**2. Material and methods**

#### **2.1. Preparation of animals**

Experiment was carried out on 24 mature crossbred ewes, Polish merino sheep weighing 32– 42 kg B.W., being *in anoestrus* period accordingly to the earlier described methods [16, 18, 20]. Food was removed 24 hours prior to the experiment. Analgesia was initiated by *i.m.* ketamine (Calypsovet, 20 mg kg−1 B.W., Gedeon Richter, Budapest, Hungary) administration, and 15 min later, *i.v.* infusion of pentobarbital anaesthesia in the dose of 20 mg kg−1 B.W. (Vetbutal, BIOWET, Pulawy, Poland) was performed. During unconsciousness, a T-shaped silicon cannula (inside diameter of 21 mm) was inserted into the duodenum (12–15 cm from pylorus). Secondly, an identical cannula was inserted into the dorsal sac of the rumen, using techniques described previously [30] on all animals. Simultaneously, under the same general anaesthesia/ analgesia, a permanent stainless steel cannula, 29 mm length and 2 mm in diameter (guide cannula), was inserted into the lateral cerebral ventricle (on the left and/or the right side) of the brain, 10 mm above the bregma and 5 mm laterally from the midline suture using stereotaxic method described by Sorraing et al [31]. After recovery from surgery, the animals were placed in metabolic cages at constant temperature (18–20°C) for at least 14 days prior to beginning of the experiment [20].

Several data show antinociceptive/antistressoric effects of organic Ca2+ inhibitors of L-VGCCs in acute duodenal pain of sheep [16, 20]. These inhibitors potentiate the analgesic action of κopioidergic receptor agonists [15], as well as morphine by decreasing opioids' tolerance [24]. It was also shown by Bongianni et al. [25] that VGCCIs suppress not only metabolic but also behavioral expression of the morphine withdrawal syndrome. In experiments performed on mice, it was shown that verapamil blocked amphetamine and also physostigmine induced footshock-induced aggression [26]. It was postulated by Michaluk et al. [27] that VGCCIs show antinociceptive properties; but they also change the territorial behavior of animals [28] and conspecific aggression in fish [29]. Such effects were probably caused by the inhibition of Ca2+ entry into neurons, preventing the appearance of synaptic vesicles in axon terminal, and release of neurotransmitter into the synaptic cleft. Davis and Bauer [14] have shown in experiments performed on rats, that activation of L-VGCCs are necessary for the long-term retention of fear

A duodenal and/or colonic distension method, provoking jejunal pain, stimulates hypothalamic-pituitary-adrenal-cortical (HPA) and sympatico-adrenal system (SAS), pathways that revealed as an increase in cortisol and CA in blood plasma [18, 20]. A different role of L-type antagonists for VGCC has been previously identified in different types of experimental and clinical pain in man and animals. Present study examined comparative role of VGCC blockers from different chemical groups—diltiazem, nifedipine, and verapamil administered *i.c.v.* in the same four different doses (0.25, 0.5, 1.0 and/or 2.0 mg *in toto*)—to estimate the comparable effect on the development of pain-related symptoms, clinical signs, plasma cortisol and catecholamine level, and the inhibition of ruminal motor activity caused by 5 min lasting

Experiment was carried out on 24 mature crossbred ewes, Polish merino sheep weighing 32– 42 kg B.W., being *in anoestrus* period accordingly to the earlier described methods [16, 18, 20]. Food was removed 24 hours prior to the experiment. Analgesia was initiated by *i.m.* ketamine (Calypsovet, 20 mg kg−1 B.W., Gedeon Richter, Budapest, Hungary) administration, and 15 min later, *i.v.* infusion of pentobarbital anaesthesia in the dose of 20 mg kg−1 B.W. (Vetbutal, BIOWET, Pulawy, Poland) was performed. During unconsciousness, a T-shaped silicon cannula (inside diameter of 21 mm) was inserted into the duodenum (12–15 cm from pylorus). Secondly, an identical cannula was inserted into the dorsal sac of the rumen, using techniques described previously [30] on all animals. Simultaneously, under the same general anaesthesia/ analgesia, a permanent stainless steel cannula, 29 mm length and 2 mm in diameter (guide cannula), was inserted into the lateral cerebral ventricle (on the left and/or the right side) of the brain, 10 mm above the bregma and 5 mm laterally from the midline suture using stereotaxic method described by Sorraing et al [31]. After recovery from surgery, the animals were

mechanical duodenal distension (DD) in the sheep.

**2. Material and methods**

228 Pain Relief - From Analgesics to Alternative Therapies

**2.1. Preparation of animals**

excitation.

Investigations were performed in four steps (groups, each of six animals for every drug). Every exercise was carried together on two unfed animals, placed in separate boxes at seven days interruption. Blood collection was performed 30 min prior to investigation, in 0 time and 5, 10, 15, 30, 60, and 120 min (**Figure 1**).

**Figure 1.** Experimental timelines for the four test group and blood sampling: Control: 0.9% solution NaCl or 20% DMSO—100 μL *i.c.v.* Diltiazem, nifedipine, or verapamil—1.0 or 2.0 mg *in toto* in 100 μL of 0.9% NaCl or 20% DMSO *i.c.v*. DD40—duodenal distension—40 mL water (temp. = 39°C) placed into rubber balloon. Duodenal distension + drug treatment. The time at which the intraduodenal balloon was inserted is marked with the letters: BP.

In the first experimental group, a 60 min recording of the ruminal motility was performed in each animals (n = 6) receiving 100 μL of 20% DMSO (control for nifedipine) or 0.9% NaCl (control for diltiazem and/or verapamil), during 1 min infusion (20 min after the 1st venous blood collection) into the lateral ventricle of the brain (*i.c.v.*); the rumen contraction was registered for 90 min (schema of the blood collection, it was respectively to **Figure 1**).

The second group of sheep (n = 6) were subjected to VGCCIs treatment alone, after 60 min control recording of rumen motility. Every animal received each dose of the substance (with 7 days interval). After the second collection of the venous blood, the sheep were *i.c.v.* given a 1 min lasting infusion of 100 μL of nifedipine in 20% DMSO solution (diltiazem or verapamil in 0.9% NaCl solution) in a dose of 0.25 mg in the first, 0.5 in the second, 1.0 mg in the third or 2.0 mg *in toto* (6.25, 12.5, 25.0 or 50.0 μg kg−1 B.W.) in 4 weeks and then the registration was maintained for the next 90 min.

In the third group of sheep (n = 6), after 30 min of control registration of the rumen motility, a rubber balloon (10 cm long) was inserted into the duodenum *via* the duodenal fistula. After placing the balloon in the jejunum, soon after the 2nd blood collection (0 time), the balloon was filled with 40 mL of warm water (DD40) and the distension was maintained for 5 min [28]. Then, the recording of ruminal contractions was continued for 60–90 min. Ten minutes before DD40, each animal received *i.c.v.* infusion of 100 μL of solvents for the drugs tested (**Figure 1**).

In the fourth group of sheep (n = 6), after 30 min of control registration of the rumen motility, a rubber balloon (10 cm long) was inserted into the duodenum and 30 min after the animals received the 100 μL *i.c.v.* infusion of diltiazem, nifedipine (in 20% DMSO solution) or verapamil at a dose of 0.25 mg in the first, 0.5 mg in the second, 1.0 in the third or 2.0 mg *in toto* in the 4th week (the same mode it was used for diltiazem, nifedipine and verapamil experimentation). After 10 min of the diltiazem, nifedipine or verapamil, 1-minute infusion duodenum was distended for 5 min with the balloon containing 40 mL of water (DD40) at body temperature. After the 5 min distension was over, the recording was continued for 60–90 min.

Experimental procedure lasted for 10 months. The doses of 1.0 and 2.0 mg diltiazem, nifedipine or verapamil *in toto* were effective in premedication contra DD40 only.
