Bone loss in a new rodent model combining spinal cord injury and cast immobilization.

OBJECTIVES: Characterize bone loss in our newly developed severe contusion spinal cord injury (SCI) plus hindlimb immobilization (IMM) model and determine the influence of muscle contractility on skeletal integrity after SCI. METHODS: Female Sprague-Dawley rats were randomized to: (a) intact controls, (b) severe contusion SCI euthanized at Day 7 (SCI-7) or (c) Day 21 (SCI-21), (d) 14 days IMM-alone, (e) SCI+IMM, or (f) SCI+IMM plus 14 days body weight supported treadmill exercise (SCI+IMM+TM). RESULTS: SCI-7 and SCI-21 exhibited a >20% reduction in cancellous volumetric bone mineral density (vBMD) in the hindlimbs (p⋜0.01), characterized by reductions in cancellous bone volume (cBV/TV%), trabecular number (Tb.N), and trabecular thickness. IMM-alone induced no observable bone loss. SCI+IMM exacerbated cancellous vBMD deficits with values being >45% below Controls (p⋜0.01) resulting from reduced cBV/TV% and Tb.N. SCI+IMM also produced the greatest cortical bone loss with distal femoral cortical area and cortical thickness being 14-28% below Controls (p⋜0.01) and bone strength being 37% below Controls (p⋜0.01). SCI+IMM+TM partially alleviated bone deficits, but values remained below Controls. CONCLUSIONS: Residual and/or facilitated muscle contractility ameliorate bone decrements after severe SCI. Our novel SCI+IMM model represents a clinically-relevant means of assessing strategies to prevent SCI-induced skeletal deficits.

Overexpression of int-5/aromatase in mammary glands of transgenic mice results in the induction of hyperplasia and nuclear abnormalities.

Our recent studies have shown that the cellular gene at the mouse mammary tumor virus integration site in the int-5 locus is aromatase. To study the role of int-5/aromatase in normal mammary development and mammary neoplasia, we have generated transgenic mice that overexpress int-5/aromatase under the control of mouse mammary tumor virus enhancer/promoter. All the transgenic virgin (n = 10) and postlactational (n = 15) females that overexpress int-5/aromatase show various histological abnormalities. Overexpression of int-5/aromatase in mammary glands of virgin females leads to the enlargement of 40% of ducts, of which 65% had hyperplastic lesions, 20% had dysplastic lesions, and 15% had fibroadenoma lesions. Overexpression of int-5/aromatase in involuted mammary glands of transgenic females induces hyperplasia in 75-80% of ducts and glands that exhibit a range of morphological abnormalities, including formation of hyperplastic alveolar nodule (10%), ductal and glandular hyperplasia (70-80%), ductal and lobular dysplasia (15%), and nuclear abnormalities (2-5%) such as multinucleation and karyomegaly, which are all indicative of preneoplastic changes. Our results show that early exposure of mammary epithelium to in situ estrogen and continued exposure to in situ estrogen as a result of overexpression of int-5/aromatase appears to predispose mammary tissue to preneoplastic changes, which may, in turn, increase the risk of developing neoplasia and increase susceptibility to environmental carcinogens. These findings support clinical and experimental data that suggest that early estrogen exposure increases breast cancer risk.

Human neurons that constitutively secrete A beta do not induce Alzheimer's disease pathology following transplantation and long-term survival in the rodent brain.

Since cultured neurons secrete beta-amyloid (A beta) peptides, and A beta forms amyloid deposits in the Alzheimer's disease (AD) brain, transplanted neurons may induce the deposition of A beta in the host brain. To assess this possibility, we studied grafted human neurons (NT2N cells) and their progenitors (NT2 cells) in the rodent brain. Although NT2N cells secrete more A beta than the NT2 cells in vitro, no A beta deposits or other AD lesions were induced in the rodent brain by grafts that survived days (NT2 and NT2N cells) to 46 weeks (NT2N cells). Thus, neither the deposition of A beta nor the induction of other AD lesions are obligatory consequences of the transplantation and long-term survival of human neurons or their progenitors in the rodent brain.

Neurons derived from a human teratocarcinoma cell line establish molecular and structural polarity following transplantation into the rodent brain.

Studies of neurons grafted into the brains of experimental animals have been limited by the lack of suitably homogeneous populations of neurons for transplantation. Here we describe the transplantation and survival of pure, postmitotic human neurons (NT2N cells) into the rat brain. NT2N cells were derived from a human teratocarcinoma line (NTera2/clone D1 or NT2 cells) in vitro by retinoic acid treatment. Approximately 5-10 x 10(4) NT2N cells (including previously frozen aliquots of NT2N cells) were injected into the neocortex, subjacent white matter, or hippocampus of adult (N = 51) or neonatal (N = 17) Sprague-Dawley rats. Cyclosporine (7-10 mg/kg) was administered daily to 13 adult rats for up to 12 weeks post-transplant prior to sacrifice. Untreated rats survived for up to 21 weeks post-transplant. Injection sites were serially sectioned and NT2N grafts were analyzed immunohistochemically using antibodies to diverse neuronal and glial proteins to assess the lineage of the grafted cells and their ability to establish molecular and structural polarity. NT2N cells transplanted into untreated adult and neonatal rat brains were committed exclusively to the neuronal phenotype and survived for as long as 8 weeks, although most were rejected after 4 weeks. However, cyclosporine prolonged survival of the NT2N grafts for up to 12 weeks. Further, grafted NT2N cells exhibited an asymmetric geometry (with long axons and simplified dendrites), as well as molecular polarity (with highly phosphorylated neurofilament proteins segregated in axons and microtubule associated protein 2 confined to perikarya and dendrites) by 4 weeks post-transplant. However, the grafted neurons did not become fully mature as evidenced by their failure to express the most highly phosphorylated heavy neurofilament proteins. Finally, previously frozen NT2N cells survived in the rat brain, and none of the grafts formed neoplasms. We conclude from these studies that transplanted NT2N cells represent a highly advantageous model system for studies of the developmental biology of neurons.

Fluorescence-activated separation of cervical abnormal cells using herpesvirus antigenic markers.

Antisera to total HSV-2 (G) viral antigens (Ra-2) and to increasingly purified viral antigenic fractions ("crude" and "pure" AG-e) specifically stain HSV-2-(G)- or HSV-1-(F)-infected HEp-2 cells in indirect immunofluorescence. Anti-"crude" AG-e sera induced by HSV-1 (F) or HSV-2 (G) produce a single precipitin band of identity when reacted in gel immunodiffusion against soluble antigenic mixtures from HSV-2-infected cells (HSV-2 (G) SAM). These reactivities are lost following adsorption of the sera with HSV-1 or HSV-2-infected cells or with pelleted or dextran gradient purified virions. Ra-2 and anti-"crude" or "pure" AG-e sera stain exfoliated cervical cells from patients with herpetic cervicitis as well as atypical cells from patients with atypia, carcinoma in situ or invasive cancer. Normal squamous cells do not stain. HSV-2 viral antigens recognized by the Ra-2 and anti-AG-e sera appear to constitute specific and sensitive markers for the separation of atypical cells in a fluorescence-activated cell sorter.