Some Pig! Terrific Science!
Safety Assessment
Regina Kelder

Some Pig! Terrific Science!

A diminutive swine's role in animal research is expanding. The fourth of a four-part series.

Diabetes, a disorder of glucose metabolism, is a major public health crisis. According to the International Diabetes Federation, about 387 million people worldwide are living with the disease and over 14 million died from it last year.

Virtually all organ systems are impaired in the face of diabetes and some therapies that provide good glycemic control have adverse effects on the musculoskeletal system. So researchers are eager to find better ways to treat it and manage the many complications that generally occur as the disease progresses.

Developing new drugs has proved challenging, however, in part because the rodent models used widely in preclinical drug development have not been good predictors of how the disease progresses in humans.

SOT2015 LogoA closer match might be the minipig, says Jason Smedley, a toxicologist at Charles River's Spencerville site who has worked extensively with minipigs. Smedley presented findings from a diabetic mouse study at SOT2015.

The International Conference on Harmonization requires the use of two species in nonclinical studies for pharmaceuticals; one rodent and one nonrodent species. With many anatomical, physiological and functional similarities to humans, the miniature pig has become a viable alternative nonrodent animal model in nonclinical safety testing.

Minipigs have been used extensively in dermal studies, in large part because the skin is about the same thickness as human. Both humans and pigs also have roughly the same sparse coat of hair on the skin. Göttingens, in particular, have lightly-pigmented skin. About five other routes of administration –from intravenous to subretinal—are also used in toxicology studies.

Ocular research also benefits from the minipig, notes Mark Vezina, Scientific Director of the Ocular and Neuroscience Department at Charles River's preclinical site in Montreal. Minipig eyes are most approximate in size to human eyes; they also have similar innervation and vascular structures. "The larger eye size permits implantation of clinical devices and allows you to conduct clinically relevant surgical procedures. This facilitates translation of preclinical results to the development and support of clinical protocols," he said during a breakfast meeting at SOT2015.

At the SOT2015, Smedley described findings from a year-long study that induced and managed type 1 diabetes in a colony of Göttingen minipigs, the smallest and most widely used of the minipig models.

Two male minipigs and two female minipigs were given a single intravenous dose of streptozotocin, a naturally occurring chemical that is toxic to the insulin-producing beta cells of the pancreas in mammals. The drug is used in research to create diabetic animal models.

The animals reached a diabetic state within three days, at which point they were administered insulin twice a day, followed by glucose checks and feeding. Their body weight, condition and eyes were also monitored, and the results compared to a control animal.

Smedley said all the animals lost weight during the first 15 days, but slowly gained weight thereafter. Within three months, one female and one male minipig had developed cataracts, and within 11 months oral glucose levels averaged just over 250 mg/dL in the morning and 200 md/dL in the afternoon. Smedley said one of the female pigs experienced a bit of wasting after her oral glucose levels skyrocketed, but the remainder of the colony mimicked what occurs in the human diabetic condition. Smedley said when they performed neck incisions and biopsied the tissue they found most of the animals had the same wound healing impairments as those seen in diabetic patients.

The nesting instinct

Research has shown that nesting material reduces thermal stress and that nesting behavior might be a useful indicator might be useful of well-being in laboratory mice. You can read more about such studies here and here on Eureka. (Studies have also shown that male scents, which we blogged about here, elevate the stress level of mice causing them, paradoxically, to feel less pain.)

But does the material that laboratory mice use to construct their elaborate nests have any impact on basic toxicology parameters, either good or bad? This is not merely an academic question. If one were to add enrichment—such as nesting material—might it skew the results of studies?

Charles River animal scientists were curious, so they looked at whether metabolic and physiological changes resulting from nesting material could potentially impact the outcome of a toxicology study for immunomodulating drugs. The findings were presented at SOT2015 by Nathalie Hébert, a scientific director in toxicology. The nesting work was conducted by Catherine Brochu, a clinical veterinarian at Charles River's PCS site in Sherbrooke, Québec.

Mouse in nest (Image credit: Brianna Gaskill, Purdue University)
(Image credit: Brianna Gaskill, Purdue University)

CD1 mice, a multi-purpose animal used a lot in toxicology studies, were housed in groups of three in eight conventional cages, with crinkle paper nesting material placed in half the cages. Clinical signs and body weight were recorded weekly. Half the mice got the drug cyclophosphamide (CY), which is used to treat cancer and autoimmune disorders, with the controls receiving saline. Fecal pellets were then analyzed four times over a 12-week period, using cortisol metabolites as a marker of stress. Tissue analysis of the spleen, thymus, kidneys, adrenal glands and mandilubar lymph nodes were also performed. And clinical signs—from posture and dental hygiene to dehydration and fur erection—a kind of "goose bump" reaction to stress—were evaluated as well.

The study found that cortisol levels declined across-the board, though they were more rapid in the nested groups, suggesting greater acclimation to stress. And although the nesting material appeared to reduce overall stress in the nested mice given saline, it did not significantly mask stress induced by CY.

CY induced microscopic changes in CY treated animals, but the differences were not significant between the nested and unnested groups.

As expected, the groups administered CY had severe reductions in the absolute numbers of T and B cells compared to controls. But the nesting material appeared to induce an increase in the relative number of B cells, regardless of the treatment provided, suggesting perhaps that adding nesting material might improve immune function.

Clinical signs were seen in nested and unnested CY groups, though fur erection was observed less frequently in the group provided nesting material.

Overall, the findings suggest that the nesting material decreases stress, improves welfare without interfering with study parameters, which appears to be win-win both for the animal and the tox study.