Construction nearby animal houses has sporadically been reported to affect various aspects of animal health. Most of the reports have focussed on the impact on stress hormone levels and the hypersensitivity of animals relative to humans. There has also been an anecdotal report on the impact of construction on autoimmune diabetes in NOD mice. Here, we describe that nearby construction significantly impedes the progression to overt diabetes in female NOD mice offspring. We demonstrate that this was not due to a genetic drift or to particularities associated with our specific mouse colony. Interestingly, although the glycemia levels remained low in mice born from mothers subject to construction stress during gestation, we detected an active autoimmune reaction towards pancreatic islet cells, as measured by both the degree of insulitis and the presence of insulin autoantibody levels in the serum. These results suggest that the external stress imposed during embryonic development does not prevent but significantly delays the autoimmune process. Together, our findings emphasize the impact of surrounding factors during
The NOD mouse was developed in the 1970s in Japan and has since become the animal model of choice to investigate the genetic, cellular, and molecular mechanisms involved in the development of autoimmune diabetes [
Two main factors are known to contribute to disease susceptibility of this complex trait, namely, genetic and environmental factors. First, autoimmune diabetes is a complex genetic trait, with over 50 genetic loci contributing to disease susceptibility [
Interestingly, environmental factors are not only at play in defining susceptibility to autoimmune diabetes in humans but also exhibit a major impact on susceptibility to disease in the inbred NOD strain. Indeed, the incidence of diabetes is known to vary depending on the barrier status of the animal house, where the incidence of disease is the highest in specific pathogen-free facilities relative to conventional facilities [
We currently hold a NOD mouse colony in our specific pathogen-free facility, for which we routinely monitor the incidence of diabetes in female mice. These data serve as a control for ongoing experiments, wherein we administer different products to female NOD mice to determine the impact of these products on the pathophysiology of the disease as well as the incidence of diabetes [
All experiments were performed in line with the rules and regulations of the Canadian Council for Animal Protection, and the experimental procedure was approved by the Maisonneuve-Rosemont Hospital Animal Care Committee.
NOD mice from the NOD/LtJ colony were purchased from Jackson Labs in 2006 and were maintained by intercrossing nondiabetic 6-week-old male and female mice at the Hôpital Maisonneuve-Rosemont (HMR) specific pathogen-free facility. Routine microbiological monitoring of dirty bedding exposed sentinel animals was performed for the following murine pathogens: mouse hepatitis virus, Sendai virus, pneumonia virus of mice, reovirus-3, Theiler’s murine encephalomyelitis virus,
The breeding couples have been renewed, on average, every 14 weeks in an attempt to minimize potential genetic drifts [
Asphalt and part of the sidewalk were repaired from September 15 to 18, 2010. The animal house where all the NOD mice are kept is located in the basement immediately adjacent to this construction site. At that time, we held three breeder pairs, for which the details are provided in Table
Description of the mothers and litters from the NOD/LtJ-HMR mouse colony used in this study.
Breeding pair (#) | Date of birth |
Became diabetic | Number of litters produced | In gestation |
Age of mothers |
Litters included in the study |
---|---|---|---|---|---|---|
142 | June 16, 2010 | Unknown | 2 litters | Yes | 15 weeks | 1st litter |
143 | June 16, 2010 | Yes | 6 litters | No | 8 weeks | N/A, not included |
144 | July 25, 2010 | Yes | 5 litters | Yes | 10 weeks | 1st litter |
Diabetes incidence was monitored daily for overt signs of diabetes (wet cage, hunched posture) and every two weeks for urine glucose levels using Diastix (Bayer) starting at between 8 to 10 weeks of age. Mice were called diabetic upon two consecutive positive urine glucose readings. Blood glucose measurements >12 mM were used to confirm diabetes. Mice presenting with >12 mM of blood glucose were sacrificed within one week. All other mice were sacrificed at 32 weeks of age. At sacrifice, the pancreas was collected and frozen in Optimal Cutting Temperature Compound (OCT, Fisher) and the serum was collected and stored at −80°C.
Frozen pancreases were cut to 7
The serum levels of IAA were measured by ELISA in a protocol adapted from previous work [
Log rank Mantel-Cox tests were performed using GraphPad Prism 5 to determine the statistical significance of the difference in the incidence of diabetes.
Jackson Labs had previously reported that nearby construction partially affected the diabetes incidence in their NOD/LtJ colony in 2006 (
Nearby construction during the gestation period impacts diabetes onset and cumulative incidence in offspring. Depicted is a typical cumulative incidence of diabetes for eighteen female NOD/LtJ-HMR mice (open squares). Two NOD/LtJ-HMR female mice were in gestation while asphalt and sidewalk repairs were being carried out immediately outside the facility. The incidence of diabetes was closely monitored in the seven female offspring from these mothers (closed circles), where the onset is observed at 22 weeks of age and the cumulative incidence reaches approximately 30% at 32 weeks of age. Log-rank test,
Two of the three female NOD mice, that had been placed in breeding pairs, were in gestation during the construction period—September 15 to 18, 2010 (Table
Notably, the incidence of diabetes in Figure
We thus went on to test the hypothesis that the diabetes onset was delayed and that the incidence was reduced in the NOD/LtJ-HMR mouse colony as a consequence of a genetic drift. We undertook a second diabetes incidence study with six female NOD mice of the NOD/LtJ-HMR colony born in December 2010, more than two months after the construction had been completed. In addition, we purchased twenty 7-week-old female NOD mice from the Jackson Labs NOD/ShiLtJ mouse colony and maintained them in the same conditions as the mice from the NOD/LtJ-HMR colony. The onset of diabetes for female NOD mice from both the NOD/LtJ-HMR and NOD/ShiLtJ mouse colonies was between 12–15 weeks of age and the cumulative incidence of diabetes also reached 70–100% in both mouse colonies by 32 weeks of age (Figure
The lower incidence of diabetes is not due to a genetic drift in our colony. The onset of diabetes was monitored in six female NOD mice from the NOD/LtJ-HMR mouse colony born more than two months after the construction had been completed (open circles) as well as from twenty female NOD/ShiLtJ mice bought from Jackson Labs and maintained at the HMR facility (closed triangles). The cumulative diabetes incidence shows no difference between these two colonies. Log-rank test,
Of interest, all of the six female NOD mice of the NOD/LtJ-HMR colony from Figure
Nearby construction affects diabetes onset and cumulative incidence in NOD mice born from the same breeder pair. The cumulative incidence of diabetes is compared for female NOD mice born from the same breeder pair at different times, namely, those who nearby construction occurred during their embryonic development (closed circles,
Islet destruction and lymphocytic infiltration in nondiabetic NOD mice subject to construction stress. The number of islets per pancreatic section was scored as exemplified by the histology sections presented (scale 200
As mentioned previously, of the seven NOD female offspring born from mothers subject to construction stress during gestation, only two mice progressed to overt diabetes. Therefore, the environmental stress imposed by the construction during embryonic development appears to delay, but not necessarily impede, the autoimmune reaction towards pancreatic islet antigens. As such, we evaluated the subclinical progression of autoimmunity by quantifying the degree of insulitis and islet cell destruction. As expected, all diabetic NOD mice presented with few pancreatic islets and heavy lymphocyte infiltrates, suggesting an active autoimmune process (Figure
To further define whether an active autoimmune response was ongoing in the five nondiabetic NOD/LtJ-HMR mice which were subject to construction stress during embryonic development, we determined the serum insulin autoantibody (IAA) levels. Serum IAA levels correlate with autoimmune diabetes onset in both humans and NOD mice and the serum IAA levels eventually decline with disease progression [
Serum IAA levels as an indication of an ongoing autoimmune response towards pancreatic islets. The serum IAA levels are shown for every mouse group and segregated according to the health status (i.e., diabetic versus nondiabetic). Note that there are no nondiabetic mice from the NOD/LtJ-HMR mice born after construction. Each dot represents one mouse.
Limits of this current study include the low number of mice analyzed in our cohorts and the difficulty in reproducing similar events. However, similar variations in diabetes incidence have previously been documented by the Jackson Laboratory in much larger NOD animal cohorts. Indeed, the Jackson Laboratory has previously reported a modest effect of nearby construction on the incidence of diabetes in their NOD/ShiLtJ mouse colony (
Another factor which could have influenced the incidence of diabetes in our study includes variations in the microbiome of the mouse colony over time. The animal house barrier status is known to influence the incidence of diabetes in NOD mice [
In addition to the microbiome and hormone levels, elevated serum IAA levels from the mothers have also been shown to significantly impact diabetes incidence [
In summary, many parameters can influence diabetes incidence in NOD mice and there are possible alternative explanations as to why the mouse cohorts subject to construction stress during gestation showed a reduced incidence of diabetes. Nevertheless, the fact that litters born from the same mother, where one litter was subject to construction stress and the other was not, exhibited a significant difference in diabetes incidence suggests that construction stress during gestation modulates biological responses. Our results thus add to the observations from the Jackson Laboratory documenting a potential influence of nearby construction on the incidence of diabetes in NOD mice.
Altogether, these results emphasize the importance of surrounding factors which should be taken into consideration when performing long-term
The authors wish to thank Adam-Nicolas Pelletier for expert help with statistical analyses. E. E. Hillhouse and R. Collin currently hold a Diabète Québec scholarship. E. E. Hillhouse was also a recipient of a CIHR Ph.D. scholarship. S. Lesage holds a CIHR New Investigator Award and is currently funded by the Canadian Foundation for Innovation and the Natural Sciences and Engineering Research Council of Canada. The weblink in the manuscript presents data obtained by The Jackson Laboratory, which was funded by The National Center for Research Resources (NCRR) at NIH.