In utero and lactational treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin impairs mammary gland differentiation but does not block the response to exogenous estrogen in the postpubertal female rat.

These experiments tested whether in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters mammary gland differentiation, estrogen receptor alpha (ERalpha) expression levels, or the response to estrogen in the female postpubertal rat mammary gland. Pregnant Holtzman rats were administered a single oral dose of 1 microg/kg TCDD or vehicle on gestation-day 15. Exposed and non-exposed female offspring were weaned on postnatal day 21 and ovariectomized at 9 weeks of age. Two weeks later, both TCDD and control animals were divided into 3 groups, receiving treatment with placebo, 0.025, or 0.1 mg 17beta-estradiol pellet implants. After 48 h, mammary tissue was removed for analysis following euthanasia. TCDD-exposed mammary glands demonstrated impaired differentiation as measured by the distribution of terminal ductal structures and increased expression levels of ERalpha. The response to exogenous estrogen was tested in TCDD-exposed animals and compared to control non-exposed animals. Estrogen stimulation of the TCDD-exposed glands induced progesterone receptor expression and mammary gland differentiation as measured by a shift in distribution from terminal end buds and terminal ducts to Types I and II lobules. Control glands were better differentiated at baseline and did not exhibit any significant changes in the distribution of terminal ductal structures following estrogen stimulation. The increase in progesterone receptor-expression levels by exogenous estrogen in control glands was similar to the TCDD-exposed glands. These experiments demonstrate that in utero and lactational exposures to TCDD impair mammary gland differentiation but that TCDD-exposed mammary glands retain the ability to differentiate in response to estrogen.

A simplified method to prepare PCR template DNA for screening of transgenic and knockout mice.

Polymerase chain reaction (PCR) amplification of DNA is the most widely used technique for screening of large numbers of genetically engineered transgenic or knockout mice (Mus musculus). In this report, we present a new DNA preparation procedure for running diagnostic PCR. In this procedure, mouse ear tissue was used directly for PCR after the tissue underwent brief digestion in a solution containing only proteinase K. Using this method, we have successfully screened several lines of single, double, and triple transgenic and knockout mice. The results are reliable and reproducible. The advantage of this new method is that DNA purification by organic extraction or isolation kit was omitted. DNA purification is the limiting factor in terms of time and money when screening transgenic and knockout mice by PCR. In addition, using ear instead of tail tissue can reduce distress of animals because the samples can be obtained when the mice are labeled by ear punch.

Ciliary neurotrophic factor stimulates nuclear hypertrophy and increases the GFAP content of cultured astrocytes.

Studies using transgenic mice that overexpress ciliary neurotrophic factor (CNTF), direct injection of CNTF into brain parenchyma, and ectopic expression of CNTF by an adenoviral vector have demonstrated that CNTF activates astrocytes. Paradoxically, studies to date have failed to show an effect of CNTF on the expression of GFAP by cultured astrocytes. Therefore, the goal of this study was to use nuclear hypertrophy and GFAP expression as indices of glial activation to compare the responsiveness of forebrain type 1 and type 2 astrocytes to CNTF. As reported by others, CNTF did not increase GFAP in type 1 astrocytes; however, it rapidly increased their nuclear size by 20%. Nuclear hypertrophy was apparent within 4 h after CNTF exposure and persisted for at least 48 h. In contrast, type 2 astrocyte GFAP increased 2-fold over the course of 48 h of CNTF treatment. During this same treatment period type 2 astroglial nuclei enlarged by 25%. We conclude that CNTF stimulates both type 1 and type 2 astrocytes directly. Together with our in vivo studies (Levison et al., 1996: Exp. Neurol. 141: 256), these data support the concept that CNTF is responsible for many of the progressive astroglial changes that appear after CNS injury and disease.

Ciliary neurotrophic factor stimulates astroglial hypertrophy in vivo and in vitro.

After insult or trauma, astrocytes become activated and endeavor to restore the brain's delicately balanced microenvironment. An index of their activated state is that they become enlarged or hypertrophic. Ciliary neurotrophic factor (CNTF), a member of the alpha helical family of cytokines, is synthesized by astrocytes and is generally regarded to be an autocrine and paracrine injury signal. To determine whether CNTF might be an endogenous signal that stimulates astrocyte hypertrophy in vivo, we intracerebrally injected 200 ng of recombinant human CNTF into the adult rat neocortex. To study the astrocytes their cytosol was stained with antibodies against S100beta and their nuclei were stained with propidium iodide (PI). Fluorescent images of astrocytic nuclei and somas were acquired using a confocal laser-scanning microscope and their areas were measured using the NIH image software. Within 24 h of treatment, CNTF induced a volume increase of the somas and nuclei of protoplasmic and fibrous astrocytes in vivo, and this effect persisted for at least 48 h. To determine whether CNTF activates astrocytes directly, glial cultures were treated with CNTF (10 ng/ml) and were evaluated by measuring the area of PI stained nuclei. CNTF stimulation increased the size of both polygonal and process-bearing astroglia. Since our studies in vivo have shown that CNTF induces other key aspects of gliosis (S. W. Levison et al., 1996; Exp. Neurol. 141, 256), we conclude that CNTF is a powerful activator of astrocytes and that it is likely responsible for the persistent glial hypertrophy observed following injuries and diseases of the CNS.