7. Normal tissue damage.

The response in all normal tissues is due to killing and subsequent depletion of the critical parenchymal cells of that organ, and that the differences in the time it takes for damage to be expressed are due simply to differences in turnover kinetics of the target cells. Tissue damage is due to depletion of critical target cells and is not an indirect result of vascular damage.

8. Categories of arrays of damaged tissue; criteria necessary to value tissue effect.

Based on the difference in turnover kinetics of the critical target cells in different tissues, normal tissues can be divided into two categories:

• acutely responding

• late responding normal tissues

The acutely responding tissues manifest their injury within a few months after radiation is completed because they are self-renewal tissues containing rapidly dividing stem cell populations. Examples are the bone marrow, skin, intestine, and testis.

The late responding normal tissues do not express injury for at least 3 months or longer because they contain slowly dividing cell populations. Two examples of the latter are lung and kidney.

Two criteria are necessary for an assay of tissue effect to be of value:

• It must be quantifiable (i.e., assign a number to it).

• The effect must increase with increasing radiation dose.

9. Clonogenic assays. In Situ Assays.

Assays of damage in organized tissues and organs can be divided into three categories:

1. Clonogenic (related to the reproductive integrity of the clono­genic stem cells in the tissue).

2. Specific tissue function.

3. Lethality.

There are a number of such "clonogenic" assays for some normal tissues: bone marrow, testis, and mammary and thyroid glands, to name just a few. In some of these, clonogenic survival is assessed in situ in the same animal that was previously irradiated (skin, basal cells of the epidermis; intestine, number of surviving crypt cells; testis, number of tubules containing spermatogenic epithelium). In others, the cells are taken from the irradiated animal (donor) and transplanted into a genetically identical mouse for assay.

Recently, Withers and his colleagues have developed an in situ clonogenic assay for kidney, a late responding normal tissue. The un­derlying hypothesis on which the assay is based is that radiation dam­age in the kidney is due to depletion of the parenchymal cells, specifically the epithelial cells of the proximal tubules.

In Situ Assays

Withers and his colleagues have been instrumental in developing in situ clonogenic assays for a number of normal tissues, including skin, intestine, testis, and kidney. The intestine will be used to illustrate this technique.

The intestine is an example of a tissue in which dividing cells are confined to one location, the crypts of Lieberkühn. These cells divide about every 12 hours, replacing their own population, plus providing a constant supply of cells for the nondividing differentiated cells that are sloughed from the villi every 24 hours. The villi are dependent on the crypts for cell replacement; if the cells in the crypt are dead, the villi become shortened, flattened, and partially or completely denuded. In the intestine, then, damage is due to killing, with subsequent depletion of the cells in the crypts of Lieberkühn followed by denudation of the epithelial lining of the mucosa. Because the crypts are easily identified using the light microscope, the number of surviving crypts can be counted and plotted as a function of dose. Since it is assumed that a crypt can be repopulated by one surviving cell, a cell survival curve can be constructed