Introduction

In 1938, the first animal-venom component, crotoxin from Crotalus durissus terrificus, a heterodimeric complex composed of a weakly toxic, basic Phospholipase A2 (PLA2) (component B or Crotoxin B, CB) and a toxic, acidic and non-enzymatic polypeptide named crotapotin (component A or crotoxin A, CA) (Aird et al., 1985, 1986; Aird et al., 1990) was purified and crystallized (Bon et al., 1989;

Slotta, 1938; Slotta and Fraenkel-Conrat, 1938b; Slotta and Fraenkel-Conrat, 1938a).

Slotta and Fraenkel-Conrat could show for the first time that toxins contained in snake venom are proteins, a milestone of toxinology (Slotta and Fraenkel-Conrat, 1938a).

Since then crotoxin became a substance of great interest for toxinologists.

Lots of studies were performed to evaluate the biological, biochemical, and pharmacological properties of this component of the venom of Crotalus durissus terrificus (South American rattlesnake [Linnaeus, 1758]). Classic effects of crotoxin include most notably neurotoxicity (Brazil, 1966) but also myotoxicity (Breithaupt, 1976; Gopalakrishnakone et al., 1984; Gutierrez et al., 2008a), nephrotoxicity (Amora et al., 2006; Hadler and Brazil, 1966), and cardiotoxicity (Hernandez et al., 2007;

Santos et al., 1990). In addition numerous studies have shown that crotoxin also exhibits analgesic (Nogueira-Neto Fde et al., 2008; Zhang et al., 2006; Zhu et al., 2008), immunomodulatory (Cardoso and Mota, 1997; Zambelli et al., 2008), anti-inflammatory (Nunes et al., 2010; Sampaio et al., 2006; Sampaio et al., 2003; Sampaio et al., 2005), anti-microbial (Diz Filho et al., 2009; Soares et al., 2001), and anti-tumor effects (Baldi et al., 1988; Cura et al., 2002; Yan et al., 2006).

Current knowledge claims that the mode of action of crotoxin is basically peripheral (Brazil and Excell, 1971; Chang and Lee, 1977; Hawgood and Smith, 1977a;

Hawgood and Smith, 1977b). CB binds to receptors of the presynaptic membrane and inhibits the release of acetylcholine. But there are also some studies that suggest that crotoxin acts postsynaptically by stabilizing the acetylcholine receptors in their inactive state (Bon et al., 1979; Brazil et al., 2000). Crotoxin leads to non-depolarizing neuromuscular blockade and in consequence in higher doses to paralysis (Kini, 2003).

CA acts like a chaperone and potentiates the toxicity of the PLA2 by conducting CB to its correct binding sites (Bon, 1982; Bon et al., 1979; Chang and Su, 1978; Hawgood

and Smith, 1977b; Hendon and Fraenkel-Conrat, 1971; Rubsamen et al., 1971).

Furthermore, crotoxin shows important effects in the central nervous system (Brazil et al., 1966b; Dorandeu et al., 1998; Habermann and Cheng-Raude, 1975; Moreira et al., 1996). Current research shows, that Crotoxin inhibits the pain-evoked discharge of neurons in rat brains after intracerebroventricular injection (Zhu et al., 2008), considered to be based on central nervous effects.

The antinociceptive potential of crotoxin has long been known (Brazil, 1934) and could be confirmed in different experimental pain models (Giorgi et al., 1993;

Gutierrez et al., 2008b). Some researchers suggested that muscarinic and opioid receptors are not involved in the analgesic effects of purified crotoxin in acute pain models after intraperitoneal (Zhang et al., 2006) and intracerebroventricular (Zhu et al., 2008) injection. In contrast, Nogueira-Neto and colleagues could show that central muscarinic receptors in combination with activation of α-adrenoceptors and 5-HT receptors are involved in the analgesic actions of topical crotoxin in a neuropathic pain model (Nogueira-Neto Fde et al., 2008). Furthermore, they investigated that eicosanoids, important signaling molecules during inflammation, modulate the action of crotoxin. Several studies have indicated that crotoxin has immunomodulatory and anti-inflammatory activities (Cardoso and Mota, 1997; Nunes et al., 2010; Sampaio et al., 2006; Sampaio et al., 2003; Sampaio et al., 2005; Zambelli et al., 2008). Most of the studies so far used behavioral tests or electrophysiological approaches to investigate the analgesic activities of crotoxin. Due to limited capabilities of these methods, the exact mechanisms of the antinociceptive action of crotoxin are not yet completely understood. Moreover, in these experiments lots of animals have to undergo painful sensations and get sacrificed for extraction of brain slices or neurons.

In the field of clinical research, Cura and coworkers could demonstrate the relevance of the administration of crotoxin in advanced cancer patients. Intramuscular administration of crotoxin led to significant decrease of painful sensations and reduction in consumption of analgesics (Cura et al., 2002). New non-invasive methods should be developed to examine these antinociceptive effects of crotoxin not only in animals but also in human patients to promote better understanding of the mode of action of this substance.

At first, in order to confirm the antinociceptive effects of crotoxin in our setup accordant with the literature (Zhang et al., 2006), four basic behavioral tests were performed: Hargreaves test and tail-flick test to prove the antinociceptive activity of crotoxin, rota-rod test to check motoric integrity and open-field test to get information about locomotor and exploration activity. Rota-rod and open-field test were important to ensure that changes in Hargreaves and tail-flick test were not a consequence of neurotoxic and myotoxic activities of crotoxin.

Secondly, fMRI investigations were performed to evaluate not only peripheral but primarily possible central nervous antinociceptive properties of crotoxin in a brain- structure-specific manner. fMRI is an efficient and extensively used method to study processing of nociception in the brain of living subjects with minimized burden and no harm for the experimental human or animal (Apkarian et al., 2005; Hess et al., 2011;

Hyder et al., 1994; Knabl et al., 2008; Neely et al., 2010; Wise and Tracey, 2006) as well as to perform drug research in anesthetized subjects (de Celis Alonso et al., 2012;

Hess et al., 2011; Neely et al., 2010; Wise and Tracey, 2006). The blood oxygenation level dependent (BOLD) signal reflects neuronal activation patterns in the brain (Logothetis and Pfeuffer, 2004; Ogawa et al., 1990a; Ogawa et al., 1990b) in a completely non-invasive way. Neurons, when activated, consume more energy.

Consequently, the local response of the vasculature is to increase blood volume and flow in order to supply more oxygen. Oxygen consumption leads to changes in local concentration of deoxyhemoglobin that are detectable as the BOLD signal due to the paramagnetic properties of deoxyhemoglobin (Logothetis, 2002; Wise and Tracey, 2006). Ploghaus and co-workers (Ploghaus et al., 1999) demonstrated that pain, in contrast to other sensory modalities, is reflected in a complex network of different brain structures which lead to the concept of the pain matrix in the central nervous systems (Ingvar, 1999; Melzack, 1990, 2001; Ploghaus et al., 1999). Consequently, in a first approach, the antinociceptive activity of pharmacological agents is reflected in reduced BOLD signals in specific brain structures of this pain matrix.

The focus of this article is twofold: first, to obtain insight into antinociceptive effects of crotoxin at the level of the central nervous system and, second, to disentangle modulatory effects of crotoxin on normal pain (analgesic effects) from

pathophysiological pain states like hyperalgesia (antihyperalgesic effects). Therefore, local unilateral hyperalgesia was induced by injecting Zymosan A subcutaneously into the left hind paw but applying painful heat stimulations to both hind paws during the fMRI experiment. Zymosan A, a glucan prepared from yeast cell walls (Saccharomyces cerevisiae), induces a strong inflammation leading to hyperalgesia when injected in living tissues (Meller and Gebhart, 1997).

Combining the results of behavioral tests with brain-structure specific readouts, provided by high-resolution MRI techniques, introduces a new way of investigating the analgesic activity of crotoxin. This leads to a deeper mechanistically inside in the mode of the antinociceptive action of crotoxin and simultaneously contribute to animal welfare (Russell, 1959) by its non-invasiveness and applying the nociceptive stimulation under anesthesia. Due to the non-invasiveness of fMRI its use in human studies is well established (Apkarian et al., 2001). Recently, our group has demonstrated, that fMRI can be utilized as a very effective translational tool enabling researchers to transfer knowledge obtained in rodent studies to patients (Hess et al., 2011). Therefore, fMRI allows gathering unique knowledge about the antinociceptive effects of crotoxin and effectively facilitates the translation to treatment of humans in the future.