Tuesday, January 23, 2007

Screening for Androgens- part 2

Combination of Whole Animal and Isolated Organs Method (ex vivo)

* First involves treating immature or adult animals according to selected dosing regimen.
* Route, duration of exposure are not constrained.
* In addition to the acute exposures, exposures for ex vivo studies can actually begin as early as gestation and continue for as long as the life expectancy of animal being tested.
* During the in-life exposure period, blood samples can be collected at specified times for serum hormone analysis.
* After the exposure period is completed, animal is killed, at which time final necropsy
specimens collected, and the testis isolated.
* The gonads are then processed according to the type of in vitro preparation that was selected to assess the test substance’s effect.

Isolated Organ Methods (in vitro)

*The organs where steroidogenesis occurs removed from animal & kept viable, thereby providing an isolated organ method for assessing substances as stimulants of the
steroidogenic pathway.
* Organs, once isolated, can be used whole or further processed into sections or minced organ preparations
* These preparations allow effect of given test substance to be measured without influence of other organs or systems/other physiological factors.
* The integrity and interrelationship of the cells and tissues within the organ remain intact.
*These preparations retain the cellular and biochemical pathways that involve the receptor and second messenger.

# way in which the organ is processed.
*Whole organ-- after the organ is removed and placed in media, no further processing occurs
*Sectioned organ --For the testes, an organ section refers to an organ that has been cut such that each section constitutes 1/8 to ½ the size of the whole organ, i.e., 50 to 250 mg (rat testis). *Minced organ -- organ that has been cut into very small sizes, i.e., 1 to 50 mg (rat testis).

The perfused testis

# Provides a method to simulate biosynthesis and secretion of testicular steroid hormones
Developed for the rabbit by Vandemark and Ewing (1963), modified for the rat by Chubb and Ewing (1979).
# Apparatus is sterilized and then assembled.
# The system must be air-free to prevent bubbles from entering the perfusate and blocking flow.
# Testis is removed, spermatic artery cannulated, organ flushed of blood with perfusate solution.
# The testis is placed in the organ chamber and perfusion initiated.
# Test substance given after a preliminary perfusion for approximately 1 hour, the temperature (370 C), rate of flow (20 mL/hr), and pH (7.4) are monitored and maintained.
# The perfusion can be maintained for 6 to 10 hours.
# Samples are collected from the perfusate for analysis.

Simple Whole Organ Incubation technique

* Involves removal of the testis and incubation of the organ in medium for testing.
* For the whole testis (Deb et al., 1980), animal anesthetized or euthanized & testes removed.
* The testes are incubated in Kreb-Ringer-bicarbonate solution (pH 7.6) at 370 C & atmosphere of 95 percent O2 and 5 percent CO2
* Testes are incubated with or without the substance being tested, as well as with or without stimulant, e.g., LH.

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