Discussion

“Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” as defined by the International Association for the Study of Pain derived from Bonica 1979 (Bonica, 1979). Several authors have shown mainly in behavioral pain models that crotoxin induces antinociceptive effects, supposed to be mediated by actions on the peripheral (Brazil and Excell, 1971; Chang and Lee, 1977; Hawgood and Smith, 1977a;

Hawgood and Smith, 1977b) but also on the central nervous system (Brazil et al., 1966b; Dorandeu et al., 1998; Habermann and Cheng-Raude, 1975; Moreira et al., 1996). In order to confirm these antinociceptive effects of crotoxin in our settings, behavioral tests were performed. The finding that intraperitoneally injected crotoxin exhibited analgesic effects in acute heat pain models like Hargreaves test and tail-flick test reproduced the results of former studies. Zhang et al. have demonstrated that the intraperitoneal injection of crotoxin in a dose of 44.3 µg/kg exhibits analgesic action in mice and rats using behavioral tests (Zhang et al., 2006). In this study crotoxin was used in a dosage of 45 µg/kg. The fact that there were no significant changes in rota-rod test, supported the presumption that the increase of paw- and tail-withdrawal latency was based on analgesia and was not a consequence of motoric disabilities due to local paralysis of the hind paws, although crotoxin is a neurotoxic agent (Brazil, 1966). Furthermore, crotoxin is known for its myotoxic activities (Breithaupt, 1976;

Gopalakrishnakone et al., 1984; Gutierrez et al., 2008a). To check the locomotion, open-field test was performed in the present study. As result, no significant changes in locomotor activity due to myotoxic effects of crotoxin could be found at this dosage.

The brain pain matrix in its classical meaning is a network of brain structures, like thalamus, primary, and secondary somatosensory cortex, prefrontal cortex, limbic system, periaqueductal gray, hypothalamus, and motor cortex that were considered to be important for pain processing in physiological and pathophysiological states (Ingvar, 1999; Melzack, 1990; Ploghaus et al., 1999; Tracey, 2005). Of note, there is recent evidence, that this matrix is not solely pain specific but detects salient sensory inputs generally (Iannetti and Mouraux, 2010; Legrain et al., 2011).

Analyzing the activity within the pain matrix by PCA revealed that (I) the crotoxin and the saline group could be separated and that (II) Euclidian distances pointed to brain structures relevant for this group separation during algesic and hyperalgesic stimulation. In particular, reticular nuclei like the dorsal medullary reticular nucleus were identified as especially susceptible to crotoxin under algesic stimulation. The reticular nuclei are part of the brain pain matrix and belong to the reticular formation of the sensory input, exercising an alert state of the brain by pain (Zambreanu et al., 2005). They project the incoming neuronal activation from the periphery to the diencephalon. The dorsal medullary reticular nucleus is supposed to be a pro-nociceptive center of supraspinal pain control (Lima and Almeida, 2002). Another integral input structure of the brain pain matrix is the thalamus, the largest structure of the diencephalon. It acts as a relay between input and higher-order processing in the cortical areas. With BOLD fMRI we could show that crotoxin decreased neuronal activation in thalamus significantly during painful algesic stimulation. This influence of crotoxin on sensory input structures like thalamus, and the finding that brainstem nuclei changed their behavior as revealed by PCA and Euclidian distances, provides so far an indication of the peripheral analgesic effects of crotoxin. This supports the results of former studies that showed that the mode of action of crotoxin is mainly peripheral (Brazil and Excell, 1971; Chang and Lee, 1977; Hawgood and Smith, 1977a; Hawgood and Smith, 1977b).

Furthermore, crotoxin decreased the BOLD amplitude and the activated volume in higher-order brain structures like retrosplenial cortex, cingulate cortex, primary, and secondary somatosensory cortex. In addition, orbital cortex was identified as especially susceptible to crotoxin during algesic stimulation. The influence of crotoxin

on cortical areas indicates that crotoxin induces not only peripheral but also central nervous antinociceptive effects. Retrosplenial cortex, cingulate cortex, and orbital cortex belong to the association system, receive input from the thalamus and are integral parts of the pain matrix. The association system is responsible for the validation of incoming signals and is important for the regulation of emotional states.

The somatosensory cortex processes incoming sensory information like painful sensations used in this study. Association and somatosensory system are strongly interconnected and are essential for pain perception and processing. Consequently, crotoxin, besides its peripheral effects, reduced the pain processing to higher-order brain structures like association and somatosensory cortex which probably is the major reflection of the analgesic but mainly antihyperalgesic effects of crotoxin. Another reason for the mostly antihyperalgesic activity could be the antiinflammatory effects of crotoxin. Several authors have indicated that crotoxin exhibits immunomodulatory and anti-inflammatory activities (Cardoso and Mota, 1997; Nunes et al., 2010;

Sampaio et al., 2003; Sampaio et al., 2005; Zambelli et al., 2008). The development of thermal hyperalgesia, induced by Zymosan A, could be inhibited by crotoxin which was reflected in lower neuronal activation after painful stimulation.

The limbic system is another functional system where crotoxin induced a decrease of the BOLD amplitude in this study. Particularly, entorhinal and perirhinal/ectorhinal cortex were identified as important for group separation by calculation of Euclidian distances. Perirhinal and ectorhinal cortex belong to the limbic system and play a role in memory forming. The entorhinal cortex is an interface between hippocampus and neocortex crucial for learning and memory. In general, the limbic system plays a role in qualitatively evaluating pain sensations and this process was inhibited by crotoxin.

Furthermore, the amygdala is a structure which was strongly influenced by crotoxin.

We found that crotoxin induced a significant increase of the BOLD-response amplitude and activated volume in the amygdala for algesic stimulation, especially in the basolateral amygdaloid nucleus, during phase I of the challenge experiment. The amygdala is part of the limbic system, located deeply in the temporal lobe of the mammalian brain, and is an important brain structure for anxiety and fear (LeDoux, 2007). Hugh vast of research indicates that the aversive stimuli particularly reach the

basolateral complexes of the amygdala during fear conditioning (LeDoux, 2007;

Schiller and Delgado, 2010). This supports the assumption of direct anxiety induction by crotoxin. Moreira could show that Crotoxin induces an increased emotional state in rats in the open-field and hole-board tests, however they used high concentrations of crotoxin (100, 250, 500 µg/kg, ip; Moreira et al., 1996). Under these conditions, crotoxin significantly increased freezing and decreased ambulation. These behavioral changes could be blocked by an anxiolytic dose of diazepam. Moreira and colleagues (1996) suggested that these high-dosage effects express an increased emotional state induced by crotoxin and consequently central nervous effects of this substance. The fact that crotoxin increases freezing and that anxiolytic drugs like diazepam can block this behavior indicates an anxiety induction through crotoxin. In the present study the lower dose of crotoxin (45 µg/kg) did not lead to detectable anxiety behavior in the experimental animals in the open-field test. However, with fMRI direct anxiety induction by crotoxin could be detected even before changes in animals` behavior occurred. Moreover, in a clinical study of Cura and colleagues, two of six patients with advanced cancer showed grade 2-3 anxiety after crotoxin treatment in the highest dose (0.03 to 0.22 mg/m² crotoxin i.m.) for 30 consecutive days (Cura et al., 2002). In rats a dose of 0.26 mg/m² would correspond to the dose used in the present study (45 µg/kg). Consequently, the fMRI technique allows direct future validation of crotoxin-induced increased activity of the amygdala in humans (cf. Hess et al., 2011) and of the central nervous effects of crotoxin.

In the present study the peripheral antinociceptive effects of crotoxin could be confirmed with the non-invasive fMRI technique. Beyond that, specific higher-order brain structures like somatosensory and association cortices that contribute to this antinociceptive effect could be identified. BOLD amplitude as well as activated volume was affected by crotoxin but each in a different manner with more significant antihyperalgesic effects. Because changes in the observed BOLD effect might also be caused by direct brain-structure specific interactions of the applied drug, future studies of the regional cerebral blood volume (rCBV, respective pharmacological MRI) should be performed (Belliveau et al., 1991; Chin et al., 2008a; Chin et al., 2008b;

Schwarz et al., 2007). The direct interactions of drugs with the central nervous system

can be visualized without any sensory stimulation using this imaging technique (Schwarz et al., 2004; Schwarz et al., 2003). This approach could disentangle potential direct interactions of crotoxin in the CNS und enhance the mechanistic understanding of the effects of crotoxin.

Finally we would like to emphasize that BOLD fMRI is an ideal method for translational drug research in the field of toxinology because (I) it is non-invasive, (II) enhances animal welfare, (III) provides the possibility to translate findings from animals to humans using the very same surrogate marker and readout technology, and thereby (IV) allows to investigate pharmacological effects of animal-venom components in humans in the future (cf. Hess et al., 2001).