Separate groups of male Swiss mice were injected with 25 μg of somatostatin (somatostatin-ucb^®, UCB Pharma, Brussels) intraperitoneally (in the abdomen) or intravenously (in the caudal vein in the tail). At defined moments after injection (10, 20, 40, 60 minutes) the animals were anaesthetized with Nembutal^® (60 mg/kg). The thoracic cavity was cut open and blood collected from the right ventricle of the heart into chilled syringes containing EDTA (1 mg/ml) and Aprotinin (500 KIU/ml blood). The collected blood was centrifuged at 3000 rpm for 15 minutes at 0°C. The plasma was immediately frozen at -80°C freezer. Untreated naïve mice were also bled to ascertain background levels of somatostatin.

The maintenance of the S. mansoni life cycle and the transcutaneous infection of mice with S. mansoni have been previously described [14]. Male Swiss mice (age 7 weeks) were anaesthetized with Nembutal^® (60 mg/kg) and the abdomen was shaved. A metal ring was placed on the abdomen and then filled with treated water containing infectious cercariae of a Puerto Rican strain of S. mansoni. The cercariae were allowed to penetrate during 20 minutes after which the water was removed and checked for remaining cercariae.

Groups of Swiss mice were infected with 100 S. mansoni cercariae each as mentioned above. Age matched mice were maintained as uninfected control animals. Groups of 10 mice composed Group 1 that were maintained till 10 weeks following infection, other 10 mice composed Group 2 that were maintained till 14 weeks of infection.

At such times animals of Groups 1 and 2 were treated with somatostatin (Somatostatine-ucb^®), that was kindly gifted by UCB Pharma, Brussels. Somatostatin was administered in two regimens – a one-day treatment or a two-day treatment. One day treatment consisted of 3 doses of 30 μg somatostatin each, two day treatment was the double of this dose, that is 6 doses of 30 μg somatostatin to each animal. Somatostatin was administered intravenously via the caudal vein in the tail and intraperitoneally at regular intervals during the day. One week after the last somatostatin administration, mice were killed, the weight of the animal, liver and spleen were noted, plasma was extracted and stored as mentioned above, and the liver frozen immediately in liquid nitrogen and then at -80°C. The overall planning is depicted in Table 1. Untreated animals of the acute and chronic stage were also sacrificed at the respective times together with their treated counterparts. Research protocols involving rodents received ethical clearance by the University of Antwerp ethical committee.

All animals of Groups I and II were controlled for liver, spleen and total body weight, before sacrifice. The adult worms were recovered from the hepatic portal system and the liver by perfusion with citrate saline (0.85% sodium chloride; 1.5% sodium citrate). The livers were cut out and snap frozen in liquid nitrogen. For cyosectioning, liver fragments were embedded in Tissue-Tek OCT compound, 4 μm thick transverse sections were cut on a cryostat, mounted on slides coated with 0.1% ploy-L-lysine and stored at -20°C until use. To study parasite egg count, hepatocyte status, granuloma size and cellularity, series of sections were stained with Haematoxylin-eosin stain.

The collagen concentration was determined by assessing hydroxyproline amount. Herein is described the protocol of Technique B for the biochemical assessment of fibrosis used by Bergman and Loxley [15]. Just as was done by Cheever [16] we neutralized our samples for the color reaction.

The measurement of somatostatin concentrations in the Swiss mice plasma was carried out in the laboratory of Gastro-intestinal Hormones, at Gasthuisberg, K.U. Leuven, by means of a radio-immuno assay (RIA). The RIA was performed by incubating the samples with 1.7 pM 3-[¹²⁵I] iodotyrosyl¹¹ somatostatin-14 (specific activity 2000 Ci/mmol, Amersham Pharmacia Biotech, Buckinghamshire, UK) and a rabbit antibody against human SOM [1-14] in a 50 mM sodium phosphate buffer (pH 7.4, 0.25% EDTA, 0.5% charcoal-BSA, 500 U/ml Trasylol) for at least two days at 4°C. At the end of the incubation period the SOM bound to the antibody was separated from the free SOM by adding 500 μl dextran-charcoal followed by centrifugation for 15 min at 3000 rpm. Both fractions were counted in a gamma counter and the results were read from a standard curve (0–250 pg/ml) included in the RIA. The minimal detectable dose was 2.5 pg/ml.

Somatostatin is a highly purified compound reflected by the fact that it has very little toxicity. Studies in mice have shown that the LD50 is comparable to 10,000 times the acute therapeutic dose used in humans. The favorable metabolic profile of somatostatin is further supported by the fact that this compound has a very short half-life, and thus any undesirable effects may be rapidly reversed. In our experiment we have used 90 μg/24 hours in the Swiss mice that weighs about 40 g. In humans the therapeutic dose is 3.5 μg/kg/hour or 6 mg/24 h for a 75 kg man. A comparison of these values tells us that we are working with about 100 times higher values in mice as compared to that used in humans.

Following an intravenous infusion of a therapeutic dose of somatostatin (250 μg/hour or 6 mg/24 hours) to healthy volunteers, the plasma profile demonstrated that the drug reaches a plateau of 300–3000 pg/ml within 15 minutes. Somatostatin has a very short half-life of 1–3 minutes in man. In animal studies the same profile has been observed in dogs, whereas in rats somatostatin is stable for up to 30 minutes in whole blood, indicating that it is broken down in tissues [17]. To determine the pharmacokinetic profile in outbred Swiss mice, 25 μg of somatostatin was administered per mouse via the intraperitoneal or intravenous route. To assess the break down in these mice, plasma was collected at regular time intervals and circulating somatostatin levels assayed (Figure 2). At 10 minutes post injection somatostatin levels fall to 30233 pg/ml (IP) and 17413 pg/ml (IV). After intravenous injection thus somatostatin appears to be more rapidly degraded than after intraperitoneal injection, however after one hour of injection using either means the levels of circulating somatostatin in blood reaches baseline control levels. Thus in our experimental set up of somatostatin treatment in S. mansoni infected mice the schedule of 3 treatments per day (morning, noon and evening), assured that for at least 3 hours per day there were significantly high levels of circulating somatostatin in vivo.

The weight of the liver increased with infection, with a significant rise at the acute (p = 0.01) and chronic stage (p = 0.01) as compared to the uninfected animals (Fig. 3.). Acute and chronic infected animals after 2 days of somatostatin treatment showed no significant change in their liver weights (p = 0.19; 0.90 respectively).

Mean spleen weights were significantly increased in acute (p = 0.01) and chronic (p = 0.01) infected animals as compared to uninfected controls (Fig. 4.). Acute and chronic infected animals after 2 days somatostatin treatment showed no significant changes in their spleen weights (p = 0.28; 0.73 respectively).

The total body weight of the acute infected animals showed a decrease as compared to uninfected animals however this difference was borderline significant (p = 0.06) (Fig. 5). There were no significant changes in body weight in acute or chronic infected animals after somatostatin treatment.

S. mansoni egg stage parasites were counted in liver sections before and after somatostatin treatment. A series of 10 slides were examined from each sub group, the sections obtained at intervals of at least 40 μm so as to avoid counting repetitive eggs. Two days of somatostatin treatment in acute infected animals significantly reduced the mean parasite egg counts form 56.01 ± 3.34 to 43.76 ± 3.56 (p = 0.03). At the chronic stage of infection, mean egg counts were only significantly (p = 0.006) reduced after 1 day of somatostatin treatment from69.82 ± 2.77 to 56.62 ± 3.02 (Fig. 6.).

Haematoxylin-eosin staining of liver sections displayed the evolution of granuloma formation in infected animals as compared to treated animals. After treatment with somatostatin hepatocyte status remained unaltered, granulomas were not remarkably changed in size or cellularity (results not shown).

Hepatic fibrosis is the process of excessive deposition of collagen in the liver. Collagen generation involves 3 polypeptide chains, each chain composed of 19 amino acids consisting of glycine (30%), proline (12%) and two other rather uncommon amino acids – hydroxyproline (10%) and to an even lesser extent hydroxylysine. Proline and hydroxyproline are responsible for the angle (kink) in the polypeptide backbone furthermore hydroxyproline stabilizes the collagen via intramolecular hydroxyl bridges. Normal hydroxylated collagen is equivalent to 10% hydroxyproline in weight. Thus 1 μmol hydroxyproline (MW = 131.13) is equivalent to 1.3113 mg collagen. Hydroxyproline concentrations in the livers (controls versus acute, chronic, treated and untreated) of the Swiss mice are depicted in Table 2 and figure 7. The differences in hydroxyproline concentrations between uninfected and infected mice with or without treatment are illustrated by non-parametric (Mann-Whitney U) or parametric (unpaired Student's t) test as shown in Table 2.

At 10 weeks post infection with 100 S. mansoni cercariae, levels of hydroxyproline were significantly higher as compared to that assayed in uninfected animals (p = 0.01) (Fig. 7.). After treatment with somatostatin for 2 days, circulating levels of this collagen at one-week post treatment time were significantly lower as compared to infected animals that were not treated (p = 0.01).

Progression of the infection from week 10 to week 14 did not result in a significant rise in hydroxyproline levels. Treatment of chronically infected animals with a two-day regimen of exogenous somatostatin resulted in a drop in hydroxyproline levels at one-week post treatment. This fall was significant (p = 0.03) as compared to chronic untreated animals.

Somatostatin concentrations in the plasma (controls versus acute, chronic, treated and untreated) of the Swiss mice are depicted in Table 3 and figure 8. The differences in somatostatin concentrations between uninfected and infected mice with or without treatment were illustrated by a non-parametric (Mann-Whitney U) test as shown in Table 3. The distribution of somatostatin levels in all groups is depicted in Figure 8. At 10 weeks post infection with 100 S. mansoni cercariae, levels of circulating somatostatin were significantly higher as compared to that assayed in uninfected animals (p = 0.01). After treatment with somatostatin for 1 or 2 days, circulating levels of this neuropeptide at one week post treatment time were not significantly different as compared to infected animals that were not treated.

Progression of the infection from acute (week 10) to chronic (week 14) stages resulted in a drop in the somatostatin levels. Comparison of somatostatin levels between these two time points depicted borderline significant differences (p = 0.06). Treatment of chronically but not of acute infected animals with a two-day regimen of exogenous somatostatin resulted in an elevated level of circulating hormone at one-week post treatment. This rise in circulating somatostatin level was borderline significant (p = 0.06) as compared to chronic untreated animals.