HIPEC, and CS + RIT groups was 18 (range 9–22), 12 (range 5–15), and 18 (range 16–19), respectively, with significant differences between the CS + HIPEC and both other treatment groups (P < .001 for both comparisons).
The survival curves of the various treatment groups are depicted in Fig. 6. Median survival of the rats that were treated with CS only was 57 days (range 36–112). Adjuvant HIPEC resulted in a median survival of 76 days (range 33–112), P = .17, when compared with CS only. Median survival of the rats that were treated with CS followed by the adjuvant administration of RIT was improved to a median survival of 97 days (range 49–112), P < .001 compared with CS only. When compared with CS followed by adjuvant HIPEC, the adjuvant administration of RIT to surgery did not result in an improved survival (P = .33).
FIG. 6.Kaplan–Meier survival curves for Wag/Rij rats with small peritoneal CC-531 tumors after cytoreductive surgery (CS), CS + RIT (RIT) or CS + HIPEC (HIPEC).
At the endpoint of the study, 16 weeks after CS, 14 animals (two animals in the CS group, five animals in the CS + HIPEC group, and seven animals in the CS + RIT group) were still alive, without any physical signs of intraperitoneal tumor growth.
Of these 14 animals that were still alive 16 weeks after surgery, one animal in the CS + HIPEC group and three animals in the CS + RIT group showed macroscopic evidence of tumor at dissection. In the remaining 10 animals (two in the CS alone group, four in the HIPEC group, and four in the RIT group) not even microscopic tumor presence was found.
DISCUSSION
In the present study, adjuvant radioimmunotherapy after cytoreductive surgery for peritoneal carcinomatosis of colorectal origin in rats significantly improved survival, whereas HIPEC did not. In addition, the application of HIPEC was associated with considerably more toxicity as compared to RIT.
The treatment of peritoneal carcinomatosis was studied with CC-531 syngeneic tumors that grew intraperitoneally in Wag/Rij rats. This model is highly reproducible, and the growth and distribution pattern throughout the abdominal cavity are similar to the human entity of PC.14 Cytoreduction performed at 7 days after tumor reduction resulted in minimal residual disease (<1 mm). In this setting, both HIPEC and RIT result in maximum therapeutic efficacy.12,13,17
The MG1 MAb preferentially localizes in the CC-531 tumors,11 with only minor localization in thymus, lymph node, salivary gland tissue, and skin.15177Lu was selected as the radionuclide for RIT of minimal residual disease because of its high tumor uptake and adequate physical properties including a medium-energy β-emission with a maximum penetration range in tissue of 2.5 mm. In our previous studies we have used the combination of 177Lu-MG1 radionuclide-antibody. These studies demonstrated the combination to be highly effective for the improvement of survival in the model of PC as described previously.11,16 Moreover, in previous experiments we have shown that radioimmunotherapy with a radiolabeled irrelevant antibody is less effective by far compared with therapy with a radiolabeled specific antibody.18 HIPEC has been studied in only a few preclinical studies.19–22 These studies showed that its use was associated with a decreased tumor load compared with control groups.19 However, in these studies, HIPEC was associated with a considerable toxicity, indicated by lethargy, marked body weight loss, and bacterial translocation.23 These results on toxicity are in corroboration with the results of our study and mimic the clinical effects of HIPEC. Clinical studies with postoperative intraperitoneal chemotherapy are associated with a high mortality and morbidity.12
Of 16 published reports on the use of HIPEC in the clinical setting, 13 reports described the administration of MMC.12 In vitro, MMC has also shown to inhibit growth of CC531 cells in a concentration a thousand-fold lower than the concentration used in the present experiment.24 The applied dose of 16 mg/L MMC in our study is within the range of doses applied in clinical practice (5–20 mg/L25) and is higher than described in other preclinical studies (2.25 mg/L19 and 4 mg/L22). In addition, in the present study HIPEC was applied as an adjuvant treatment to cytoreduction, whereas Pelz et al.19 applied HIPEC as monotherapy with subsequent killing of the animals after only 10 days.
The intraperitoneal distribution of MMC during the perfusion was studied before the start of the actual experiment and showed equal distribution among all quadrants. The effect of the perfusion technique on distribution differences and their associated differences in survival have never been studied clinically. Glehen and colleagues performed a large clinical study in 506 patients that were treated with both the open and closed abdomen perfusion technique. The authors reported no differences in survival between both perfusion techniques.6 This observation was confirmed by the study of Sugarbaker and colleagues.26
RIT with 74 MBq of the 177Lu-MG1 radionuclide-antibody in 3 mL has previously been shown to be an effective treatment for experimentally induced PC of colonic origin when administered intraperitoneally.11,16 The biodistribution of intraperitoneally injected 111In-labeled MG1 was studied by Koppe et al. and showed a preferential uptake of the radiolabeled antibody in the tumor.11
The extraperitoneal temperature of 48°C necessary to obtain inflow temperatures of 44°C would not have had a negative influence on the cytotoxicity of MMC, since Ahrar et al.27 showed that only temperatures exceeding 60°C decreased its cytotoxic effect. On the other hand, one has to bear in mind the fact that the additive effect of hyperthermia in HIPEC in the clinical setting has not yet been proven in a randomized trial. Elias et al. and Glehen et al. both reported the use of early postoperative chemotherapy (EPIC), without hyperthermia, ranging from day 1 to day 5 after surgery, and HIPEC. Elias et al. found no significant difference in survival. Similarly, in the multicenter study in 506 patients of whom 53.3 and 24.3 % underwent HIPEC and EPIC alone, no significant difference was found in survival between the two treatment groups.6 The duration of the heated perfusion, 60 min. in this experiment, is in concordance with the recent consensus statement regarding Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy in the Management of Peritoneal Surface Malignancies of Colonic Origin, stating that the perfusion should last 60–120 minutes.13 These data on the dose of MMC, the used perfusion time and temperature, together with our results that showed antitumor effect in the model of induced PC from CC531 cells (significantly lower PCI at HEP in favor of CS + HIPEC), we can conclude that the HIPEC model used in our study was able to induce regression of PC of colonic origin.
In the latest reported clinical trial on adjuvant RIT in the setting of colon cancer, Liersch et al. reported results of a Phase II trial with 131I-labeled anti-CEA antibody labetuzumab administered to patients after complete resection of colorectal liver metastases. This study, where RIT was applied in an adjuvant setting to complete resection, resulted in a promising 5-year survival rate of 51.5%.10 The results of this study is in accordance with the conclusion of a recent review on the use of RIT to treat colon cancer.17 In this review, the authors state that the time may have come for clinical trials in which RIT is added to standard regimens to establish the place of this treatment modality as an adjuvant treatment after CS.
To our knowledge, the present study is the first study comparing the use of RIT and HIPEC in an adjuvant setting to CS for the treatment of PC in colorectal cancer. Our preclinical studies indicate that the application of RIT immediately following CS can improve survival in rats with PC of CRC. Moreover, from the present study we conclude that the use of adjuvant RIT is an effective treatment with low toxicity. When compared with today’s standard of care, HIPEC, RIT was at least as effective. RIT consisted of an activity dose of 74 MBq of 177Lu-labeled MG1per rat, resulting in only minor toxicity, whereas the theoretical MTD of 177Lu-labeled antibodies in 250 g rats could be approximately 150 MBq.11
There are, however, some challenges to the clinical applications of adjuvant RIT. For example, after cytoreductive surgery, patients are transferred to the intensive care unit. Optimal patient care has to be balanced with radiation safety issues for the medical staff. Previously, we reported on the optimal time interval between RIT and CS.16 In that study, we showed that RIT should be administered as soon as possible after CS, with a window of opportunity for RIT administration up to 4 days after surgery. It can therefore be envisioned that for radiation safety reasons the therapy should be given not before discharge of the patient from ICU and removal of the abdominal drains.
Our study therefore justifies the consideration of intraperitoneal radioimmunotherapy after cytoreductive surgery in case of peritoneal carcinomatosis of colorectal cancer. In clinical studies, this approach should be compared with HIPEC.
CONCLUSION
This study showed that RIT adjuvant to CS significantly improved survival compared to CS alone in a rat model of PC of CRC, whereas the contemporary gold standard, HIPEC, did not cause a significant improvement in survival. This improvement of survival was associated with a decreased level of treatment-related toxicity compared with HIPEC. Adjuvant radioimmunotherapy might therefore be an alternative adjuvant treatment after cytoreductive surgery of PC of colorectal origin in a clinical trial setting.

radioimmunotherapy
peritoneal carcinomatosis
cytoreductive surgery
heated intraperitoneal chemotherapy
adjuvant
colon cancer