A Practical Approach, Second Edition=Ronald D. Ho.pdf

MATERNALLY MEDIATED EFFECTS ON DEVELOPMENT 113defects in various components of pathways or systems activated by stressors or toxic substances (e.g.,the LHPA axis 218 ). Use of such animal models could allow inferences to be made regarding the possibleinfluences of alterations in maternal physiology on offspring development and survival.B. Induction of Maternal Restraint Stress as a Model SystemPhysical restraint, the partial or complete limitation of movement, has been used extensively as aninducer of stress in laboratory animals, and Paré, Glavin, and their colleagues have reviewed itsuse. 213,219 Restraint has advantages as a stress inducer: It can be applied relatively consistently, itcan be used in a manner that is not likely to cause pain to the experimental subjects, it does notrequire elaborate or expensive equipment, and it does not involve toxicity, so purely stress-relatedeffects can be separated from maternal toxicity effects. Nevertheless, results seem to differ dependingon the exact methodology employed for restraint (as this determines the degree to whichmovement is restricted) and the length of the restraint period.Chernoff and coworkers have established maternal restraint as a relatively consistent inducerof supernumerary ribs (SNR) in the CD-1 strain mice. 58,59,69 Exact timing is critical, as fetuses frommice restrained on gestation day 8 (copulation plug day is day 0 throughout this chapter) from9:00 a.m. to 9:00 p.m. had a significantly elevated incidence of SNR, while those from damsrestrained during the following 12 h did not. When pregnant mice were restrained for 12 h on oneof gestation days 6 through 14, increased incidences of SNR were found only in fetuses from damsrestrained on days 7 or 8. 69 Rib fusion was seen in a few fetuses from mice immobilized (i.e.,completely restricted in their movement) in a supine position by use of Johnson and JohnsonElastikon ® surgical tape, but not in those from dams restrained in padded conical holders, whichallow some movement. This suggestion that the exact method of restraint may be important wasconfirmed by Rasco and Hood, 65 who found that a slight modification of the taping procedure couldconsistently influence the incidence of fused ribs in CD-1 mice.The CD-1 mouse is an appropriate animal model for the investigation of effects of maternalimmobilization stress on offspring development. The restraint method must be applied consistently,and it should be employed on gestation day 7 or 8 if SNR are to be used as an endpoint. The initialstudies generally employed 12-h restraint periods; shorter restraint periods of 8 or 9 h may not beconsistently effective. The most acute stress effects on the conceptus appear to result from maternalrestraint in the supine position by some means such as immobilization with surgical tape, but othermethods may prove to be adequate.Plastic conical tipped screw cap 50-ml centrifuge tubes, such as the Falcon brand produced byBecton Dickinson Labware, act as inexpensive, easily obtained restraint devices. They have frostedareas useful for marking numbers on the tubes for identification of the restrained animals andassignment to treatment protocols. Holes for breathing should be drilled in the closed end andelsewhere as desired. The holes can be made using an ordinary electric power drill, but the bitshould be as sharp as possible to minimize melting the plastic from friction during the drilling, asthis leaves rough edges. The location of the holes should be kept consistent among different tubes.This can be facilitated by designating the appropriate locations with a marking pen prior to drilling.However, investigators should take care to use tubes from the same manufacturer to maintainconsistency within a given study or laboratory, as 50-ml centrifuge tubes from various supplierscan vary in their inner diameter.Pregnant mice can be introduced to the open end of the tube, and with a little encouragementwill generally enter it. They will quickly begin to attempt to back out, but they can be kept in byuse of the screw cap. Other means of keeping mice in the tubes include drilling two holes onopposite sides near the open end, placing a bolt through both holes, and securing it in place witha nut. Similarly, one can place a long hypodermic needle through both holes and stick the sharpend into a cork to secure the needle in place. Use of the screw cap allows the mouse to move backand forth in the tube, while use of the bolt or needle can either allow such movement or, if placed© 2006 by Taylor & Francis Group, LLC