SummaryIn the past several decades, the therapeutic use of anabolic androgenic steroids (AAS) has been overshadowed by illicit use of these drugs by elite athletes and a growing number of adolescents to enhance performance and body image. As with adults, AAS use by adolescents is associated with a range of behavioral effects, including increased anxiety and altered responses to stress. It has been suggested that adolescents, especially adolescent females, may be particularly susceptible to the effects of these steroids, but few experiments in animal models have been performed to test this assertion. Here we show that chronic exposure of adolescent female mice to a mixture of three commonly abused AAS (testosterone cypionate, nandrolone decanoate and methandrostenolone; 7.5 mg/kg/day for 5 days) significantly enhanced anxiety-like behavior as assessed by the acoustic startle response (ASR), but did not augment the fear-potentiated startle response (FPS) or alter sensorimotor gating as assessed by prepulse inhibition of the acoustic startle response (PPI). AAS treatment also significantly increased the levels of corticotropin releasing factor (CRF) mRNA and somalassociated CRF immunoreactivity in the central amygdala (CeA), as well as neuropil-associated immunoreactivity in the dorsal aspect of the anterolateral division of the bed nucleus of the stria terminalis (dBnST). AAS treatment did not alter CRF receptor 1 or 2 mRNA in either the CeA or the dBnST; CRF immunoreactivity in the ventral BNST, the paraventricular nucleus (PVN) or the median eminence (ME); or peripheral levels of corticosterone. These results suggest that chronic AAS treatment of adolescent female mice may enhance generalized anxiety, but not sensorimotor gating or learned fear, via a mechanism that involves increased CRF-mediated signaling from CeA neurons projecting to the dBnST.

Stimulation of postnatal neurogenesis in the subventricular zone (SVZ) and robust migration of neuroblasts to the lesion site in response to traumatic brain injury (TBI) is well established in rodent species; however, it is not yet known whether postnatal neurogenesis plays a role in repair after TBI in gyrencephalic species. Here we describe the anatomy of the SVZ in the piglet for the first time and initiate an investigation into the effect of TBI on the SVZ architecture and the number of neuroblasts in the white matter. Among all ages of immaturity examined the SVZ contained a dense mesh network of neurogenic precursor cells (doublecortin+) positioned directly adjacent to the ependymal cells (ventricular SVZ, Vsvz) and neuroblasts organized into chains that were distinct from the Vsvz (abventricular SVZ, Asvz). Though the architecture of the SVZ was similar among ages, the areas of Vsvz and Asvz neuroblast chains declined with age. At postnatal day (PND) 14 the white matter tracts have a tremendous number of individual neuroblasts. In our scaled cortical impact model, lesion size increased with age. Similarly, the response of the SVZ to injury was also age dependent. The younger age groups that sustained the proportionately smallest lesions had the largest SVZ areas, which further increased in response to injury. In piglets that were injured at 4 months of age and had the largest lesions, the SVZ did not increase in response to injury. Similar to humans, swine have abundant gyri and gyral white matter, providing a unique platform to study neuroblasts potentially migrating from the SVZ to the lesioned cortex along these white matter tracts. In piglets injured at PND 7, TBI did not increase the total number of neuroblasts in the white matter compared to uninjured piglets, but redistribution occurred with a greater number of neuroblasts in the white matter of the hemisphere ipsilateral to the injury compared to the contralateral hemisphere. At 7 days after injury, less than 1% of neuroblasts in the white matter were born in the 2 days following injury. These data show that the SVZ in the piglet shares many anatomical similarities with the SVZ in the human infant, and that TBI had only modest effects on the SVZ and the number of neuroblasts in the white matter. Piglets at an equivalent developmental stage to human infants were equipped with the largest SVZ and a tremendous number of neuroblasts in the white matter, which may be sufficient in lesion repair without the dramatic stimulation of neurogenic machinery. It has yet to be determined whether neurogenesis and migrating neuroblasts play a role in repair after TBI and/or whether an alteration of normal migration during active postnatal population of brain regions is beneficial in species with gyrencephalic brains.

The piglet scaled cortical impact model creates a focal contusion using a skull-mounted, spring-loaded blunt indentation device scaled to achieve identical tissue strains in subjects with different brain sizes. Preliminary data showed that contusion size increased proportional to subject age. This study details the results from a new, larger series of subjects of three ages, and compares the effect of age and additional host and physiologic variables on injury response. Sixty-seven subjects, including infant (5- to 7-day-old), "toddler" (1-month-old), and early adolescent (4-month-old) swine underwent scaled cortical impact under strict anesthetic protocols. Serum glucose, testosterone, and 17beta-estradiol levels were measured. Lesion size was measured at 1 week post injury, as the ratio of the lesion area over the area of the contralateral hemisphere. Adolescent subjects had lesions over eight times larger than infants (p < 0.0001). Lesion volumes were larger in toddlers than in infants, most significantly for males (p < 0.05). Adolescent subjects were warmer on average, but there was no correlation between temperature and lesion volume within any age group. Serum glucose did not differ among ages. Infant males had the highest levels of circulating sex steroids. In this model, age was the most robust predictor of lesion size. Temperature had an effect, but did not explain all the variability seen among age groups. There was an interaction among gender, hormone levels, and lesion size in younger subjects. Characterization of these variables allows use of this model for treatment trials for subjects at different stages of maturation.

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Anabolic-androgenic steroids (AAS) are synthetic derivatives of testosterone that are illicitly selfadministered for enhancement of performance and body image, but which also have significant effects on the brain and on behavior. While the stereotypical AAS user is an adult male, AAS abuse in women is rapidly increasing, yet few studies have examined AAS effects in female subjects. We have assessed the effects in female mice of a combination of commonly abused AAS on neuronal activity and neurotransmission mediated by γ-aminobutyric acid type A (GABA A ) receptors in the medial preoptic nucleus (MPN); a nexus in the circuits of the hypothalamus and forebrain that are critical for the expression of social behaviors known to be altered in AAS abuse. Our data indicate that chronic exposure to AAS resulted in androgen receptor (AR)-dependent upregulation of α 5 , β 3 and δ subunit mRNA. Acute application of the α 5 subunit-selective inverse agonist, L-655,708, indicated that a significant fraction of the synaptic current is carried by α 5 -containing receptors and that AAS treatment may enhance expression of α 5 -containing receptors contributing to synaptic, but not tonic, currents in the MPN. AAS treatment also resulted in a significant decrease in action potential frequency in MPN neurons that was also correlated with an increased sensitivity to L655,708. Our data demonstrate that chronic exposure to multiple AAS elicits significant changes in GABAergic transmission and neuronal activity that are likely to reflect changes in the expression of α 5 -containing synaptic receptors within the MPN. Keywordsreal time PCR; patch clamp; medial preoptic area; L-655; 708; flutamide Anabolic-androgenic steroids (AAS) are synthetic derivatives of testosterone originally devised for the treatment of hypogonadal dysfunction in men, initiation of delayed puberty, and growth promotion (Basaria et al., 2001;Shahidi, 2001), but are now predominantly selfadministered to enhance performance or body image (for review, Kochakian and Yesalis, 2000;Llewellyn, 2007). Humans administer a myriad of AAS in complex regimes © 2009 IBRO. Published by Elsevier Ltd. All rights reserved.Corresponding author: Leslie P. Henderson, Departments of Physiology and of Biochemistry, Dartmouth Medical School, Hanover, NH 03755 USA, Phone: 603-650-1312, FAX: 603-650-1128, Email: leslie.henderson@dartmouth.edu. Section Editor: Dr. Yoland Smith, Yerkes National Primate Research Center, Emory University, 954 Gatewood Road NE, Atlanta, GA 30329, USA * These authors contributed equally to the work Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers th...

We have developed the first immature large animal translational treatment trial of a pharmacologic intervention for traumatic brain injury (TBI) in children. The preclinical trial design includes multiple doses of the intervention in two different injury types (focal and diffuse) to bracket the range seen in clinical injury and uses two post-TBI delays to drug administration. Cyclosporin A (CsA) was used as a case study in our first implementation of the platform because of its success in multiple preclinical adult rodent TBI models and its current use in children for other indications. Tier 1 of the therapy development platform assessed the short-term treatment efficacy after 24 h of agent administration. Positive responses to treatment were compared with injured controls using an objective effect threshold established prior to the study. Effective CsA doses were identified to study in Tier 2. In the Tier 2 paradigm, agent is administered in a porcine intensive care unit utilizing neurological monitoring and clinically relevant management strategies, and intervention efficacy is defined as improvement in longer term behavioral endpoints above untreated injured animals. In summary, this innovative large animal preclinical study design can be applied to future evaluations of other agents that promote recovery or repair after TBI. AbstractWe have developed the first immature large animal translational treatment trial of a pharmacologic intervention for traumatic brain injury (TBI) in children. The preclinical trial design includes multiple doses of the intervention in two different injury types (focal and diffuse) to bracket the range seen in clinical injury and uses two post-TBI delays to drug administration. Cyclosporin A (CsA) was used as a case study in our first implementation of the platform because of its success in multiple preclinical adult rodent TBI models and its current use in children for other indications. Tier 1 of the therapy development platform assessed the short-term treatment efficacy after 24 h of agent administration. Positive responses to treatment were compared with injured controls using an objective effect threshold established prior to the study. Effective CsA doses were identified to study in Tier 2. In the Tier 2 paradigm, agent is administered in a porcine intensive care unit utilizing neurological monitoring and clinically relevant management strategies, and intervention efficacy is defined as improvement in longer term behavioral endpoints above untreated injured animals. In summary, this innovative large animal preclinical study design can be applied to future evaluations of other agents that promote recovery or repair after TBI.

Experiments were conducted to determine whether exogenous estradiol-17beta (E2) and oxytocin (OT) can be used to improve transcervical (TC) embryo transfer (ET) procedures for sheep. Our concerns that the E2-OT treatment may alter luteal function prompted Exp. 1, in which 32 ewes were assigned to treatments in a 2x2 factorial array. On d 7 after onset of estrus, ewes received i.v. either 100 microg of E2 or diluent; 12 h later, ewes received i.v. either 400 USP units of OT or saline. To monitor luteal function, progesterone was measured in jugular blood collected from d 7 to 18. The treatments did not affect progesterone concentrations. Two trials were conducted in Exp. 2. In Trial 1, ewes were assigned to one of three treatments: TC transfer with E2-OT treatment to dilate the cervix, laparoscopic ET with E2-OT treatment, or laparoscopic ET with an equivalent diluent that did not dilate the cervix. In Trial 2, ewes were assigned to treatments in a 2x2 factorial array: TC or laparoscopic ET on d 6; E2-OT treatment for cervical dilation or diluents on d 6. Transferred embryos were recovered on d 12 in Trial 1 and d 14 in Trial 2, evaluated morphologically for development, and scored. Treatments did not affect the percentage of transferred embryos recovered. However, mode of transfer decreased (P<.01) the mean embryo development score. The E2-OT treatment increased (P<.01) the development score of embryos transferred transcervically, indicating that cervical dilation may improve the chances of embryos surviving after TC transfer. In conclusion, E2-OT treatment did not affect luteal function, and the E2-OT treatment can be used to enhance the success of TC embryo transfer in sheep.

Object Traumatic brain injury (TBI) is the leading cause of acquired disability in children, yet innate repair mechanisms are incompletely understood. Given data from animal studies documenting neurogenesis in response to trauma and other insults, the authors investigated whether similar responses could be found in children of different ages after TBI. Methods Immunohistochemistry was used to label doublecortin (DCX), a protein expressed by immature migrating neuroblasts (newborn neurons), in specimens from patients ranging in age from 3 weeks to 10 years who had died either after TBI or from other causes. Doublecortin-positive (DCX+) cells were examined in the subventricular zone (SVZ) and periventricular white matter (PWM) and were quantified within the granule cell layer (GCL) and subgranular zone (SGZ) of the dentate gyrus to determine if age and/or injury affect the number of DCX+ cells in these regions. Results The DCX+ cells decreased in the SVZ as patient age increased and were found in abundance around a focal subacute infarct in a 1-month-old non-TBI patient, but were scarce in all other patients regardless of age or history of trauma. The DCX+ cells in the PWM and dentate gyrus demonstrated a migratory morphology and did not co-localize with markers for astrocytes, microglia, or macrophages. In addition, there were significantly more DCX+ cells in the GCL and SGZ of the dentate gyrus in children younger than 1 year old than in older children. The density of immature migrating neuroblasts in infants (under 1 year of age) was significantly greater than in young children (2–6 years of age, p = 0.006) and older children (7–10 years of age, p = 0.007). Conclusions The main variable influencing the number of migrating neuroblasts observed in the SVZ, PWM, and hippocampus was patient age. Trauma had no discernible effect on the number of migrating neuroblasts in this cohort of patients in whom death typically occurred within hours to days after TBI.