Effects of crotoxin in fMRI

PCA showed that the responses of the crotoxin group can be fully separated from the saline group (Fig. 3). For the stimulation of the naive paw (algesia), a clear time-dependent effect of crotoxin was visible, because the distance between the center of gravity of phase I and phase II for crotoxin is remarkably large (Fig. 3, A). In addition, a clear separation between crotoxin and saline could be seen for the stimulation of Zymosan-injected paw (hyperalgesia) (Fig. 3, B). No clear separation could be seen between the center of gravity of phase I and phase II for saline during algesic stimulation (Fig. 3, A). A slightly better separation was observed between phase I and phase II of the saline group during hyperalgesic stimulation (Fig. 3, B).

Abb. 27: Fig. 3: Group separation between crotoxin and saline 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 crotoxin (45 µg/kg ip) and saline. A) PCA for algesic stimulation. B) PCA for hyperalgesic stimulation. Data points are centers of corresponding scatter plots. Grey arrows = distances between centers of crotoxin and saline group; Diagonal black line in Figure B = separation between crotoxin and saline group; n = 202 single brain structures of 8 animals in all fMRI groups

PCA factor load values showed that the parameters BOLD amplitude, activated volume and count are important for the obtained group separation and consequently were focused on in the further analysis. The factor load values at 50°C/ 55°C for algesic response were: BOLD amplitude (0.39/0.42), activated volume (0.41/0.41), probability (0.41/0.38), peak time (0.13/0.11). Factor load values for hyperalgesic response were: BOLD amplitude (0.37/0.40), activated volume (0.39/0.42), probability (0.42/0.39), peak time (0.19/0.10)). Obviously, the parameter peak time was not important for separation between crotoxin and saline group. By calculating Euclidian distances, functional systems and single brain structures being most important for the separation between crotoxin and saline were identified. The sensory input within the brainstem, thalamus, parts of association cortex, parts of limbic system and cerebellum had Euclidian distances larger than mean value plus standard deviation for algesic stimulation. In particular, reticular nuclei (MdD dorsal medullary reticular nucleus, GI gigantocellular reticular nucleus, PnM and PnO pontine reticular nuclei), orbital cortex (cxOrb), and perirhinal/ectorhinal cortex (cxPrh/Ect) could be identified (Fig. 4, A).

For hyperalgesic stimulation associative and limbic cortical structures and amygdala showed Euclidian distances larger than mean value plus standard deviation. In particular, entorhinal (cxEnt) and perirhinal/ectorhinal cortex (cxPrh/Ect), cortical and basolateral amygdaloid nuclei (amCo/amBL) and hip area of amygdala (amHa) expressed large distances (Fig. 4, B).

Abb. 28: Fig. 4: Important brain structures for group separation: For details see legend page 80

Euclidian distances for phase II of the challenge measurement (45 µg/kg ip) under painful stimulation (50°C/55°C). Calculation was performed over all principal components. 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. Ass. cx. = association cortex, LS = limbic system, BG = basal ganglia, motor out = motor output. Horizontal black line = mean value; Horizontal dashed black line = STDEV. n = 202 single brain structures of 8 animals in all fMRI groups

The probability of obtaining BOLD responses was 100% at 55°C for all groups indicating reliably measurements across all animals. The parameters BOLD amplitude and activated volume were both influenced by crotoxin, but each in a different manner:

Statistically significant decrease of BOLD amplitude averaged over all brain structures occurred under the influence of crotoxin during both, painful algesic and hyperalgesic stimulation (Fig. 5, A). BOLD amplitude values for the different functional systems are shown in Table 1. Significant lower activations under influence of crotoxin occurred for algesic stimulation in thalamus, sensory (primary and secondary somatosensory cortex) and association cortex (retrosplenial and cingulate cortex), in parts of the limbic system, and in limbic output structures. For hyperalgesic stimulation significant reductions due to crotoxin were found in sensory cortex, association cortex, in parts of the limbic system, and motor output structures (Table 1).

Tab. 8: Table 1: Analgesic/antihyperalgesic effects of crotoxin 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 (45 µg/kg ip) compared to saline.

Significant differences between crotoxin and saline are highlighted in gray (saline > crotoxin: dark gray;

crotoxin > saline: 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

In contrast, significant decrease of activated volume averaged over all brain structures occurred only for hyperalgesic stimulation (Fig. 5, B).

Abb. 29: Fig. 5:Analgesic and antihyperalgesic effects of crotoxin in the brain

Quantification of A) percent of BOLD changes and B) activated volume (in voxels) for the averaged brain structures during algesic and hyperalgesic heat stimulation for phase II of the challenge measurement with crotoxin (45 µg/kg ip) compared to saline. 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)

The statistical parametric maps were visualized to demonstrate these significant changes of activated volume (Fig. 6). Crotoxin reduced the activated volume compared to saline especially in retrosplenial cortex, cingulate cortex, primary and secondary somatosensory cortex, and caudate putamen. In contrast crotoxin produced significantly higher activation than saline in the pallidum, septum, and zona incerta.

Abb. 30: Fig. 6: Antihyperalgesic effects of crotoxin in statistical parametric maps

Statistical parametric maps showing hyperalgesic stimulation-induced (55°C) brain activation of crotoxin (45 µg/kg ip) and saline for phase II of the challenge measurement. Activation was assessed by BOLD fMRI. The blue/green scale indicates high activation for saline; the red/yellow scale indicates lower activation for crotoxin. Arrows point to brain structures with significant differences between crotoxin and saline. Th = thalamus, cxRS = retrosplenial cortex, cxCg = cingulate cortex, S1 = primary somatosensory cortex, S2 = secondary somatosensory cortex, ZI = zona incerta, Sep = septum, Pal = pallidum, CPu = caudate putamen. Data are presented as t-values. n = 8 animals in all fMRI groups

Another finding was that the activated volume and in part the BOLD amplitude of the amygdaloid nuclei were significantly higher for algesic stimulation under influence of crotoxin during phase I of the challenge experiment (activated volume: Saline 2.8 ± 4.60 / cro 20.50 ± 10.71, p-value: 0.001; BOLD amplitude: saline 0.09 ± 0.13 / cro 0.49 ± 0.33, p-value: 0.01). Especially the basolateral amygdaloid nucleus was involved in this phenomenon. This effect decreased in the second part of the experiment (phase II).