Effects of the venoms in functional MRI

To get a general idea about raw-venom effects, principal component analysis was performed. PCA showed that the datasets of NhZ, NanZ and control can be clearly separated both, in phase I and phase II of the challenge experiment (Fig. 3). Especially obvious separation by the first and the second principle component could be seen for the stimulation of the Zymosan-injected paw (hyperalgesia; Fig. 3, B and D).

Abb. 34: Fig 3: Group separation between NhZ/NanZ and control with PCA

Values included in the PCA matrix were BOLD amplitude, activated volume, peak time (time to BOLD amplitude maximum) and probability (probability of voxel activation) at 50°C/55°C for the challenge measurement in phase I (Ph I) and phase II (Ph II) of NhZ (90 µg/kg ip.), NanZ (200 µg/kg ip.), and control. A) PCA for algesic stimulation. B) PCA for hyperalgesic stimulation. Data points are centers of corresponding scatter plots. n = 202 single brain structures of 8 animals in all fMRI groups

Abb. 34: Fig 3: Continued; For details see legend page 104 C) PCA for algesic stimulation. D) PCA for hyperalgesic stimulation

To evaluate which functional systems and brain structures contribute the most to this separation, Euclidian distances were calculated (Fig. 4). In the NhZ group sensory input structures, association cortex, perirhinal, ectorhinal (cxPrh/Ect) and piriform cortex (cxPir), visual cortex (cxVis), structures of hypothalamus (medial and paraventricular hypothalamic nucleus (hyM/hyArc)), caudate putamen (Cpu), and cerebellum (Cb) showed the largest differences compared with control for algesic stimulation (Fig. 4.1, A). For hyperalgesic stimulation fewer structures showed differences larger than mean ± STDEV. Only perirhinal, ectorhinal (cxPrh/Ect) and visual cortex (cxVis), caudate putamen (Cpu) and cerebellum (Cb) were conspicuous (Fig. 4.1, B). In the NanZ group also sensory input structures, parts of sensory and association cortex, perirhinal, ectorhinal (cxPrh/Ect), piriform cortex (cxPir), caudate putamen (Cpu) and cerebellum (Cb) showed the largest differences compared with control during algesic and hyperalgesic stimulation (Fig. 4.2, A and B). In addition, some structures of hypothalamus (arcuate and paraventricular hypothalamic nucleus (hyArc/hyPV)) showed large differences between NanZ and control mainly during algesic stimulation (Fig. 4.2, A).

Abb. 35: Fig 4: Important brain structures for group separation For details see legend page 109

Abb. 35: Fig 4: Continued For details see legend page 109

Euclidian distances for phase II of the challenge measurement of I) NhZ (90 µg/kg ip.) and II) NanZ (200 µg/kg ip.) under painful stimulation (50°C/55°C). Brain structures are divided belonging to right and left hemisphere. A) For stimulation of the algesic paw, the structures of the right hemisphere are ipsilateral, structures of the left hemisphere contralateral. B) For stimulation of the hyperalgesic paw, the structures of the left hemisphere are ipsilateral, structures of the right hemisphere contralateral. LS = limbic system, BG = basal ganglia, MO = motor output. Horizontal black line = mean value; Horizontal dashed black line = STDEV. n = 202 single brain structures of 8 animals in all fMRI groups

In addition, PCA factor loads for NhZ showed that the parameters BOLD amplitude, activated volume and count are important for group separation and consequently were focused on in the further analysis. The factor load values for algesic stimulation at 50°C/ 55°C were: Probability (0.42/0.39), BOLD amplitude (0.40/0.38), activated volume (0.38/0.38), peak time (0.22/0.16). For hyperalgesic stimulation at 50°C/ 55°C all parameters showed homogenous factor loads: Probability (0.41/0.34), peak time (0.35/0.33), activated volume (0.29/0.37), BOLD amplitude (0.27/0.35). PCA factor load values for NanZ also showed that the parameters BOLD amplitude, activated volume and count are important for group separation. Factor load values for algesic response at 50°C/ 55°C were: Probability (0.44/0.40), BOLD amplitude (0.38/0.39), activated volume (0.39/0.38), peak time (0.23/0.03); For hyperalgesic response:

Probability (0.42/0.42), BOLD amplitude (0.36/0.47), activated volume (0.33/0.43), peak time (0.08/0.07).

The probability of brain structure activation over all animals was 100% at 55°C indicating a very reliable measurement.

BOLD response amplitude and activated volume were calculated over all brain structures to provide a global group comparison. Statistically significant decreases in BOLD signal amplitude and activated volume under influence of NhZ occurred for painful algesic and hyperalgesic stimulation (Fig. 5, A and B). Statistically significant decreases in BOLD amplitude under influence of NanZ occurred only for painful hyperalgesic stimulation (Fig. 5, C) but in activated volume for painful algesic and hyperalgesic stimulation (Fig. 5, D). In summary, NhZ showed relatively most notably analgesic, NanZ antihyperalgesic effects.

Abb. 36: Fig 5: Analgesic and antihyperalgesic effects of NhZ and NanZ in the brain

Quantification of percent of BOLD changes (A: Page 110; C: Page 111) and activated volume in voxels (B: Page 110, D: Page 111) for the averaged brain structures during algesic and hyperalgesic heat stimulation for phase II of the challenge measurement with NhZ (90 µg/kg ip.) and NanZ (200 µg/kg ip.) compared to control. The different stimulation temperatures are indicated. Data are presented as differences between mean values for each temperature and the group-specific corresponding value for 40°C ± SEM. n = 8 animals in all fMRI groups. * p < 0.05; ^ns p > 0.05 (Student`s t-test)

Abb. 36: Fig 5: Continued; For details see legend page 110

BOLD signal amplitude values averaged for each functional group are shown in Table 1. Significantly lower activations under influence of NhZ occurred for painful algesic and hyperalgesic stimulation in sensory input, thalamus, sensory and association cortex, in parts of the limbic system, and in limbic and motor output structures. After injection of NanZ, thalamus, cortical structures, and parts of the limbic system showed lower BOLD response amplitudes. Structures of the limbic output showed significant increase of BOLD amplitude (Tab. 1).

Tab. 9: Table 1: Analgesic/antihyperalgesic effects of NhZ/NanZ in functional systems

Percent of BOLD changes for the functional systems during algesic and hyperalgesic painful stimulation (50°C/55°C) for phase II of the challenge measurement with NhZ (90 µg/kg ip.) and NanZ (200 µg/kg ip.) compared to control. Significant differences between venom and control are highlighted in gray (control > venom: dark gray; venom > control: light gray). LS = limbic system, LO = limbic output, BG = basal ganglia. Data are presented as mean values ± STDEV; n = 8 animals in all fMRI groups

Spatial activation patterns are shown as second-order statistical parametric maps (Fig.

6 and 7). Thalamic structures were still activated under influence of both venoms.

Under influence of NhZ and NanZ the activated volume of the cingulate cortex was significantly reduced. Neuronal activation in cortical structures, like primary somatosensory cortex, was reduced under influence of NhZ and NanZ for algesic and hyperalgesic stimulation.

In the NhZ group, activation for hyperalgesic stimulation in the secondary somatosensory cortex was significantly reduced (p = 0.02), but not highly significant like in the primary somatosensory cortex (p = 0.00006).

In the NanZ group, activations in the primary and secondary somatosensory cortex were reduced similarly. For hyperalgesic stimulation, in activated volume (Fig 7.2) as well as in BOLD signal response (Tab. 1), basal ganglia were still active and did not differ significantly between NanZ and control.

Abb. 37: Fig. 6: Analgesic/antihyperalgesic effects of NhZ in SPMs

Statistical parametric maps showing algesic 1) and hyperalgesic 2) stimulation-induced (55°C) brain activation of NhZ (90 µg/kg ip.) and control for phase II of the challenge measurement. Activation was assessed by BOLD fMRI. The blue/green scale indicates high activation for control; the red/yellow scale indicates lower activation for NhZ. Arrows point to brain structures with significant differences between NhZ and control. Th = thalamus, cxCg = cingulate cortex, S1 = primary somatosensory cortex, S2 = secondary somatosensory cortex, cxPir = piriform cortex, am = amygdala. Data are presented as t-values. n = 8 animals in all fMRI groups

Abb. 37: Fig 6: Continued; For details see legend page 114

Abb. 38: Fig. 7: Analgesic/antihyperalgesic effects of NanZ in SPMs

Statistical parametric maps showing algesic 1) and hyperalgesic 2) stimulation-induced (55°C) brain activation of NanZ (200 µg/kg ip.) and control for phase II of the challenge measurement. Activation was assessed by BOLD fMRI. The blue/green scale indicates high activation for control; the red/yellow scale indicates lower activation for NanZ. Arrows point to brain structures with significant differences between NanZ and control. Th = thalamus, cxCg = cingulate cortex, S1 = primary somatosensory cortex, S2 = secondary somatosensory cortex, Sep = septum, BNST = bed nucleus of stria terminalis, VP = ventral pallidum, Cpu = caudate putamen. Data are presented as t-values. n = 8 animals in all fMRI groups

Abb. 38: Fig 7: Continued; For details see legend page 116

5.2.5 Discussion

In this study four different raw snake venoms (Egyptian cobra: Naja haje; Black tiger snake: Notechis ater niger; Desert cobra: Walterinnesia aegyptia; Dugite: Pseudonaja affinis) were tested for their putative analgesic and antihyperalgesic effects in an acute heat-pain model.

In the behavioral tests only the venoms of Naja haje and of Notechis ater niger showed statistically significant antinociceptive effects. The fact that there were no significant changes in rota-rod and open-field test supports the presumption that the increase of paw- and tail-withdrawal latency is based on analgesia and is not a consequence of motoric disabilities due to local paralysis. The raw venom of Walterinnesia aegyptia and Pseudonaja affinis did not show any antinociceptive potential in Hargreaves or tail-flick test. Therefore, fMRI experiments were performed only with NhZ and NanZ.

It is important to mention that the venom of Naja haje was derived from 2, 2, 0 animals, the venom of Walterinnesia aegyptia only from 0, 1, 0 animals and that no information existed about the animals` geographical origin and exact age. In the literature it is known that the composition of venoms differs between male and female snakes (Chippaux et al., 1991). This fact could be a reason why there were no antinociceptive effects found in the venom of Walterinnesia aegyptia. Furthermore, there are large venom-composition variations in different habitats and geographical localizations (Chippaux et al., 1991).

Recent studies have shown that the venom of the Indian Cobra (Naja naja atra) shows analgesic and antihyperalgesic effects in behavioral tests after intraperitoneal injection (Jiang et al., 2008; Liang et al., 2009).

In the present study intraperitoneally injected venom of the Egyptian Cobra (Naja haje, NhZ), showed antinociceptive effects in behavioral tests and in functional MRI.

The reduction of neuronal activation nearly throughout the whole brain but particularly in cingulate cortex (cxCg), primary, and secondary somatosensory cortex (S1, S2) could be an indication of analgesia and antihyperalgesia. This effect was mainly observed during painful algesic and hyperalgesic stimulation at the highest temperature (Fig. 5). A strong analgesic effect was found for NhZ (Fig. 5, A and B).

This could also be seen in Euclidian distances during algesic stimulation in form of various brain structures with wide distances indicating differences between NhZ and control group. The BOLD signal amplitudes for NhZ were significantly reduced in almost all functional systems (Tab. 1). Although the thalamus was partially reduced in BOLD signal amplitude under influence of NhZ, there were still brain structures with significantly activated volume. The BOLD parameters signal amplitude and activated volume respond differently to changes in the brain. Neuronal excitations still led to activations in the thalamus, but the strength of the BOLD signal was reduced. The thalamus is the largest structure of the diencephalon and acts as an input structure for sensory systems and as a relay between input and higher-order processing in the cortical areas. The fact that neuronal activation was still present in the thalamus but was reduced in somatosensory and cingulate cortex could represent central nervous effects of snake venoms or their metabolites. The cingulate cortex plays an important role in pain processing. It is part of the medial pain system and receives projections from the medial thalamus. NhZ reduced the activated volume in the cingulate cortex statistically significant. If the raw venom of NhZ would act only in the periphery one would expect decreases in BOLD signal mainly in brain input structures like thalamus.

The fact that NhZ led to decreases also in higher-order brain structures like primary and secondary somatosensory cortex argues for brain-structure specific influences of components contained in this venom.

Venom of the Black Tiger Snake (Notechis ater niger, NanZ) , significantly decreased both BOLD amplitude and activated volume in BOLD measurements and showed a clear antihyperalgesic effect under painful stimulation (Fig. 5, C and D). Especially cortical structures like primary and secondary somatosensory cortex (S1, S2) were inhibited by the venom (Fig. 7). Similar to NhZ effects, activated volume of thalamic structures was still measurable. The venom of the Black tiger snake consists of potent neurotoxins but also of procoagulants (Rao et al., 2003; Williams and White, 1989;

Williams et al., 1988). The BOLD response is based on local blood oxygenation and is an indirect way to measure neuronal activity (Logothetis and Wandell, 2004). In consequence, another possibility for the strong reductions of BOLD response could be a change in cerebral blood flow. Contrary to this possibility is the fact that not all brain

structures were significantly reduced in BOLD amplitude under influence of NanZ.

For example, structures of the limbic output were significantly increased under influence of NanZ for hyperalgesic stimulation at 50°C (Tab. 1). Further studies are necessary as additional control to exclude influences of procoagulatory components in Notechis-venom and their putative biasing effects in BOLD measurements in detail.

Furthermore, studies with other dosages of the venoms as well as with other pain models (e.g. neuropathic pain) could be performed in the future because it could be that the venoms act in a totally different way in other experimental setups.

In this study the central nervous antinociceptive effects of raw venom of the Egyptian cobra (Naja haje) and the Black tiger snake (Notechis ater niger) could be confirmed with the non-invasive fMRI technique. Beyond that, specific higher-order brain structures like cingulate and somatosensory cortices could be identified that contribute to this antinociceptive effect. BOLD amplitude as well as activated volume were affected by the venoms but in a different manner. Because changes in the observed BOLD effect might also be caused by cerebral blood flow changes or direct brain-structure specific interactions of the applied venoms, future studies of the regional cerebral blood volume (rCBV), respective pharmacological MRI (Belliveau et al., 1991; Chin et al., 2008a; Chin et al., 2008b; Schwarz et al., 2007), should be performed. With this method, the direct interactions of drugs with the central nervous system can be visualized without any sensory stimulation and the mechanistic understanding of their effects can be enhanced (Schwarz et al., 2004; Schwarz et al., 2003).

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., 2011).

5.2.6 Conclusion

In conclusion, the raw venom of the Desert Cobra (Walterinnesia aegyptia) and the Dugite (Pseudonaja affinis) did not show any antinociceptive potential in behavioral

tests. In contrast, the venom of the Egyptian cobra (Naja haje) and the Black tiger snake (Notechis ater niger) showed statistically significant antinociceptive effects in behavioral tests and in functional Magnetic Resonance Imaging (BOLD-Imaging).

Venom of the Egyptian cobra relatively showed analgesic, venom of Black tiger snake antihyperalgesic effects in fMRI. These results demonstrate that raw snake venoms show selective pharmacological effects in the brain and are interesting substances for further pharmacological imaging studies in animals and humans. They might have important impact in future discovery of drugs for specifically modulating brain structures in acute and/or inflammatory pain.

5.2.7 Acknowledgements

This work was supported by the DFG Research Group 661/TP4 Preclinical Imaging.

We wish to extend special thanks to the Doerenkamp foundation for innovations in animal and customer welfare, VENOM SUPPLIES PTY LTD Australia and last but not least Dr. Markus Baur and Dr. Tobias Friz from the animal collection station for reptiles in Munich.