General Restraint Procedures and Experimental Design

Three-week-old CBA mice (Harlan Labs, Indianapolis, IN) were used in the initial studies because they are of intermediate susceptibility to the BeAn strain of TMEV, with a disease incidence of 70% (Welsh et al., 1987, 1989). Thus, any alterations in disease incidence due to the effects of stress could be readily detected from this baseline. In addition, the neuropathol-ogy, rates of viral clearance, and immune response to Theiler's virus have been previously characterized in this strain (Welsh et al., 1987,1989; Blakemore et al., 1988). Additional studies were performed with SJL mice, which are highly susceptible to TVID.

The BeAn strain of Theiler's virus (obtained from Dr. H. L. Lipton, Department of Neurology, Northwestern University, Chicago, IL) was propagated and amplified in BHK-21 cells. The culture supernatant containing infectious virus was aliquoted and stored at -70°C before use (Welsh et al., 1987).

2.1.3. Restraint Stress Protocol

Mice were handled for several minutes each day for 1 week prior to the initiation of restraint stress in order to habituate each mouse to human contact in an attempt to diminish stress due to handling during bleeding, cage changes, and any other contacts that might otherwise have altered stress levels.

Five-week-old mice were randomly assigned to one of three groups, 10 mice per group according to a previously reported protocol (Sheridan et al., 1991, Campbell et al., 2001) and treated as follows: (1) a control group where mice remained undisturbed in their home cages; (2) a group in which food and water (FWD) was withheld for 12h each of 5 nights per week over a 4-week period; (3) a group in which each mouse was placed in a well-ventilated restraining tube for 12h each of 5 nights per week. Half the mice in each of the three groups were either infected intracerebrally with Theiler's virus or similarly inoculated with virus-free BHK cell supernatant. Daily food and water deprivation or restraint began 1 day prior to infection and 5 days per week for 1 month postinfection. After the first series of experiments, we did not observe any differences between the food and water-deprived mice and the nonrestrained mice so in the following experiments the design was simplified to four groups noninfected/ nonrestrained; noninfected/restrained; infected/nonrestrained; and infected/ restrained.

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